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Submitted 21 February 2022
Accepted 5 August 2022
Published 23 September 2022
Corresponding authors
Stephane Betzi,
stephane.betzi@inserm.fr
Fabienne Priam,
fabienne.priam@univ-antilles.fr,
fabiennepriam@gmail.com
Academic editor
Blanca Figuerola
Additional Information and
Declarations can be found on
page 17
DOI 10.7717/peerj.13955
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2022 Piron et al.
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OPEN ACCESS
Antimicrobial and cytotoxic effects of
marine sponge extracts Agelas clathrodes,
Desmapsamma anchorata and Verongula
rigida from a Caribbean Island
Julie Piron1, Stephane Betzi2, Jessica Pastour1, Audrey Restouin2, Rémy
Castellano2, Yves Collette2, Niklas Tysklind3, Juliette Smith-Ravin1,4and
Fabienne Priam1,4
1Groupe de Recherche BIOSPHERES, Université des Antilles, Campus de Schoelcher, Martinique, France
2Centre de Recherche en Cancérologie de Marseille (CRCM) - Aix-Marseille Université, Inserm, CNRS,
Institut Paoli Calmettes, Marseille, France
3INRAE - UMR 0745 ECOFOG, Campus Agronomique CEDEX, Kourou, Guyane, France
4Association AREBio Immeuble Bellevue, Fort de France, Martinique, France
ABSTRACT
Although marine sponges are known for their antimicrobial, antifungal and cytotoxic
activity, very few studies have been carried out on endemic species of Martinique.
Martinique is part of the Agoa Sanctuary, a marine protected area that includes the
exclusive economic zones (EEZ) of the French Caribbean islands, making it an abundant
source of marine species. To highlight the potential of this area for the discovery
of marine biomolecules with antipathogenic and antitumor activities, we tested the
aqueous and ethanolic extracts of sponge species Agelas clathrodes,Desmapsamma
anchorata and Verongula rigida. Five bacterial strains: Bacillus cereus (CIP 78.3),
Escherichia coli (CIP 54.127), Pseudomonas aeruginosa (CIP A22), Staphylococcus aureus
(CIP 67.8) and Staphylococcus saprophyticus (CIP 76125) were evaluated, as well as four
tumor cell lines: breast cancer (MDA-MB231), glioblastoma (RES259) and leukemia
(MOLM14 and HL-60). Antimicrobial activity was evaluated using the disc diffusion
technique by determining the minimum inhibitory and minimum bactericidal concen-
trations. Tumor cytotoxic activity was determined in vitro by defining the minimum
concentration of extracts that would inhibit cell growth. Ethanolic extracts of Agelas
clathrodes were bactericidal for Staphylococcus aureus and Staphylococcus saprophyticus
strains, as well as strongly cytotoxic (IC50 <20 µg/mL) on all cancer cell lines. Verongula
rigida also showed strong cytotoxic activity on cell lines but no antimicrobial activity.
These results are innovative for this species on these bacterial lines, highlighting the
potential of sponge extracts from this area as bioactive compounds sources.
Subjects Biochemistry, Cell Biology, Marine Biology, Microbiology, Oncology
Keywords Marine sponges, Antimicrobial activity, Cytotoxic activity, Agelas clathrodes,Desmap-
samma anchorata,Verongula rigida, Natural products, Martinique, Tumoral cell lines
How to cite this article Piron J, Betzi S, Pastour J, Restouin A, Castellano R, Collette Y, Tysklind N, Smith-Ravin J, Priam F. 2022.
Antimicrobial and cytotoxic effects of marine sponge extracts Agelas clathrodes,Desmapsamma anchorata and Verongula rigida from a
Caribbean Island. PeerJ 10:e13955 http://doi.org/10.7717/peerj.13955
INTRODUCTION
Martinique is part of the AGOA Sanctuary, a marine protected area recognized under
the Specially Protected Areas and Wildlife (SPAW) protocol that includes the entire
exclusive economic zones (EEZ) of the four French Caribbean islands (Saint-Martin,
Saint-Barthelemy, Guadeloupe and Martinique), making it one of the ‘‘hot spots’’ of
worldwide marine diversity (https://sanctuaire-agoa.fr/editorial/vast-territory) (Fig. 1).
Indeed, new sponge species are regularly discovered on the 47,000 km2EEZ of the island
by the active study of its biodiversity (Grenier et al., 2020;Griffiths et al., 2021;Impact Mer,
2008;Impact Mer, 2012;Perez et al., 2017;Perez & Ruiz, 2018). However, these marine
organisms are poorly valued (Laville et al., 2009). Therefore, the goal of this study is to
improve knowledge of the island’s sponge diversity as a potential source of novel marine
natural products with antibacterial activity on drug-resistant pathogens or anticancer
activity on common cancer cell lines.
