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RESEARCH ARTICLE
What determines sclerobiont colonization on
marine mollusk shells?
Vanessa Ochi Agostini
1,2☯
, Matias do Nascimento Ritter
3☯
*, Alexandre Jose
´Macedo
4‡
,
Erik Muxagata
1‡
, Fernando Erthal
5‡
1Laborato
´rio de Zoopla
ˆncton, Instituto de Oceanografia, Universidade Federal do Rio Grande (FURG), Rio
Grande, Rio Grande do Sul, Brazil, 2Programa de Po
´s-Graduac¸ão em Oceanografia Biolo
´gica, Instituto de
Oceanografia, Universidade Federal do Rio Grande (FURG), Rio Grande, Rio Grande do Sul, Brazil,
3Programa de Po
´s-Graduac¸ão em Geociências, Instituto de Geociências, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil, 4Faculdade de Farma
´cia and Centro de
Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil,
5Departamento de Paleontologia e Estratigrafia, Instituto de Geociências, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
☯These authors contributed equally to this work.
‡ These authors also contributed equally to this work.
*mnritter@gmail.com
Abstract
Empty mollusk shells may act as colonization surfaces for sclerobionts depending on the
physical, chemical, and biological attributes of the shells. However, the main factors that
can affect the establishment of an organism on hard substrates and the colonization pat-
terns on modern and time-averaged shells remain unclear. Using experimental and field
approaches, we compared sclerobiont (i.e., bacteria and invertebrate) colonization pat-
terns on the exposed shells (internal and external sides) of three bivalve species (Anadara
brasiliana,Mactra isabelleana, and Amarilladesma mactroides) with different external shell
textures. In addition, we evaluated the influence of the host characteristics (mode of life,
body size, color alteration, external and internal ornamentation and mineralogy) of sclero-
bionts on dead mollusk shells (bivalve and gastropod) collected from the Southern Brazilian
coast. Finally, we compared field observations with experiments to evaluate how the bio-
logical signs of the present-day invertebrate settlements are preserved in molluscan death
assemblages (incipient fossil record) in a subtropical shallow coastal setting. The results
enhance our understanding of sclerobiont colonization over modern and paleoecology per-
spectives. The data suggest that sclerobiont settlement is enhanced by (i) high(er) biofilm
bacteria density, which is more attracted to surfaces with high ornamentation; (ii) heteroge-
neous internal and external shell surface; (iii) shallow infaunal or attached epifaunal life
modes; (iv) colorful or post-mortem oxidized shell surfaces; (v) shell size (<50 mm
2
or
>1,351 mm
2
); and (vi) calcitic mineralogy. Although the biofilm bacteria density, shell size,
and texture are considered the most important factors, the effects of other covarying attri-
butes should also be considered. We observed a similar pattern of sclerobiont colonization
frequency over modern and paleoecology perspectives, with an increase of invertebrates
occurring on textured bivalve shells. This study demonstrates how bacterial biofilms may
influence sclerobiont colonization on biological hosts (mollusks), and shows how ecological
PLOS ONE | https://doi.org/10.1371/journal.pone.0184745 September 13, 2017 1 / 27
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OPEN ACCESS
Citation: Ochi Agostini V, Ritter MdN, Jose
´Macedo
A, Muxagata E, Erthal F (2017) What determines
sclerobiont colonization on marine mollusk shells?
PLoS ONE 12(9): e0184745. https://doi.org/
10.1371/journal.pone.0184745
Editor: Se
´bastien Duperron, UPMC, FRANCE
Received: November 4, 2016
Accepted: August 30, 2017
Published: September 13, 2017
Copyright: ©2017 Ochi Agostini et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This study was partially supported by the
FAPERGS (1982-2551/13-7). Additional funds
were covered by the CNPq (141217/2014-6 to VOA
and 140568/2014-0 to MNR), and by the
International Ocean Discovery Program (CAPES
0195/2016-02-BEX to MNR). The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
relationships in marine organisms may be relevant for interpreting the fossil record of
sclerobionts.
Introduction
The biological remains of invertebrates and vertebrates (shells, carapace, skeletons, and bones)
may act as colonization surfaces for invertebrates, especially on continental shelves covered by
unconsolidated substrates. Similarly, those remains act as colonization islands in these envi-
ronments and provide a supply of invertebrate larvae, which are essential for population per-
sistence in such regions. These biological remains are dominated by mollusk shells that can
remain for long time intervals at the sediment-water interface due to their relatively high dura-
bility (or in a safe zone of the taphonomically-active, [1]). Thus, mollusk shells provide a valu-
able archive of current and past generations of organisms and preserve the biological signals
despite the time-averaging of generations and taphonomic bias ([2] and references therein).
The intriguing relation in sclerobiont colonization (encrustation and bioerosion caused by
epi- and endobiont organisms, respectively, [3]) between a host and its colonizers has been
widely debated by several studies concerning the modern marine environments as well as
those related to the fossil record (e.g., [4–9] and references therein). On a paleontological per-
spective, the encrusting communities on hard substrates changed throughout the Phanerozoic
(since the Ordovician when were first expressed [9]), which provides a straightforward record
of competition and interactions (e.g., [10]). As a large proportion of sclerobiont species possess
highly preservable skeletons, they exhibit relatively good fossilization potential and retain the
spatial structure of the encrusting communities [9]. Additionally, the ecological and taphono-
mical relationships of modern encrusting organisms have been the focus of numerous studies
(e.g., [11–13], and references therein). Ancient biological interactions have also been explored
to understand the evolutionary relationships modulated by predation [14–16], and how
encrustation and bioerosion affect the interpretation of the fossil record (e.g., [8;17–19] and
references therein).
The invertebrates associated with sclerobiont colonization can be found in the zooplankton
community and are mostly represented by organisms with a meroplanktonic life-cycle (i.e.,
barnacles, some mollusks). Meroplankton expend part of their lives in the water column as lar-
vae drifting with ocean currents and the other part as adults in benthic or nektonic environ-
ments [20]. Holoplanktonic (i.e., some copepods) and thycoplanktonic (i.e., amphipods)
invertebrates can also be recorded on hard substrates and are classified as vagile or fouling
companion fauna [21–24].
There are many studies that have compared sclerobiont colonization patterns between dif-
ferent taxa and substrates [11]. However, there is still no consensus on the main factors that
can affect invertebrate colonization on biological substrates such as shells, carapaces, and
bones. However, the surface texture has frequently been cited [25–31] together with biological
factors, such as competition by recourses [32,33], conspecific presence [34,35], and ecological
inter-specific interactions [11,36], to induce or repulse settlement. Experimental arrays con-
ducted on non-biological hard substrates such as steel and concrete have demonstrated that
invertebrate settlement might be positively [37–39] or negatively [39–41] influenced by bacte-
rial biofilm. These biofilms are composed of multiple species of bacteria attached to a substra-
tum covered by an extracellular polymeric matrix, and their development can change the
Bacterial biofilm influences sclerobiont colonization
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Competing interests: The authors have declared
that no competing interests exist.
attractiveness of a hard substrate to periphyton, protozooplankton, seaweed and invertebrates
[11,42–44].
In this study, an experimental approach was used to compare the zooplankton and bacterial
biofilm colonization potentials on the shell of three species of bivalves with different external
textures. Furthermore, we evaluated the encrustation and bioerosion of a marine subtropical
deposit to assess the possible selectivity of sclerobionts in the fouling process on time-averaged
shells (accumulation of non-contemporaneous individuals in an assemblage; see review in
[2]), which simulated the upper limit of the taphonomically active zone (TAZ) [1]. The goals
were to assess the main factors that affect the colonization process on shells and observe how
much of the biological signal from present-day invertebrate larvae settlement is preserved in
the empty molluscan shells (death assemblage–incipient fossil record) over ecological and
paleoecological perspectives.
Materials and methods
Ethics statement
“Concheiros Beach” is located on the coast of Southern Brazilian, and is not included in the list
of sites of natural interest protected by law. Endangered mollusk taxa have not been reported
at the sampled location. Consequently, the field study did not involve endangered or protected
species. Live molluscan specimens were not collected in this study, and special permits were
not required to obtain empty shell material for scientific research in the study area. This study
is supported by the “Biofouling process under subtropical coastal conditions”, project super-
vised by Dr. Erik Muxagata and approved by PROPESP/FURG (http://www.propesp.furg.br)
(process 673520/2013, 06/2013 to 06/2017). The collect of zooplankton is permitted under the
Instituto Chico Mendes de Conservação da Biodiversidade (Sistema de Autorização e Informação
em Biodiversidade) permanent authorization number 1907371. The data from this study have
been archived as a PLoS One online-access appendix (S1,S2 and S3 Data).
Experiment observations: Zooplankton colonization
Shells of Anadara brasiliana (Lamarck, 1819), Mactra isabelleana d’Orbigny 1846 and Amaril-
ladesma mactroides (Reeve, 1854) (S1 Fig) were chosen for this experiment since they were
abundant and had distinct external textures with similar colors (white = natural or reduced
color). All shells (36 specimens, 12 of each species) were gathered from Concheiros Beach, RS,
Brazil (Fig 1B). The shells were immersed in sterile water in the laboratory, and three pulses of
20 kHz of a Cole-Parmer
1
4710 ultrasonic homogenizer were applied for 15 seconds on each
side of the shell [45] to detach the biofilm. Each shell was previously observed under a dissect-
ing microscope (Olympus BH-2) to ensure that there were no unique marks (i.e., predation,
bioerosion, encrustation, fragmentation), and categorized using their external ornamentation
(0 = A.mactroides; 2 = M.isabelleana; 3 = A.brasiliana) using criteria taken from the literature
(references in Table 1).
Later, the shells were placed in six bowls (20 cm in diameter, 18 cm in height) filled with
estuarine water (filtered through 20 μm mesh) to a height of 10 cm and kept at a constant salin-
ity (23±2), temperature (25˚C) and photoperiod (14L:10D). These conditions were chosen to
simulate the current subtropical conditions found in this region. A 5 cm-thick layer of natural
estuarine sediment was included as substrate at the bottom of each bowl to simulate the rein-
troduction of the shells to the upper part of the taphonomically active zone [1]. The shells were
inserted in the sediment (~2 cm) in a way that allowed both the internal (concave) and the
external (convex) sides to be exposed to the six replicates, and the shells were arranged in an
interleaved manner (S2 Fig). The sizes of the shells belonging to the same species were similar,
Bacterial biofilm influences sclerobiont colonization
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Fig 1. Study area on the southern Brazilian coast. (A) Patos Lagoon estuary where the experimental step was
conducted. (B) “Concheiros Beach” where the samples were collected.
https://doi.org/10.1371/journal.pone.0184745.g001
Bacterial biofilm influences sclerobiont colonization
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but the sizes were different among species (21 to 22 mm
2
for A.mactroides, 7 to 8 mm
2
for A.
brasiliana and 9 to 10 mm
2
for M.isabelleana). Thus, the zooplankton colonization density on
shells was standardized to 25 mm
2
. Once a week, the seawater was partially renewed (50%),
and the zooplankton community was also replaced. A supply of fresh plankton for the experi-
mental study was collected from the channel the Patos Lagoon estuary, which is located in Rio
Grande on the southern Brazilian coast (32˚08’53”S– 52˚06’03”W) (Fig 1A). Two samples
were collected using a conventional conical plankton net (200 μm of mesh) equipped with a
flowmeter. After collection, the plankton samples were filtered through a 500 μm mesh net to
remove the large planktonic predators. One sample was split into six equal parts (Motoda split-
ter) and placed into the bowls, while the other sample was fixed (formaldehyde 4%) to analyze
the potential of the zooplankton to colonize the shells.
To assess the zooplankton potential (the relationship between the invertebrates present in
the water column and the colonizers on available substrates), the composition in each zoo-
plankton sample was estimated from aliquots (1–5% of the sample) counted on a Bogorov
chamber, and the results were compared to the occurrence on the shells. A General Linear
Model (GLM) analysis was performed to evaluate the differences between the density of the
settled zooplankton and the richness of the bivalve shell species and the exposed shell side
(internal and external). A post hoc Tukey test followed the analyses. A simple regression was
applied to evaluate the correlation between the settled zooplankton densities on the different
shells textures.
Experiment observations: Microbial biofilm colonization
To evaluate shell colonization by bacterial biofilms, five shells of each bivalve species (A.bra-
siliana,M.isabelleana and A.mactroides) were sterilized (see the section Experiment Observa-
tions: zooplankton colonization section) and attached to a pier located in the channel of the
Patos Lagoon estuary (Fig 1B) during the austral summer of 2014 (salinity 23±2, temperature
25˚C and photoperiod 14L:10D) (S2 Fig). The sizes of the shells were the same as those used
in the laboratory experiment. The shells were recovered after five weeks of exposure and
immersed in a sterile formaldehyde 4% solution (50 Ml) to fix the biofilm. In the laboratory,
Table 1. Categorical variables measured in this study.
Ecological variables Key More information/
Methodology
Class 0 = Gastropoda; 1 = Bivalvia Rios [47]
Surface size class (mm
2
)<50; 51–150; 151–450; 451–1350; >1350 Rodland et al. [19]
Habitat of origin 0 = deep infaunal; 1 = shallow infaunal; 2 = attached infaunal; 3 = free-living epifaunal Rios [47], Mikkelsen and
Bieler [48]
Mineralogy 1 = calcite; 2 =aragonite; 3 = bimineralic Mikkelsen and Bieler [48]
Sclerobionts (bioerosion or
encrustation)
0 = absent; 1 = present; 1.1 = drill; 1.2 = sponge; 1.3 = worn; 1.4 = bryozoan; 1.5 =
‘fungae’; 1.6 = polychaete; 1.7 = bivalve; 1.8 = barnacle; 1.9 = foraminifera; 1.10 = algae;
1.11 = hydrozoan; 1.12 = unidentified
Lecinsky et al. [49]
Secondary color (or color
alteration)
a
0 = color lost; 1 = natural; 2 = oxidized color; 3 = reduced color Callender et al. [4] and
Best [50]
External ornamentation
(complexity degree)
0 = absent; 1 = low; 2 = average; 3 = high Carl et al. [30]
Internal ornamentation 0 = absent; 1 = present Carl et al. [30]
a
oxidized colors (cream, yellow, ochre, and red); reduced colors (white, gray, and black)
https://doi.org/10.1371/journal.pone.0184745.t001
Bacterial biofilm influences sclerobiont colonization
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the biofilm was detached using three pulses of 20 kHz for 15 seconds on each side of the shells
with a Cole-Parmer
1
4710 –ultrasonic homogenizer [45].
The biofilm bacteria density (bact cm
-2
) was estimated using a flow cytometer (BD FACS-
Verse™). The comparative sizes (μm) and complexities of the cells were measured using a For-
ward Light Scatter (FSC-A) and a Light Side Scatter (SSC-A), using spherical beads as the
pattern [51–53]. However, the precise value of bacteria cell size was also estimated using epi-
fluorescence microscopy, which is considered a more accurate technique than flow cytometry
[54]. A total of 100 bacterial cells were measured for each bivalve species. The bacterial biomass
(pg C cell
-1
) was calculated using the allometric biovolume (μm
3
) conversion factors proposed
by Norland [55] and Sun and Liu [56].
To evaluate the microbial community, the biological material in suspension obtained from
each shell was filtered (1 mL) through polycarbonate filters (darkened with Irgalan Black),
stained with acridine orange (1%) and viewed under an epifluorescence microscope (Zeiss
Axioplan) at 1000X magnification. The bacterial morphotypes were classified according to
Zaritski [57]. The observations of the presence or absence of fungi and periphyton followed
the same methodology.
The GLM analysis was performed to evaluate the biofilm bacterial density on the different
bivalve shells. The model was adapted to the Poisson distribution with a “log” link function.
Post hoc Tukey tests followed the analyses. Simple and multiple regressions were applied to
evaluate the correlation between the settled zooplankton density and the biofilm bacteria den-
sity on the different shell textures.
Field observations: Mollusk assemblages
To quantify the biofouling on the time-averaged mollusk assemblages, samples were collected
from Concheiros Beach (Fig 1B; 33˚32’6” S– 53˚5’37” W) on the Southern Brazilian coast in
December 2013. This locality is well known to have dense bioclastic concentrations formed by
shells mobilized from the inner continental shelf during storm events. Five to seven replicate
quadrats (300 x 300 cm) were delimited, and the uppermost 5-cm sediment layer was collected.
A total of 11 transects were sampled. Two transects were placed at a distance of 20 meters
from the lowest sea level height in the upper supralittoral zone parallel to the shoreline; two
were placed in the intertidal area perpendicular to the coastline, and the remaining seven tran-
sects were placed in the lower supralittoral zone parallel to the shore (Fig 1C).
All shell remains collected from each quadrat were identified and stored in plastic bags and
taken to the laboratory, where they were washed in fresh water and sieved using 500 μm
meshes. Host and fouling organisms were identified to the lowest possible taxonomic level
according to Roland et al. [5], Brett et al. [8,7], Rios [47], Buckup and Bond-Buckup [58],
Lopes [59], Barclay et al. [60]. Host organisms were characterized according to their (i) life
modes (deep infauna, shallow infauna, free-living epifauna, or attached epifauna), (ii) orna-
mentation complexity, both internal (present or absent) and external, with complexity varying
from absent, little, average to high, (iii) predominant mineralogy (aragonite, calcite, biminera-
lic) and (iv) categorical color (natural, reduced, oxidized) (Table 1). The marks left by fouling
organisms were also considered (bioerosion); they were identified and quantified under a ste-
reoscopic microscope to determine presence or absence, coverage percentage, and the location
of the colonization on the shell (internal or external). Taphonomic analyses were also carried
out on all shells (S3 and S4 Data, S1 Table).
The area-size and shell data were transformed into categorical variables used to observe the
occurrence frequency (%) of sclerobionts (bioerosion + encrustation) between different life
modes, shell sizes, colors, ornamentations, and mineralogy. The GLM analysis was carried out
Bacterial biofilm influences sclerobiont colonization
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to test for significant differences. The model was adapted to the data using a binomial/multi-
nomial distribution with a “logit” link function. Post hoc Tukey tests followed the analyses. A
Spearman rank correlation was performed to verify the relationship between the different cate-
gorical variables and identify any possible covariances among them. All analyses were carried
out in R [61].
Results
Experiment observation: Zooplankton colonization
The meroplanktonic components represented 25% (3,434 organisms m
-3
) of the zooplankton
samples collected from the channel in the Patos Lagoon estuary. Holoplankton components
represented 74% of the samples and thycoplankton represented 1%. However, the mero-
plankton contained a higher number of groups than the other components (Fig 2A). The
dominant meroplanktonic organisms were gastropods (339±426 org m
-3
), followed by
bivalves (190±228 org m
-3
), barnacles (139±87 org m
-3
), hydromedusae (29±36 org m
-3
),
Fig 2. Zooplankton potential colonization on shells. (A) Total occurrence frequency (%) of holoplankton, thycoplankton, and meroplankton in
zooplankton samples. (B) Zooplankton potential on sampled colonizing shells. (C) Settled zooplankton (%) on shells.
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Bacterial biofilm influences sclerobiont colonization
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polychaetes (22±22 org m
-3
) and decapods (10±17 org m
-3
). During the experiment, the natu-
ral zooplankton community changed their composition, although copepods always repre-
sented the highest fraction (Fig 2B). Slight differences in the settled zooplankton composition
on shells were observed between the different substrates. Bivalves, gastropods, and barnacles
were all present on all shells. However, decapods were only recorded on Anadara brasiliana,
while copepods were only recorded on A.brasiliana and Mactra isabelleana shells, and
hydrozoan polyps were only found on Amarilladesma mactroides (Fig 2C). On the shells, we
observed significant differences in the zooplankton colonization density (p<0.001) (Fig 3A).