Indeed, bioactive natural products are increasingly sought after, with a specific interest
in pharmacology, where they account for about 70% of approved drugs (Cortadellas et
al., 2010). In 2017, seven pharmaceuticals derived from marine substances were validated
for clinical uses by the Food and Drug Administration (FDA) (Dyshlovoy & Honecker,
2018). Marine sponges are of particular interest because of the abundance of secondary
metabolites they produce and their highly diversified chemical nature (El-Amraoui et al.,
2010;Mayer et al., 2013;Nweze et al., 2020;Sipkema et al., 2005).
The discovery of novel antibiotics from these organisms is one of the main goals of
current research. This is due to the increasing resistance of many strains to commercially
available antibiotics such as Pseudomonas aeruginosa or Staphylococcus aureus which are
ranked among the ‘‘priority pathogens’’ resistant to antibiotics. Marine organisms are a
historically rich source of compounds active against these drug-resistant pathogens such
as those found in bacteria, fungi, algae or invertebrates (Nweze et al., 2020).
Cancer treatment has also benefited from marine natural products with notable examples
like cytarabine, eribulin, and trabectedin. The anti-tumor and cytotoxic activity of sponges
have been known and exploited for years with the emergence of chemical derivatives used
in cancer treatments such as cytarabine (ARA-C) for the treatment of leukemia or eribulin
mesylate as treatment for breast cancer (Dyshlovoy & Honecker, 2018). Despite the existence
of these and other treatments, the National Estimates of Cancer Incidence and Mortality
in Metropolitan France states that women’s breast cancer incidence increased by 0.6% per
year since 2010. During this same period, the incidence rate for acute leukemia increased
by an average of 114.5% (Defossez et al., 2019). In light of this, there is an urgent need for
new anti-tumor drugs with novel targets and novel modes of action that could be filled by
novel marine sources.
This study focused on marine sponges collected on the Caribbean coast of Martinique.
We evaluated the activity of aqueous and ethanolic extracts of the three sponge species
Agelas clathrodes,Desmapsamma anchorata and Verongula rigida for their antimicrobial
and antitumor activity. We highlight the antibiotic and anticancer activity of several sponge
extracts after evaluation on five bacterial strains (Bacillus cereus (CIP 78.3), Escherichia coli
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 2/21
Figure 1 Identification of the sponges. Map of the sampling location (top) and morphological analy-
sis of each sponge (bottom) showing: (A, D, G) photos after freezing; (B, E, H) micrographs of the skele-
ton x100; (C, F, I) micrographs of the skeleton x4000. The analysis shows: (C) whorled achantostyles and
whorled achantoxes in formation; (F) oxes, isocheles, tripods and spheriaster; (I) thick and short oxes.
Full-size DOI: 10.7717/peerj.13955/fig-1
(CIP 54.127), Pseudomonas aeruginosa (CIP A22), Staphylococcus aureus (CIP 67.8) and
Staphylococcus saprophyticus (CIP 76125)), and on four tumor cell lines (breast cancer
(MDA-MB231), glioblastoma (RES259) and leukemia (MOLM14 and HL-60)), and are
able to link some of our findings to previously reported data.
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 3/21
MATERIAL AND METHODS
Sampling and identification
The samples Agelas clathrodes,Desmapsamma anchorata and Verongula rigida were
collected on the ‘‘Fond Boucher’’ site (14◦39021.1200N−61◦9021.2200 W) on the north
Caribbean side of Martinique (Fig. 1. and Table 1). The samples were collected by dives
between 15 and 20 m deep during two campaigns, on October 7, 2017 by Dr. Romain
Ferry (2019 Fishing Order No. R02-2019-04-08-004, Martinique Sea Department). The
samples were conditioned in individual plastic bags and immediately stored in a cooler
for transport to the laboratory of the Université des Antilles (UA). They were then washed
with fresh water and directly stored at −20 ◦C before extraction.
Sponges have been identified on the basis of their external morphology and skeletal
composition, in particular the type of spicule (Boury-Esnault & Rützler, 1997;Custódio &
RJ, 2007;ImpactMer, 2008;Impact Mer, 2012;Perez et al., 2017;Perez & Ruiz, 2018). The
sponge skeletons were studied at the laboratory on UA campus in Martinique, by extraction
of the spicules and by longitudinal and transverse sectioning of the tissue. Extractions were
carried out by three washes with bleach after freeze-drying 5 mg of sample. Observation was
conducted using an optical microscope equipped with a ZEISS camera. The pictures were
analyzed with the ZEN2012 software. The size of the spicules was estimated by calculation
according to the lens size. The determination of the species was realized with the external
anatomic description and observed spicules, as well as the bibliography analysis based
on the inventories already published in the area. No comparison was made with similar
samples or holotypes. Internal anatomy and cytology were not determined in this study as
well.