However, the richness was not affected (p= 0.243) (Fig 3B). No differences in the coloniza-
tion on the internal and external sides of shells were observed (density p= 0.280; richness
p= 0.111), although this factor may affect the invertebrate settlement density when interact-
ing with the substrate (p<0.041). A.brasiliana followed by M.isabelleana showed higher
densities and richness values of the zooplankton colonization on average compared to
A.mactroides (Fig 3). A positive (r = 0.806) and significant (F
(1,13)
= 24.132; p<0.001) correla-
tion between zooplankton colonization density and the different external ornamentation was
observed, with higher ornamentation values being more attractive.
Overall, regardless of the invertebrate’s composition, differences between the zooplankton
colonization of the internal and external surfaces of A.brasiliana shells were observed. The
inner surface had the highest average richness and was composed of primarily sedentary and
vagile invertebrates. For all shell species, the sedentary and vagile fauna showed the highest
density on the inner surfaces (Fig 3C).
Experiment observation: Microbial biofilm colonization
Significant differences (p<0.001) were observed in the bacterial densities (bact cm
-2
) of the
various bivalve species: A.brasiliana had the highest biofilm bacteria density (16.3×10
6
±2.885)
followed by M.isabelleana (4.6×10
6
±32.951) and A.mactroides (1.2×10
6
±473.448) (Fig 4A). A
positive (r = 0.896) and significant (F
(1,13)
= 49.278; p<0.001) correlation between the biofilm
bacteria density and the different external ornamentations of the shells was observed.
The bacterial biofilm community showed variations in cell sizes throughout the experiment
(Fig 4B). Amarilladesma mactroides had larger bacterial cells (~0.7 μm) than the other shells.
Bacteria from A.brasiliana and M.isabelleana showed an average cell size of ~0.63 and
~0.67 μm, respectively. However, the SSC-A axes from the cytometer graphs (see Fig 4B)
revealed that the bacteria cells on A.brasiliana and M.isabelleana shells were more complex
than the bacteria cells found on A.mactroides shells. Higher average bacterial biovolume
(μm
3
) and biomass (pg C cell
-1
) values were noted on A.mactroides at 13.18 and 0.114, respec-
tively. Anadara brasiliana and M.isabelleana had bacterial biovolumes of 11.87 and 12.62 μm
3
,
respectively, and biomasses of 0.112 and 0.113 pg C cell
-1
, respectively. Bacterial rods and coc-
cus shapes were observed on A.mactroides while bacterial coccus and diatoms (cf. Nitzschia)
were observed on M.isabelleana and A.brasiliana. Filamentous fungi were also recorded on
A.brasiliana (Fig 4C). A positive (r = 0.878) and significant (F
(2,27)
= 28.352; p<0.001) correla-
tion between settled zooplankton density, biofilm bacteria density and external shell ornamen-
tation was observed (S4 Fig).
Field observations: Mollusk assemblages
Of the 1,965 time-averaged mollusk shells (58 gastropods and 1,907 bivalves) collected from
Concheiros Beach, only 828 showed sclerobionts (encrustation or bioerosion). Encrusting
organisms were recorded on only 87 shells, but traces of these organisms were apparent on
741 shells. A significant difference was observed on the total sclerobiont colonization between
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the Bivalvia and Gastropoda classes (p<0.001). Table 2 presents a complete list of the bivalve
and gastropod species with their relative abundances.
The sclerobiont colonization was significantly different between the Gastropoda (p<0.001)
(Fig 5A) and Bivalvia species (p<0.001) (Fig 5B). The shells of Crepidula spp. and Glycymeris
spp. exhibited the highest number of sclerobionts among the Gastropoda and Bivalvia,
Fig 3. Zooplankton colonization on shells. (A) The colonization density on the internal and external surfaces of different shells. (B) The richness of
colonizers on internal and external surfaces. (C) Settled zooplankton composition (%) on different shells sides. The vertical lines denote the 95%
confidence intervals (standard error*1.96), and the lowercase letters indicate similarities (the same letters) or significant differences (different letters)
between the shells (Tukey test).
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Fig 4. Biofilm community on shells. (A) Bacterial biofilm density (bact cm
-2
) on different shells. (B) The relative size (FSC-A)
and complexity (SSC-A) of the bacterial cells measured by a flow cytometer. Each point represents a bacterial cell. The lighter
colors (central part) are related to higher density cells with a determined feature (size ×complexity) being characterized as one
population. (C) Microorganism communities stained with acridine orange under epifluorescence microscopy (1000X). The
vertical lines denote the 95% confidence intervals (standard error*1.96), and the lowercase letters indicate similarities (the
same letters) or significant differences (different letters) between the shells (Tukey test).
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respectively. Shells from the gastropods Epitonium sp. and Sinum sp., as well as the bivalves
Amarilladesma mactroides,Brachidontes sp., Laevicardium sp., and Perna perna, showed no
encrusting or bioeroding organisms.
The life modes and host sizes significantly (p<0.048) influenced the occurrence of sclero-
biont colonization (encrusters and bioeroders) on gastropods (Fig 6A and 6C) and bivalves
(p<0.001) (Fig 6B and 6D). The shallow infaunal and attached epifaunal mollusks showed
greater levels of colonization, which contrasted with the deep infaunal bivalves, which had
fewer sclerobionts. Apparently, color alteration of the substrate affects sclerobiont colonization
on gastropod (p<0.050; Fig 6E) and bivalve (p<0.001; Fig 6F) shells, as the oxidized (cream,
yellow, ochre, or red) shells were preferentially colonized.
The varying levels of external ornamentation in Gastropoda did not show any remarkable
influence on sclerobiont colonization (p= 0.581) (Fig 7A). In contrast, the ornamentation of
bivalve shells seems to be a key factor controlling the colonization process. Shells with average
and high degrees of external ornamentation complexity have significantly (p<0.001) more
sclerobionts than the bivalve shells with low degrees ornamentation complexity (Fig 7B), and
the same pattern was recorded on the internal surfaces, (p<0.001; Fig 7C). The shell mineral-
ogy also influenced colonization, with significantly more encrustation and bioerosion occur-
ring on bivalve shells composed predominantly of calcite (p<0.001; Fig 7D).
Despite these vital roles of these differences, most of the factors analyzed are covariates
(Table 3). Size is a key factor, which is significantly correlated with all variables, including
Table 2. Categorical classification of external ornamentation, mineralogy (1 = calcite; 2 = aragonite; 3 = bimineralic) and frequency of occurrence
(FO) data.
Taxonomic classification External ornamentation Mineralogy FO (%)
GASTROPODA
Pisania sp. 3 2 31
Buccinanops cochlidium 1 2 1
Sinum sp. 0 2 5
Adelomelon brasiliana 1 2 3
Crepidula protea 1 2 3
Olivancillaria urceus 0 2 2
Epitonium georgettinum 3 2 2
Unidentifiable not applicable not applicable 32
BIVALVIA
Mactra sp. 2 1 45.8
Pitar sp. 1 1 10.6
Glycymeris sp. 2 1 4.7
Perna perna 2 3 4.4
Ostrea sp. 2 2 1.6
Anadara brasiliana 3 1 1.4
Amiantis purpurata 2 1 0.8
Donax sp. 1 1 0.8
Crassostrea sp. 2 2 0.7
Chlamys sp. 3 2 0.3
Amarilladesma mactroides 0 1 0.1
Brachidontes rodriguezii 2 3 0.1
Laevicardium sp. 1 1 0.1
Pholas sp. 2 1 0.1
Unidentifiable not applicable not applicable 28.5
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Bacterial biofilm influences sclerobiont colonization
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Fig 5. Sclerobionts coverage on mollusks. (A) Gastropoda genera: Ade.: Adelomelon, Buc.: Buccinanops, Cre.: Crepidula, Epi.: Epitonium, Oli.:
Olivancillaria, Psa.: Psania, Sin.: Sinum. (B) Bivalvia genera: Ama.: Amalarillodesma, Ami.: Amiantis, Ana.: Anadara, Bra.: Brachidontes, Chls:
Chlamys, Cra.: Crassostrea, Don.: Donax, Gly.: Glycymeris, Lae.: Laevicardium, Mac.: Mactra, Ost.: Ostrea, Per.: Perna, Pho.: Pholas, Pit.: Pitar.
Und.: Unidentifiable.
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Fig 6. The occurrence of sclerobionts exposed to distinct life modes, sizes, and colors of thehost substrates. (A) Gastropod life modes. (B)
Bivalvia life modes. (C) Gastropod sizes (D) Bivalvia sizes. (E) Gastropod color. (F) Bivalvia color. Und.: Unidentifiable. The vertical lines denote the 95%
confidence intervals (standard error*1.96), and the lowercase letters indicate similarities (the same letters) or significant differences (different letters)
between the factors evaluated (Tukey test).
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taphonomic damage. Size is positively correlated with color and total taphonomic grade, while
it is negatively correlated with external ornamentation and mineralogy. Thus, for both gastro-
pods and bivalves, a higher average colonization was observed on shells larger than 1,351 mm
2
(gastropods p<0.037; bivalves p<0.019), while no significant differences were observed in the
smaller size classes (51–150 mm
2
for gastropods and <50 mm
2
for bivalves). When bioerosion
Fig 7. The occurrence of sclerobionts exposed to distinct ornamentation and mineralogy of the host substrates. (A) Gastropod external
ornamentation. (B) Bivalvia external ornamentation. (C) Bivalvia internal ornamentation. (D) Bivalvia mineralogy. Und.: Unidentifiable. The vertical lines
denote the 95% confidence intervals (standard error*1.96), and the lowercase letters indicate similarities (the same letters) or significant differences
(different letters) between the factors (Tukey test).
https://doi.org/10.1371/journal.pone.0184745.g007
Table 3. Spearman rank correlations between the shell factors evaluated (Table 1), and the total taphonomic grade (TTG) (see also S1 Table).
VARIABLES Size Color External ornamentation Mineralogy TTG
Life mode r = -0.460 r = -0.001 r = 0.768 r = 0.871 r = 0.162
p<0.001 p<0.938 p<0.001 p<0.001 p<0.001
Size r = 0.116 r = -0.407 r = -0.445 r = -0.111
p<0.001 p<0.001 p<0.001 p<0.001
Color r = 0.070 r = -0.015 r = 0.454
p= 0.002 p<0.509 p<0.001
External ornamentation r = 0.866 r = 0.376
p<0.001 p<0.001
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of the molluscan size classes was analyzed separately from encrustation, this pattern remained
the same. However, encrustation occurred preferentially on large (>1351 mm
2
) gastropod
shells (p<0.001), but no significant difference was observed for bivalve shells (p = 0.876) (Fig
8). The size frequency distributions of each taxonomic group (S3 Fig) and the taphonomic out-
comes (S5,S6,S7 and S8 Figs) are displayed in the supplementary data.
Several sclerobiont taxa were found colonizing the shells: Ostrea equestris Say, 1834; serpu-
lid polychaetes; Phragmatopoma caudata Krøyer in Mo¨rch, 1863; Amphibalanus improvisus
(Darwin, 1854); Stramonita haemastoma (Linnaeus, 1767) eggs; Crassostrea spp.; Pododesmus
rudis (Broderip, 1834), mytilid byssus; seaweed; Hydrozoa; Foraminifera; Bryozoa; and bioer-
oding Bryozoa, Porifera, Polychaeta and Bivalvia (Fig 9).
Discussion
Are zooplankton and biofilm bacteria colonization affected by different
shells?
The zooplankton richness potential corresponded to the settled organisms on the shells, with
the meroplanktonic larvae being the most representative (Fig 2A, 2B and 2C). However, the
settlement quantity did not reflect the meroplankton supply. As previous studies have
Fig 8. Sclerobiont occurrence on different shell size. (A) Encrustation occurrence on Gastropoda. (B) Bioerosion occurrence on Gastropoda. (C)
Encrustation occurrence on Bivalvia. (D) Bioerosion occurrence on Bivalvia.
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Fig 9. Some examples of sclerobionts that colonized molluscan shells gathered from Concheiros Beach, on the
Southern coast of Rio Grande do Sul, Brazil. (A) The inside of a Buccinanops gastropod that contained sclerobionts, such
as serpulid polychaete, bryozoans, and an oyster. On the external side of the shell, there is evidence of bioerosion by
Spionidea polychaeta (arrow). (B) A gastropod shell with a sand structure made by the polychaete Phagmatopoma caudata.
(C) A gastropod covered by bryozoans. (D) A fragment of a gastropod that was fouled by eggs of the bivalve Stramonita
Bacterial biofilm influences sclerobiont colonization
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demonstrated, larvae are attracted to light during settlement [62,63]. Therefore, due to the
glass walls of the bowls used in our study, some larvae (i.e., barnacles) settled on the glass,
thereby reducing the colonization potential on the shells.
Additionally, one meroplanktonic group that was available in the water column, the poly-
chaetes, did not settle on the shells. This phenomenon may relate to the spatial competition
with other groups that are more efficient in the settlement process [64], preference for other
substrate types [46] or orientations [65], absence of conspecifics [66] or even succession of eco-
logical needs [67], all of which can also explain the different group colonization between the
bivalve shells species. For example, Marshall and Keough [68] observed that smaller larvae
attach faster and are less selective than larger ones, and once one organism is present, it may
influence the active choice of the substrate by others using chemical signaling.
In the experimental approach, the highest encrustation density was observed on Anadara
brasiliana shells, which was probably due to the larger number of microhabitats available for
zooplankton colonization compared to Amarilladesma mactroides and Mactra isabelleana
shells. According to Carl et al. [30], surface microtopography can either induce or repel the lar-
val settlement of many marine organisms. We observed higher invertebrate colonization on
bivalve shells with texture (Fig 7). The microtopography had a strong effect in mytilids, where
400 μm (lower heterogeneity) textures enhanced the settlement at a rate of >90%. On the
other hand, larger or smaller topographies led to a much-reduced colonization, which corrob-
orated the work of Berntsson et al. [26,27] who showed that microtopographies between 30
and 45 μm inhibited colonization by barnacle cyprids (Cirripedia) by up to 92%. This pattern
might explain the absence of sclerobiont colonization on the shells of the Epitonium spp. gas-
tropod, which have a high topography (ridges a few μm away from each other). Additionally,
this genus is an epifaunal predator that spends time buried in the sand between feedings (i.e.,
partially infaunal). Intrinsic shell features, such as lower morphological heterogeneity (external
ornamentation), may also account for this phenomenon.
The presence of sclerobionts on shell interiors is related to post-mortem colonization [5],
which confirms the importance of this kind of substrate on sandy shelves. The encrusting
taxon richness showed no differences among shells, possibly because larvae can settle on any
surface type. The higher settlement densities of encrusting species recorded on A.brasiliana
shells suggest that larvae have a stronger affinity to heterogeneous substrates (presence of
microtopographies), and consider the external surface of the shell. In contrast, vagile/sedentary
species were recorded at higher densities on shell interiors. The concave position (internal
area exposed) of molluscan shells may provide protection to settling organisms since they do
not attach firmly to the substrate when compared to encrusting forms.
The substrate texture can also affect the bacteria colonization [69,70]. Thus, a similar pat-
tern was observed regarding the shells external ornamentation when the bacterial biofilms
were analyzed. A positive relationship was observed between bacteria and settled zooplankton
on bivalve shells. Heterogeneous substrata (e.g., A.brasiliana shells) exhibit higher bacteria
densities. It is known that surface roughness increases bacterial adhesion [71], as surface fea-
tures are essential to microbiological binding to a surface [72] and bacterial attachment is inde-
pendent of groove size and is greatest in the valley areas of the grooves [73]. However, in the
current study, the observed bacterial densities values for the three bivalve species are within an
haemastoma (arrow). (E) An external view of a Pholas bivalve shell with encrusting Ostreidae and bryozoans and a
sediment-tube made by a polychaete (arrow). (F) Internal view of another Pholas shell with several oysters (Ostrea
equestris) and bryozoans. (G) External view of an Anadara brasiliana bivalve shell with a boring sponge. (H) Internal view of
aMactra bivalve shell encrusted by Ostreidae. The hole was made by a spionidea polychaete. Scale bars: 10 mm.
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order of magnitude, being further studies needed to corroborate this pattern. Additionally, a
more complex microbial community, with diatoms and fungi were also observed on A.brasili-
ana, indicating a more mature biofilm, with greater bacterial biomass compared to those pres-
ent on A.mactroides and M.isabelleana shells. The small differences in the bacterial sizes
observed on the shells of A.mactroides,M.isabelleana, and A.brasiliana can be explained by
the space competition (related to bacterial density) between bacteria cells, which allows for the
various size increments of the bacteria cells [74]. Thus, the higher the bacterial density, the
smaller the bacterial size.
The relationship between bacterial biofilms and the colonization of invertebrates on hard
substrates (e.g., vessels, pipelines, piers) is already known [75,76;42]. This relationship, how-
ever, has not been proposed for settlement on shells. Contrary to the statement on biofilm pro-
duction Rodland et al. [18], the formation of a polymeric matrix over the internal and external
surface of a shell may attract zooplankton, and consequently enhance the colonization proba-
bilities of sclerobioic organisms (S4 Fig). According to Tamburri et al. [77], some oyster larvae
species prefer natural substrates (e.g., other oyster shells) covered with biofilms for settlement.
In addition, old shells are probably less attractive to larvae for settlement rather than fresh
shells, as described as the “fresh shell syndrome” by Brett et al. [7].
The shell texture influenced both the zooplankton and the bacteria colonization. However,
we believe that the bacteria biofilm exerts a greater effect on the settlement of invertebrates
(r = 0.828; r
2
= 0.687) than the substrate texture (r = 0.806; r
2
= 0.660), given the correlation
values obtained.
Is the experimental sclerobiont colonization pattern preserved on
mollusk assemblages?
The taphonomic alteration on mollusk beach assemblages could be a substantial bias concern-
ing the preservation of sclerobiont frequency [50]. However, the sclerobiont colonization in
dead (as in fossil) molluscan shells appears to remain almost intact despite the taphonomic
biases [9,18]. Thus, taphonomic alteration in our data also does not play a significant role in
sclerobiont colonization preservation on hosts (S5,S6,S7 and S8 Figs).
Sclerobiont colonization was more intense on shells with oxidized color, which was likely
due to their taphonomic alteration (see Tables 1and 3). Except for this study, information
about the effect of the color of the substrate on bacteria and zooplankton colonization has
been limited. Dobretsov et al. [78] investigated the effects of substratum color (black and
white) on the formation of micro and macrofouling communities and verified that higher den-
sities were observed on black hosts. Yule and Walker [79] and Monteforte and Garcia-Gasca
[80] described the same patterns in barnacles and oysters, respectively. These findings can be
explained as a result of the negative phototaxis of larvae [81], or the quantity of energy
(absorbed or reflected) and the consequent temperature of the substratum [78]. These works
emphasized the importance of substratum color on the formation of micro and macrofouling
communities, as corroborated in this study (see Fig 6E and 6F). In the experimental approach,
we only tested the colonization on white (reduced or natural colors) shells, which proved that
the sclerobiont colonization could occur even in this situation. However, these results do not
confirm the preference by oxidized shells from a modern perspective.