Method for calculating the size of the spicules: size on the photo/magnification
(lens*objective).
Preparation of sponge extracts
The extracts were prepared by maceration in two solvents, distilled water and 100% ethanol.
The sponges were dried in a freeze-dryer, then 2 g were cut into small pieces, crushed with
a mortar, placed in a 15 mL falcon tube, then 10 mL of solvent were added. After repeated
manual turning and swirling, the tubes were placed under agitation for 24 h at room
temperature. The extracts were then filtered on standard filter paper. The process was
repeated three times. The extracts were then pooled and stored at −20 ◦C before drying.
The ethanolic extracts (E) were dried in a rotary evaporator at 45 ◦C. one mL of solvent
was added to the flask twice and passed through an ultrasonic bath to recover the residues
and placed in glass vials. The solvent residues were evaporated in a fume hood and the dry
extract was stored at −20 ◦C before testing. The aqueous extracts (A) were freeze-dried
and stored at −20 ◦C.
Antimicrobial activity
Bacterial strains
The five bacterial strains belong to group 1 and 2 of the classification of microorganisms
by risk groups (Article R4421-3 of the Decree no. 2008-244 of March 7, 2008-art. (V)).
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 4/21
Table 1 List of species collected and GPS localization data.
Species Site GPS data
Agelas clathrodes Fond Boucher 61◦9021.2200W 14◦39021.1200 N
Desmapsamma anchorata Fond Boucher 61◦9021.2200W 14◦39021.1200 N
Verongula rigida Fond Boucher 61◦9026.5500 W 14◦39023.6700N
Strains came from the Institut Pasteur Collection (CIP): Paris: Bacillus cereus (CIP 78.3),
Staphylococcus saprophyticus (CIP 76125T), Escherichia coli (CIP 54.127) strains are listed in
group 1 (non-pathogenic) and Pseudomonas aeruginosa (CIP A22), Staphylococcus aureus
(CIP 67.8) strains are listed in group 2 (pathogenic). Inoculums were prepared from strains
cultivated in agar nutrient at 37 ◦C for all strains except for Pseudomonas aeruginosa (CIP
A22) incubated at 30 ◦C.
Antimicrobial assay
Antimicrobial activity was tested on Mueller Hinton agar using the disc diffusion method
according to Majali et al. (2015). Pure 18 h culture inoculums on agar nutrient, were seeded
at a concentration of 107UCF/mL. Sterile six mm diameter discs were soaked with 20 µL
of extract (at 500 µg/mL) and dried for 15min. The discs were then stored at 4 ◦C before
being placed on the seeded agar and the petri dishes were incubated for 24 h at 37 ◦C for
all strains except for Pseudomonas aeruginosa (CIP A22)incubated at 30 ◦C with agitation.
The inhibition diameter was then measured. Tests were performed in triplicates for each
species. The standard antibiotic ampicillin (10 µg, lot 7B5479) was used as a positive
control for S. saprophyticus,S. aureus and E. coli; chloramphenicol (30 µg, lot 5C5220) was
used as a positive control for B. cereus and fosfomycin (50 µg, lot 4L5252) was used as
a positive control for P. aeruginosa. Discs impregnated with 20 µL of solvent (H2O and
ethanol) were also used as negative controls.
An inhibition diameter greater than nine mm around the disc indicated positive activity
according to the analysis of Cita et al. (2017).
Minimal inhibitory concentration (MIC)
MIC was determined by the successive liquid microdilution method according to Majali et
al. (2015), modified for ethanolic extract of Agelas clathrodes. Two-in-two dilutions of the
crude extract were performed in a 96-well plate. The plates were then incubated 24 h at
37 ◦C for all strains except for Pseudomonas aeruginosa (CIP A22) incubated at 30 ◦C. The
optical density was measured on a plate reader at 600 nm. The concentration range used
was 0.488 µg/mL to 1,000 µg/mL.
Minimal bactericidal concentration (MBC)
MBC was also evaluated for the ethanolic extract of Agelas clathrodes by counting the
surviving bacteria in tubes with no visible growth using a method adapted from Majali
et al. (2015) and Marinho et al. (2010). A streak of a 10 µL aliquot was seeded onto PCA
plates using a calibrated plater, and incubated for 24 h at 37 ◦C for all strains except for
Pseudomonas aeruginosa (CIP A22) incubated at 30 ◦C. After incubation, colonies were
counted for each streak. Only streaks between 30 and 300 colonies were considered. The
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 5/21
extract was considered having a bactericidal effect for MBC/MIC =1 and a bacteriostatic
effect for MBC >MIC (Majali et al., 2015).