These shells have therefore revealed a complex taphonomic profile of preservation: oxidized
shells were related to ancient shorelines and shallow areas [82]. Thus, most of the oxidized
shells were produced by subaerial exposure during the sea level oscillations that have occurred
since the Last Glacial Maximum [82]. Hence, this pattern might be related to the durability of
the shells in the TAZ [1], which should increase the probabilities of larvae settlement, posterior
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encrustation and bioerosion. However, the relationship between color and temporal mixing
has not been empirically demonstrated. Furthermore, high frequency of encrustation is not
inevitably related to the colonization window time (but see Rodland et al. [18]). Thus, our
results indicate that oxidized color exhibited higher frequencies of encrustation and bioero-
sion, or shells with color alterations were more prone to preserve the encrusters/bioeroders on
their shells than those displaying reduced colors.
The mollusk assemblages are time-averaged and display the present-day ages up to ~56
kyrs on the adjacent inner shelf (but, the Holocene shells are numerically dominant; [83]).
Thus, these shells have experienced different time-windows regarding the sclerobiont coloni-
zation process. Obviously, the larvae pool has not been constant or taxonomically homogenous
along the time-averaging windows present in these death assemblages. Although encrustation
is considered an instantaneous event (snapshot) (limited-exposure scenario sensu Rodland
et al. [18]), older shells do not exhibit the current higher encrustation intensities or richness
when compared to younger shells [18]. However, it is difficult to determine at what moment
in this time-averaging window each sclerobiont settled since it is theoretically possible to find
an almost infinite number of non-contemporaneous organisms. However, long-term experi-
ments have shown that encrustation is established mainly in the first year, and the addition of
new taxa decreases with time [7].
Additionally, due to the “fresh shell syndrome” [7], shells attain much of their potential cov-
erage in the first few months; then the possibility of time-averaging of the biotic communities
is probably reduced. Thus, even the settling process is a geologically instantaneous event, and
the temporal acuity is limited to the host age, due to the analytical time-averaging [84]. Theo-
retically, any shell in a death assemblage possesses the same colonization potential when avail-
able at the seafloor, regardless of its age and taphonomic condition. Therefore, we believe that
these factors will have a null effect when the encrustation on shells with a wide age range [83]
is empirically tested.
The surface area plays a different role on colonization, as seen in Fig 6 and corroborates the
findings in Rodland et al. [18]. We observed no differences between the shells with small (<50
mm
2
for bivalves and 150 mm
2
for gastropods) and large areas (greater than 1351 mm
2
) when
considering encrustation and bioerosion together, or these factors separately (Fig 8). When
considering encrustation, the pattern observed for gastropods was the same as that detected by
Rodland et al. [5], where larger shells exhibited more severe encrustation. However, larger
bivalve shells are not necessarily susceptible to greater colonization because of their larger sur-
face areas. On the other hand, it remains unknown to what degree encrustation affects smaller
or fragmented shells, as this evidence may be erased due to taphonomic processes that occur
during the (wide) time-averaging window, as noted by Rodland et al. [18]. In addition, it was
difficult to state that bioerosion acted directly on small shells and fragments; larger bioclasts
may be bioeroded, encrusted and further fragmented, thereby losing their encrusters and only
retaining their record of bioerosion. This phenomenon may explain why either smaller (frag-
mented shells) or larger sizes displayed the greater frequencies of sclerobiont colonization (Fig
6C and 6D). In the experiment, all shells were smaller than 50 (mm
2
), which made a compari-
son impossible. However, the highest invertebrate densities and richness values were found
on the smallest A.brasiliana shells while the biggest shells (A.mactroides) had the lowest colo-
nization, which was explained by their lack of external texture related to their life mode
(covariables).
As shell size plays a major role in sclerobiont colonization, the significant correlation of
shell size with all other factors highlights that size class is negatively correlated with tapho-
nomic damage (Table 3). However, bigger shells showed slightly higher alterations than small
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shells (S5 Fig). Thus, since sclerobiont colonization is higher in bigger shells with slightly
higher taphonomic bias, it confirms that taphonomic alteration does not negatively influence
the preservation of sclerobiont traces on shells. Meanwhile, small fragments also displayed
high intensities of sclerobionts. This finding is probably due to the fragmentation of the colo-
nized bigger shells.
Regardless of these biases, the Anadara shells had the third highest occurrence of sclero-
bionts (%) (Fig 5A), thus, reinforcing the results of this experiment. Therefore, shell size is one
of the most crucial factors [19], with external ornamentation also playing a secondary role, as
experimentally demonstrated. It is difficult to account for this key element (except for shell
size) since mineralogy and life modes are also correlated with external ornamentation.
Remarkably, calcitic bivalves are more prone to encrustation or bioerosion. This difference
may be due to the high occurrences of Ostreidae colonization by other species of the same fam-
ily. Additionally, the occurrence of sclerobionts is greater in shallow infaunal species rather
than epifaunal species. Some of the shallow infaunal bivalve species, such as Glycymeris and
Pitar, showed a higher frequency of sclerobionts than Amarilladesma, a deep infaunal and rela-
tively unornamented bivalve. Nevertheless, veneroid and myoid bivalves evolved siphons in
the early Mesozoic and invaded the deep infauna [85] and are well represented in this study by
the relatively ornamented genus Pholas. However, the shells of Pholas displayed an occurrence
of sclerobionts comparable to Anadara shells, an epifaunal bivalve. Counter-intuitively, the
mode of life and the mineralogy are unlikely to play key processes alone. In the experiment, we
observed bacteria and zooplankton colonization on all bivalve shells, and all of these shells also
show aragonite mineralogy.
Interestingly, after the Marine Mesozoic Revolution (MRV) [86], bivalves declined in the
sediment column, which is well known as an infaunalization trend due to gastropod preda-
tion [87,88]. Meanwhile, external ornamentation probably also reflects the mode of life
on infaunal bivalves, which enhances its stability near the sediment-water interface [89].
External ornamentation also showed a positive correlation with a taphonomic alteration
(Table 3,S5 Fig). This correlation may be an indication of a megabias in the fossil record, as
relatively more ornamented species do not have higher preservability [90], but they also
presented greater occurrence of sclerobionts, thus diminishing their preservability potential
due to bioerosion. This finding could indicate that either shallow infaunal bivalve species
are more prone to be not preserved or that sclerobiont colonization is a negatively taphono-
mical bias that reduces the preservability of those species. However, encrustation could be a
positive bias, which increases the preservability of ornamented species. Thus, sclerobiont
colonization could be a two-way bias in the fossil record needing more attention in the
future.
Bivalve and gastropods shells showed differences in the factors that affected the sclerobiont
occurrence. For example, a larger external texture on the gastropod shells did not proportion-
ally reflect a greater colonization observation, nor did its mineralogy. One of the hypotheses in
this study proposed a relationship between these factors and other factors (mode of life, color,
taphonomic damage). These factors were hypothesized to that overlap with one another as
covariates affecting the invertebrate colonization. The other hypothesis raised is related to the
use of gastropods shells as housing for the vagile fauna (i.e., hermit crabs). Shell used as hous-
ing for vagile fauna are in constant movement, thereby preventing meroplankton settlement.
This pattern is already observed for different substrates and is associated with hydrodynamic
stress [91]. According to Walker [92], crab-inhabited shells show more encrusting organisms
which could also be explained by the possible alterations caused by the hermit crabs on the gas-
tropod shells that repel sclerobiont colonization.
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Conclusions
1. Zooplankton colonizes different shells, but the density and richness values are affected by
the attributes of Amarilladesma mactroides,Anadara brasiliana, and Mactra isabelleana
shells. Additionally, fouling invertebrates seem to be more associated with the external shell
sides, while vagile and sedentary fauna are more associated with the internal side.
2. The external shell texture seems to directly affect the bacteria biofilm density, as most
ornate surfaces are more attractive. Zooplankton colonization seems to respond directly to
bacteria density, the microbial biofilm community, and consequently to the external orna-
mentation of the shells.
3. Shell size is one of the most significant variables regarding sclerobiont colonization, as pre-
vious studies have documented. External ornamentation also plays at least a secondary role,
as experimentally demonstrated. However, all factors may have a covarying effect on sclero-
biont occurrence on the shells.
4. The sclerobiont occurrence patterns observed for bivalves do not apply in the same way to
gastropods (external ornamentation and life mode), which is probably related to other fac-
tors that were not evaluated.
5. Similar sclerobiont patterns were also found in experimental and assemblage deposit obser-
vations, despite the taphonomic biases. These observations allowed us to infer that an
experiment might be used to explain the paleontological patterns. However, as our study
has covered only three bivalve species experimentally, broader studies are still necessary.
Supporting information
S1 Data. Raw data on zooplankton abundance used in the analyses in this study.
(XLSX)
S2 Data. Biofilm density data used in this paper.
(XLSX)
S3 Data. Taphonomic scores of all shells from “Concheiros” Beach, Southern Brazil. The
table presents the raw data of the taphonomic scores of 1,965 shells (58 gastropods and 1,907
bivalve shells) used in this paper. See also S4 Data and S1 Table.
(CSV)
S4 Data. A more detailed description of the methods used (taphonomic analyses).
(DOCX)
S1 Table. Taphonomic protocol utilized in this study.
(DOCX)
S1 Fig. Species employed in the study screening for different external textures. (A) Amaril-
ladesma mactroides (Reeve 1854), external view. (B) Amarilladesma mactroides, internal view.
(C) Mactra isabelleana d’Orbigny 1846, external view. (D) Mactra isabelleana, internal view.
(E) Anadara brasiliana (Lamarck 1819), external view. (F) Anadara brasiliana, internal view.
Scale bars: 5 cm.
(TIF)
S2 Fig. Experimental diagrams employed in both the laboratory and the experimental field
steps of the current study. (A) Zooplankton colonization experiment. Each bowl (20 cm in
Bacterial biofilm influences sclerobiont colonization
PLOS ONE | https://doi.org/10.1371/journal.pone.0184745 September 13, 2017 21 / 27
diameter, 18 cm in height) was filled with estuarine water up to a height of 10 cm and kept at a
constant salinity (23±2), temperature (25˚C), and photoperiod (14L:10D). These conditions
were preferred to simulate the subtropical conditions found in this region. A 5 cm-thick layer
of natural sediment was included as substrate at the bottom of each bowl to simulate the upper
limit of the taphonomically active zone. (B) The field experiment in the channel of the Patos
Lagoon estuary in Southern Brazilian.
(TIF)
S3 Fig. Size-frequency distributions for each mollusk class collected. (A) Gastropoda.
(B) Bivalvia.
(TIF)
S4 Fig. Multiple regression analysis between bacterial density (bact cm
-2
) and zooplankton
colonization density (org 25 cm
-2
) regarding the external ornamentation of shells.
(TIF)
S5 Fig. Total taphonomic grade (percentage damage index) of intrinsic measured variables
in Bivalvia. The box plots are showing interquartile range, the 95% confidence intervals
and the outliers. (A) Size class. (B) External ornamentation. (C) Mineralogy. (D). Life mode.
All p-values were obtained from the Kruskal-Wallis Test. Und.: undetermined.
(TIF)
S6 Fig. Total taphonomic grade (percentage damage index) of the intrinsically measured
variables in Gastropoda. The box plots are showing the interquartile range, the 95% confi-
dence intervals and the outliers. (A) Size class. (B) Life mode. (C) External ornamentation.
All p-values were obtained from the Kruskal-Wallis Test. Und.: undetermined.
(TIF)
S7 Fig. Total taphonomic grade (percentage damage index) among Bivalvia species. The
box plots are showing the interquartile range, the 95% confidence intervals and the outli-
ers. Bivalvia genera: Ama.: Amalarillodesma, Ami.: Amiantis, Ana.: Anadara, Bra.: Brachi-
dontes, Chls: Chlamys, Cra.: Crassostrea, Don.: Donax, Gly.: Glycymeris, Lae.: Laevicardium,
Mac.: Mactra, Ost.: Ostrea, Per.: Perna, Pho.: Pholas, Pit.: Pitar. Und.: Unidentifiable. p-value
was obtained from the Kruskal-Wallis Test.
(TIF)
S8 Fig. Total taphonomic grade (percentage damage index) among Gastropod species. The
box plots are showing the interquartile range, the 95% confidence intervals and the outli-
ers. Gastropoda genera: Ade.: Adelomelon, Buc.: Buccinanops, Cre.: Crepidula, Epi.: Epitonium,
Oli.: Olivancillaria, Psa.: Psania, Sin.: Sinum. (B). p-value was obtained from the Kruskal-Wal-
lis Test.
(TIF)
Acknowledgments
The authors thank Michał Kowalewski and Claudio De Francesco for their useful comments
and discussion on the earlier version of the manuscript. We also would like to thank Fla
´vio
Lopes (UFRGS), who took the photographs in Fig 9.
Author Contributions
Conceptualization: Vanessa Ochi Agostini.
Bacterial biofilm influences sclerobiont colonization
PLOS ONE | https://doi.org/10.1371/journal.pone.0184745 September 13, 2017 22 / 27
Formal analysis: Vanessa Ochi Agostini.
Investigation: Vanessa Ochi Agostini, Matias do Nascimento Ritter.
Methodology: Vanessa Ochi Agostini, Matias do Nascimento Ritter.
Visualization: Vanessa Ochi Agostini, Matias do Nascimento Ritter.
Writing – original draft: Vanessa Ochi Agostini, Matias do Nascimento Ritter.
Writing – review & editing: Vanessa Ochi Agostini, Matias do Nascimento Ritter, Alexandre
Jose
´Macedo, Erik Muxagata, Fernando Erthal.
References
1. Olszewski TD. Modeling the influence of taphonomic destruction, reworking, and burial ontime-averag-
ing in fossil accumulations. Palaios 2004; 19: 39–50, https://doi.org/10.1669/0883-1351(2004)
019<0039:MTIOTD>2.0.CO;2
2. Kidwell SM. Time-averaging and fidelity of modern death assemblages: building a taphonomic foundation
for conservation paleobiology. Palaeontology 2013; 56: 487–522, https://doi.org/10.1111/pala.12042
3. Taylor PD., Wilson MA. Palaeoecology and evolution of marine hard substrate communities. Earth Sci
Rev 2003; 62: 1–103, https://doi.org/10.1016/S0012-8252(02)00131-9
4. Callender WR, Staff GM, Parsons-Hubbard KM, Powell EN, Rowe GT, Walker SE, Brett CE, Raymond
A, Carlson DD, White S, Heise EA. Taphonomic trends along a forereef slope: Lee Stocking Island,
Bahamas. I. Location and Water Depth. Palaios 2002; 17: 50–65, https://doi.org/10.1669/0883-1351
(2002)017<0050:TTAAFS>2.0.CO;2
5. Rodland DL, Kowalewski M, Simões MG, Carroll M. Colonization of a ‘‘Lost World”: encrustation pat-
terns in modern subtropical brachiopod assemblages. Palaios 2004; 19: 381–395, https://doi.org/10.
1669/0883-1351(2004)019<0381:COALWE>2.0.CO;2
6. Bromley RG, Heinberg C. Attachment strategies of organisms on hard substrates: A palaeontological
view. Palaeogeogr Palaeoclimatol Palaeoecol 2006; 232: 429–453, https://doi.org/10.1016/j.palaeo.
2005.07.007
7. Brett CE, Parsons-Hubbard KM, Walker SE, Ferguson C, Powell EN, Staff G, Ashton-Alcox KA, Ray-
mond A. Gradients and patterns of sclerobionts on experimentally deployed bivalve shells: synopsis of
bathymetric and temporal trends on a decadal time scale. Palaeogeog Palaeoclim Palaeoecol 2011;
321: 278–304, 635 https://doi.org/10.1016/j.palaeo.2011.05.019
8. Brett CE, Smrecak TT, Hubbard KP, Walker SE. Marine sclerobiofacies: encrustring and endolithic
communities on shells through time and space. In: Talent JA, editor. Earth and Life: global biodiversity,
extinction intervals and biogeographic perturbations through time. Springer Netherlands; 2012, pp.
129–157, https://doi.org/10.1007/978-90-481-3428-1_6
9. Taylor PD. Competition between encrusters on marine hard substrates and its fossil record. Palaeontol-
ogy 2016; 59: 481–497, https://doi.org/10.1111/pala.12239
10. Liow LH, Di Martino E, Voje KL, Rust S, Taylor PD. Interspecific interactions through 2 million years: are
competitive outcomes predictable? Proc R Soc Lond B Biol Sci 2016; 283: 20160981, https://doi.org/
10.1098/rspb.2016.0981 PMID: 27581885
11. Freckelton M-L, Nedved BT, Hadfield MG. Induction of invertebrate larval settlement; different bacteria,
different mechanisms? Sci Rep 2017; 7: 42557, https://doi.org/10.1038/srep42557 PMID: 28195220
12. Taylor PD, Wilson MA. A new terminology for marine organisms inhabiting hard substrates. Palaios
2002; 17: 522–525, https://doi.org/10.1669/0883-1351(2002)017<0522:ANTFMO>2.0.CO;2
13. Santos A, Mayoral E. Bioerosion versus colonisation on Bivalvia: A case study from the Upper Miocene
of Cacela (southeast Portugal). Geobios 2008; 41: 43–59, https://doi.org/10.1016/j.geobios.2007.01.
009
14. Kelley PH, Hansen TA. The fossil record of drilling predation on bivalves and gastropods. In: Kelley PH,
Kowalewski M, Hansenn TA, editors. Predator—Prey Interactions in the Fossil Record. Kluwer Aca-
demic/Plenum Press; 2003, pp. 113–139, https://doi.org/10.1007/978-1-4615-0161-9_6
15. Harper EM. Assessing the importance of drilling predation over the Palaeozoic and Mesozoic. Palaeo-
geog Palaeoclim Palaeoecol 2003; 201: 185–198, https://doi.org/10.1016/S0031-0182(03)00624-2
16. Klompmaker AA, Kowalewski M, Huntley JW, Finnegan S. Increase in predator-prey size ratios
throughout the Phanerozoic history of marine ecosystems. Science 2017; 356: 1178–1180, https://doi.
org/10.1126/science.aam7468 PMID: 28619943
Bacterial biofilm influences sclerobiont colonization
PLOS ONE | https://doi.org/10.1371/journal.pone.0184745 September 13, 2017 23 / 27
17. Kowalewski M. The fossil record of predation: an overview of analytical methods. In: Kowalewski M, Kel-
ley PH, editors. The Fossil Record of Predation. New Haven: The Paleontological Society; 2002, pp.
3–42.
18. Rodland DL, Kowalewski M, Carroll M, Simões MG. (2006) The temporal resolution of epibiont assem-
blages; are they ecological snapshots or overexposures? J Geol 2006; 114: 313–324, https://doi.org/
10.1086/501221
19. Rodland DL, Simões MG, Krause RA, Kowalewski M. Stowing away on ships that pass in the night:
sclerobiont assemblages on individually dated bivalve and brachiopod shells from a subtropical shelf.
Palaios 2014; 29: 170–183, https://doi.org/10.2110/palo.2013.033
20. Johnson WS, Allen DM. Zooplankton of the Atlantic and Gulf Coasts: A Guide to Their Identification and
Ecology. Baltimore, Johns Hopkins University Press; 2012, 472 p.
21. Tsuchiya M. Faunal structures associated with patches of mussels on East Asian coasts. Helgol Mar
Res 2002; 56: 31–36, https://doi.org/10.1007/s10152-001-0099-2
22. Gollner S, Zekely J, Van Dover C, Govenar B, Le Bris N, Nemeschkal HL, Bright M. Benthic copepod
communities associated with tubeworm and mussel aggregations on the East Pacific Rise. Cah. Biol.
Mar. 2006; 47: 397–402. http://archimer.ifremer.fr/doc/00000/3611/
23. Abdallah LGB, Chargui T, Abidli S, Trigui N, Menif E. Associated and digenean fauna of the mussel
Mytilus galloprovincialis cultured on shellfish tables in the lagoon of Bizerta (Tunisia). Transit. Waters
Bull. 2012; 6: 20–33, https://doi.org/10.1285/i1825229Xv6n1p20
24. Sarmento VC, Lage LM, Santos PJP. Copepoda Harpacticoida community of a rocky shore under the
influence of upwelling (Arraial do Cabo, southeastern Brazil). J Mar Biol Assoc U.K. 2012; 92: 1117–
1126, https://doi.org/10.1017/S0025315411001718
25. Hills JM, Thomason JC. The effect of scales of surface roughness on the settlement of barnacle (Semi-
balanus balanoides) cyprids. Biofouling 1998; 12: 57–69, https://doi.org/10.1080/08927019809378346
26. Berntsson KM, Jonsson PR, Lejhall M, Gatenholm P. Analysis of behavioural rejection of micro-textured
surfaces and implications for recruitment by the barnacle Balanus improvisus. J Exp Mar Bio Ecol 2000;
251: 59–83, https://doi.org/10.1016/S0022-0981(00)00210-0 PMID: 10958901
27. Berntsson KM, Andreasson H, Jonsson PR, Larsson L, Ring K, Petronis S, Gatenholm P. Reduction of
barnacle recruitment on micro-textured surfaces: analysis of effective topographic characteristics and
evaluation of skin friction. Biofouling 2000; 16: 245–261, https://doi.org/10.1080/08927010009378449
28. Bers AV, Wahl M. The influence of natural surface microtopographies on fouling. Biofouling 2004; 20:
43–51, https://doi.org/10.1080/08927010410001655533 PMID: 15079892
29. Scardino AJ, Guenther J, De Nys R. Attachment point theory revisited: the fouling response to a micro-
textured matrix. Biofouling 2008, 24: 45–53, https://doi.org/10.1080/08927010701784391 PMID:
18066730
30. Carl C, Poole AJ, Sexton BA, Glenn FL, Vucko MJ, Williams MR, Whalan S, Nys R. Enhancing the set-
tlement and attachment strength of pediveligers of Mytilus galloprovincialis by changing surface wetta-
bility and microtopography, Biofouling 2012; 28: 175–186, https://doi.org/10.1080/08927014.2012.
662676 PMID: 22332795
31. Whalan S, Wahab MAA, Sprungala S, Poole AJ, Nys R. Larval settlement: the role of surface topogra-
phy for sessile coral reef invertebrates. PLoS One 2015; 10: e0117675, https://doi.org/10.1371/journal.
pone.0117675 PMID: 25671562
32. Ferguson N, White CR, Marshall DJ. Competition in benthic marine invertebrates: the unrecognized
role of exploitative competition for oxygen. Ecology 2013; 94: 126–135, https://doi.org/10.1890/12-
0795.1 PMID: 23600247
33. Agostini VO, Ozorio CP. Colonization record of Isognomon bicolor (Mollusca: Bivalvia) on pipeline
monobuoys in the Brazilian south coast. Marine Biodiversity Records 2016; 9:84, https://doi.org/10.
1186/s41200-016-0061-2
34. Dreanno C, Kirby RR, Clare AS. 2007. Involvement of the barnacle settlement-inducing protein complex
(SIPC) in species recognition at settlement. J Exp Mar Bio Ecol 2007; 351: 276–282, https://doi.org/10.
1016/j.jembe.2007.07.003
35. Petrone L, Aldred N, Emami K, Enander K, Ederth T, Clare AS. Chemistry-specific surface adsorption
of the barnacle settlement-inducing protein complex. Interface Focus 2015; 5: 20140047, https://doi.
org/10.1098/rsfs.2014.0047 PMID: 25657832
36. Ko SK, Hur SB. Effects of microalgal species on the settlement and survival of Haliotis discus hannai lar-
vae. Fish Aquat Sci 2011; 14: 339–345, https://doi.org/10.5657/FAS.2011.0339
37. Hadfield MG. Biofilms and marine invertebrate larvae: what bacteria produce that larvae use to choose
settlement sites. Ann Rev Mar Sci 2011; 3: 453–470, https://doi.org/10.1146/annurev-marine-120709-
142753 PMID: 21329213
Bacterial biofilm influences sclerobiont colonization
PLOS ONE | https://doi.org/10.1371/journal.pone.0184745 September 13, 2017 24 / 27
38. Khandeparker L, Kumar KS. Significance of biofilm proteins in modulating cyprid metamorphosis of
Balanus amphitrite (Cirripedia: Thoracica). Mar Ecol 2011; 32: 509–520, https://doi.org/10.1111/j.
1439-0485.2011.00439.x
39. Tait K, Havenhand J. Investigating a possible role for the bacterial signal molecules N-acylhomoserine
lactones in Balanus improvisus cyprid settlement. Mol. Ecol 2013; 22: 2588–602, https://doi.org/10.
1111/mec.12273 PMID: 23506419
40. Faimali M, Garaventa F, Terlizzi A, Chiantore M, Cattaneo-Vietti R. The interplay of substrate nature
and biofilm formation in regulating Balanus amphitrite Darwin, 1854 larval settlement. J Exp Mar Biol
Ecol 2004; 306: 37–50, https://doi.org/10.1016/j.jembe.2003.12.019
41. Dobretsov S, Abed RMM, Teplitski M. Mini-review: inhibition of biofouling by marine microorganisms.
Biofouling 2013; 29: 423–441, https://doi.org/10.1080/08927014.2013.776042 PMID: 23574279
42. Bao W, Satuito C, Yang J-L, Kitamura H. Larval settlement and metamorphosis of the mussel Mytilus
galloprovincialis in response to biofilms. Mar Biol 2007; 28: 249–256, https://doi.org/10.1007/s00227-
006-0383-4
43. Mieszkin S, Callow ME, Callow JA. Interactions between microbial biofilms and marine fouling algae: a
mini review. Biofouling 2013; 29: 1097–1113, https://doi.org/10.1080/08927014.2013.828712 PMID:
24047430
44. Martı
´n-Rodrı
´guez AJ, Babarro JMF, Lahoz F, Sanso
´n M, Martı
´n VS, Norte M, Ferna
´ndez JJ. From
broad-spectrum biocides to quorum sensing disruptors and mussel repellents: antifouling profile of alkyl
triphenyl phosphonium salts. PLoS One 2015; 10: e0123652, https://doi.org/10.1371/journal.pone.
0123652 PMID: 25897858
45. Oliveira SS, Wasielesky JRW, Ballester ELC, Abreu PC. Caracterizac¸ão da assembleia de bacte
´rias
nitrificantes pelo me
´todo "Fluorescent in situ Hybridization" (FISH) no biofilme e a
´gua de larvicultura do
Camarão-rosa Farfantepenaeus paulensis. Atla
ˆntica 2006; 28: 33–45.
46. Azevedo FBB, Carloni GG, Carvalheira LV. Colonization of benthic organisms on different artificial sub-
stratum in Ilha Grande bay, Rio de Janeiro, Brazil. Braz Arch Biol Techn 2006; 49: 263–275, https://doi.
org/10.1590/S1516-89132006000300012
47. Rios EC. Compendium of Brazilian Sea Shells. Rio Grande: Evangraf; 2009, 668 p.
48. Mikkelsen PM, Bieler R. Seashells of Southern Florida: living marine mollusks of the Florida Keys and
adjacent regions. Bivalves. New Jersey: Princeton University; 2008, 503 p.
49. Lescinsky HL, Edinger E, Risk MJ. Mollusc shell encrustation and bioerosion rates in a modern epeiric
sea: taphonomy experiments in the Java Sea, Indonesia. Palaios 2002; 17: 171–191, https://doi.org/
10.1669/0883-1351(2002)017<0171:MSEABR>2.0.CO;2
50. Best MMR. Contrast in preservation of bivalve death assemblages in siliciclastic and carbonate tropical
shelf settings. Palaios 2008; 23: 796–809, https://doi.org/10.2110/palo.2005.p05-076r
51. Herzenberg LA, Tung J, Moore WA, Herzenberg LA, Parks DR. Interpreting flow cytometry data: a
guide for the perplexed. Nat. Immunol 2006; 7: 681–685, https://doi.org/10.1038/ni0706-681 PMID:
16785881
52. Bouvier T, Troussellier M, Anzil A, Courties C, Servais P. Using light scatter signal to estimate bacterial
biovolume by flow cytometry. Cytometry 2001; 44: 188–194, https://doi.org/10.1002/1097-0320
(20010701)44:3<188::AID-CYTO1111>3.0.CO;2-C PMID: 11429769
53. Picot J, Guerin CL, Le Van Kim C, Boulanger CM. Flow cytometry: retrospective, fundamentals and
recent instrumentation. Cytotechnology 2012; 64: 109–130, https://doi.org/10.1007/s10616-011-9415-
0PMID: 22271369
54. Heldal M, Norland S, Bratbak G, Riemann B. Determination of bacterial cell number and cell volume by
means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy. J Micro-
biol Methods 1994; 20: 255–263, https://doi.org/10.1016/0167-7012(94)90050-7
55. Norland S. The relationship between biomass and volume of bacteria. In: Kemp PF, Sherr BF, Sherr
EB, Cole JJ, editors. Handbook of Methods in Aquatic Microbial Ecology. USA: Lewis Publishers;
1993, 777 p.
56. Sun J, Liu D. Geometric models for calculating cell biovolume and surface area for phytoplankton. J
Plankton Res 2003; 25: 1331–1346, https://doi.org/10.1093/plankt/fbg096
57. Zaritski A. On dimensional determination of rod-shaped bacteria. J Theor Biol 1975; 54: 243–248,
https://doi.org/10.1016/S0022-5193(75)80129-9 PMID: 1207136
58. Buckup L, Bond-Buckup G. Os Crusta
´ceos do Rio Grande do Sul. Porto Alegre: Editora da Universi-
dade Federal do Rio Grande do Sul; 1999, 647 p.
59. Lopes RP. Bioerosion and bioincrustation in body fossils from the Coastal Plain of Rio Grande do Sul
State, Southern Brazil. In: Netto RG, Carmona NB, Tognoli FMW, editors. Ichnology of Latin America—
Bacterial biofilm influences sclerobiont colonization
PLOS ONE | https://doi.org/10.1371/journal.pone.0184745 September 13, 2017 25 / 27
Selected Papers. Porto Alegre: Sociedade Brasileira de Paleontologia; 2012, pp. 179–194 (Monogra-
fias 2).
60. Barclay KM, Schneider CL, Leighton LR. Palaeoecology of Devonian sclerobionts and their brachiopod
hosts from the Western Canadian Sedimentary Basin. Palaeogeog Palaeoclim Palaeoecol 2013; 383–
384: 79–91, https://doi.org/10.1016/j.palaeo.2013.05.007
61. R Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Sta-
tistical Computing; 2017 (www.R-project.org).
62. Chia FS, Koss R. Fine structural studies of the nervous system and the apical organ in the planula larva
of the sea anemone Anthopleura elegantissima. J. Morphol 1979; 160: 275–297, https://doi.org/10.
1002/jmor.1051600303
63. Svane I, Dolmer P. Perception of light at settlement: a comparative study of two invertebrate larvae, a
scyphozoan planula and a simple ascidian tadpole. J Exp Mar Biol Ecol 1995; 187: 51–61, https://doi.
org/10.1016/0022-0981(94)00171-9
64. Jenkins SR, Hawkins SJ. Barnacle larval supply to sheltered rocky shores: a limiting factor? Hydrobiolo-
gia 2003; 503: 143–151, https://doi.org/10.1023/B:HYDR.0000008496.68710.22
65. Glasby TM. Surface composition and orientation interact to affect subtidal epibiota. J Exp Mar Biol Ecol
2000; 248: 177–190, https://doi.org/10.1016/S0022-0981(00)00169-6 PMID: 10771301
66. Keough MJ. Responses of settling invertebrate larvae to the presence of established recruits. J Exp
Mar Biol Ecol 1998; 231: 1–19, https://doi.org/10.1016/S0022-0981(98)00073-2
67. Absalão RS. Colonizac¸ão prima
´ria e sequência de substituic¸ões, em substrato artificial, na comunidade
epibêntica da Baı
´a da Guanabara, RJ, Brasil. Ph.D. Thesis, Universidade de São Paulo; 1993, 230 p.
68. Marshall DJ, Keough MJ. Variation in the dispersal potential of non-feeding invertebrate larvae: the des-
perate larva hypothesis and larval size. Mar Ecol Prog Ser 2003; 255: 145–153, https://doi.org/10.
3354/meps255145
69. Hsu LC, Fang J, Borca-Tasciuc DA, Worobo RW, Moraru CI. Effect of micro- and nanoscale topography
on the adhesion of bacterial cells to solid surfaces. Appl Environ Microbiol 2013; 79: 2703–2712,
https://doi.org/10.1128/AEM.03436-12 PMID: 23416997
70. Feng G, Cheng Y, Wang S-Y, Borca-Tasciuc DA, Worobo RW, Moraru CI. Bacterial attachment and
biofilm formation on surfaces are reduced by small-diameter nanoscale pores: how small is small
enough? NPJ Biofilms Microbiomes 2015; 1: 15022, https://doi.org/10.1038/npjbiofilms.2015.22 PMID:
28721236
71. Vladkova T. Surface modification approach to control biofouling. In: Flemming H-S, Murthy PS, Venka-
tesan R, Cooksey K, editors. Marine and Industrial Biofouling. Heidelberg: Springer Berlin; 2008, pp.
135–163. https://doi.org/10.1007/978-3-540-69796-1_7
72. Whitehead KA, Rogers D, Colligon J, Wright C, Verran J. Use of the atomic force microscope to deter-
mine the effect of substratum surface topography on the ease of bacterial removal. Colloids Surf B
Biointerfaces 2006; 51: 44–53, https://doi.org/10.1016/j.colsurfb.2006.05.003 PMID: 16822658
73. Scheuerman TR, Camper AK, Hamilton MA. Effects of substratum topography on bacterial adhesion. J
Colloid Interface Sci 1998; 208: 23–33, https://doi.org/10.1006/jcis.1998.5717 PMID: 9820746
74. Ju¨rgens K, Pernthaler J, Schalla S, Amann R. Morphological and compositional changes in a planktonic
bacterial community in response to enhanced protozoan grazing. Appl Environ Microbiol 1999; 65:
1241–1250 PMID: 10049890
75. Unabia CRC, Hadfield MG. Role of bacteria in larval settlement and metamorphosis of the polychaete
Hydroides elegans. Mar Biol 1999; 133: 55–64, https://doi.org/10.1007/s002270050442
76. Dobretsov S, Qian P-Y. Facilitation and inhibition of larval attachment of the bryozoan Bugula neritina in
association with mono-species and multi-species biofilms. J Exp Mar Biol Ecol 2006; 333: 263–274,
https://doi.org/10.1016/j.jembe.2006.01.019
77. Tamburri MN, Luckenbach MW, Breitburg DL, Bonniwell SM. Settlement of Crassostrea ariakensis lar-
vae: effects of substrate, biofilms, sediment and adult chemical cues. J Shellfish Res 2008; 27: 601–
608, https://doi.org/10.2983/0730-8000(2008)27[601:SOCALE]2.0.CO;2
78. Dobretsov S, Abed RM, Voolstra CR. The effect of surface colour on the formation of marine micro and
macrofouling communities. Biofouling 2013; 29: 617–627, https://doi.org/10.1080/08927014.2013.
784279 PMID: 23697809
79. Yule AB, Walker G. The temporary adhesion of barnacle cyprids—effects of some differing surface
characteristics. J Mar Biol Assoc U.K. 1984; 64: 429–439, https://doi.org/10.1017/
S0025315400030101
80. Monteforte M, Garcia-Gasca A. Spat collection studies on pearl oysters Pinctada mazatlanica and
Pteria sterna (Bivalvia, Pteriidae) in Bahia de La Paz, South Baja California, Mexico. Hydrobiologia
1994; 291: 21–24, https://doi.org/10.1007/BF00024236
Bacterial biofilm influences sclerobiont colonization
PLOS ONE | https://doi.org/10.1371/journal.pone.0184745 September 13, 2017 26 / 27
81. Visscher JP, Luce RH. Reactions of the cyprid larvae of barnacles to light with special reference to spec-
tral colors. Biol Bull 1928; 54: 336–350, https://doi.org/10.2307/1537021
82. Corrêa ICS, Ponzi VRA. Depo
´sitos de calca
´rio biodetrı
´tico das regiões 654 do Albardão e Mostardas na
plataforma interna do Rio Grande do Sul. Recife: Anais do XXX Congresso Brasileiro de Geologia 2;
1978, pp. 851–866.
83. Ritter M.N., Erthal F., Kosnik M., Coimbra J.C., Kaufman D.S. Spatial variation in the temporal resolu-
tion of subtropical shallow-water molluscan death assemblages. Palaios 2017; https://doi.org/10.2110/
palo.2017.003
84. Kowalewski M, Bambach RK. The limits of paleontological resolution. In: Harries PJ, editor. Approaches
in high-resolution stratigraphic paleontology. New York: Springer Science; 2003, pp. 1–48 (Topics in
Geobiology 21). https://doi.org/10.1007/978-1-4020-9053-0_1
85. Stanley SM. Post-Paleozoic adaptive radiation of infaunal bivalve molluscs: a consequence of mantle
fusion and siphon formation. J Paleontol 1968; 42: 214–29.
86. Vermeij GJ. The Mesozoic marine revolution: evidence from snails, predators, and grazers. Paleobiol-
ogy 1977; 3: 245–58.
87. Madin JS, Alroy J, Aberhan M, Fu¨rsich FT, Kiessling W, Kosnik MA, Wagner PJ. Statistical indepen-
dence of escalatory ecological trends in Phanerozoic marine invertebrates. Science 2006; 312:897–
900, https://doi.org/10.1126/science.1123591 PMID: 16690862
88. Bush AM, Bambach RK. Paleoecologic megatrends in marine metazoan. Annu Rev Earth Planet Sci
2011; 39: 241–269, https://doi.org/10.1146/annurev-earth-040809-152556
89. Stanley SM. Relation of shell form to life habits of the Bivalvia (Mollusca). GSA Memoirs 1970; 125: 1–
282.
90. Behrensmeyer AK, Fu¨rsich FT, Gastaldo RA, Kidwell SM, Kosnik MA, Kowalewski M, Plotnick RE, Rog-
ers RR, Alroy J. Are the most durable shelly taxa also the most common in the marine fossil record?
Paleobiology 2005; 31: 607–623, https://doi.org/10.1666/0094-8373(2005)031[0607:ATMDST]2.0.
CO;2
91. Caceres-Martinez J, Robledo JAF, Figueras A. Settlement and post-larvae behaviour of Mytilus gallo-
provincialis: field and laboratory experiments. Mar Ecol Prog Ser 1994; 112: 107–117, https://doi.org/
10.3354/meps112107
92. Walker SE. Taphonomy of modern and fossil intertidal gastropod associations from Isla SantaCruz and
Isla Santa Fe, Galapagos Islands. Lethaia 1995; 28: 371–382, https://doi.org/10.1111/j.1502-3931.
1995.tb01829.x
Bacterial biofilm influences sclerobiont colonization
PLOS ONE | https://doi.org/10.1371/journal.pone.0184745 September 13, 2017 27 / 27