Cytotoxic evaluation
Cell lines
Four cell lines were selected for the cytotoxic evaluation. A human breast cancer cell line
MDA-MB-231, a glioblastoma cell line (RE259) and two leukemia cell lines: MOLM-14
acute myeloid leukemia (more precisely, a MOLM14 luc cell line, expressing the luciferase-
GFP gene) and HL-60 acute human promyelocytic cell line. Cell lines came from ATCC
(CCL-240) for HL-60; ECACC (cat no. =92020424) for MDA-MB-231; MOLM-14
GFP/Luc =MOLM-14 were obtained from JE. Sarry and engineered to express luciferase
(Stuani et al., 2021) and Res259 (grade II, diffuse astrocytoma) were kindly provided by
Chris Jones (The Institute of Cancer Research, Sutton, UK. (Rakotomalala et al., 2021).
Cell lines were cultivated according to the following protocol established by the TrGET
facility in the Marseille Cancer Research Center (CRCM).
All cancer cell lines were tested negative for mycoplasma contamination. RES259 cells
were grown in MEM medium supplemented with 10% heat-inactivated foetal bovine
serum and 1% non-essential amino acids. MDA-MB-231 cells were cultured in RPMI
supplemented with 10% FCS, 1% L-Glutamine and 1% sodium pyruvate. MOLM-14 were
maintained at a concentration of 0.5 M/mL in MEM alpha medium supplemented with
10% FCS at 37 ◦C 5% CO2. The HL-60 cell line was grown using the same procedure in
IMDM (Iscove’s Modified Dulbecco’s Medium, Gibco 12440053) supplemented with 20%
FCS. Adherent cells were seeded in Corning 3903 clear-bottom 96-well plates overnight
prior to treatment at 1250 cells for RES259 and 5000 cells for MDA MB231 in 90 µL of
their respective medium. MOLM-14 and HL-60 cells were seeded at 10,000 cells per well
on the day of testing.
Dilution of the extracts
During cytotoxic tests, only ethanolic extracts were analyzed because contaminations
have been noticed for aqueous extracts in the cell cultures. For each sponge sample, two
extractions were evaluated (X1 and X2) for a total of 3 performed experiments labelled as
#1, #2 or #3. A 10X concentrated cascade dilution of the compounds was performed in
medium at 10% constant DMSO concentration and then 10 µL of these dilutions were
added to the 90 µL of the wells in triplicate, in order to get concentrations ranging from
500 µg/mL to 0.06 µg/mL (1% final DMSO concentration).
Cytotoxic assay
Experiments were performed as triplicates (except marked otherwise in the result table).
Doxorubicin was used as positive control for the MDA-MB231 assay, and Aracytin was
used for the three other cell lines. Plates were incubated for 72 h at 37 ◦C with 5% CO2,
then after a 30 min room temperature reset, 50 µl of cell titer Glo (Promega, Madison,
WI, USA) were added. Cell lysis was induced for 2 min on an orbital shaker, then after 10
min of incubation at RT to allow the signal to stabilize, the luminescence was measured on
a Berthold centro LB960 luminometer. Curves depending on the doses were plotted and
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 6/21
the median inhibitory concentration corresponding to the lowest concentration of active
compound allowing to inhibit the growth of the cells by 50% in vitro (IC50) was calculated
using the GraphPad PRISM software, as well as a confidence interval range (CRI). For
IC50 measurements, values were normalized and fitted with GraphPad Prism (least squares
regression) using the following equation Y=100/(1+((X/IC50)∧Hillslope)).
RESULTS
Sponges identification
Sponges were identified according to two criteria: their external morphology and the
composition of their skeleton.
The first sample was identified as Agelas clathrodes (Table 2). For this sample, the external
morphology was massive and the color was bright orange (Fig. 1A). Consistency was hard
and oscula were often fused in a comma shape. The skeleton was composed of spongin
fibers, achantostyles and achantoxes verticillates megascleres, mostly between 70 µm and
500 µm in size (Fig. 1B). No microscleres were observed (Fig. 1C) (Hooper & VanSoest,
2002a;Hooper & VanSoest, 2002b;VanSoest & Hooper, 2002;Van Soest, 2002).
The second sample was identified as Desmapsamma anchorata (Table 2). External
morphology presented a branching of pink-lilac colour with oscules scattered at the surface
(Fig. 1D). Consistency was soft and the skeleton consisted of diactinal oxea megascleres
(Fig. 1E) of around 80 µm to 100 µm as well as miscrocleres: sigmas, isochelas, spherasters
and tripods of less than or equal to 20 µm (Fig. 1F) (Hooper & VanSoest, 2002a;Hooper &
VanSoest, 2002b;Van Soest, 2002).
Finally, the last sample was identified as Verongula rigida (Table 2). External morphology
was massive with a charcoal black exterior and a sulphur yellow interior (Fig. 1G).
Consistency was hard and oscula were located in crevices. They were round, wide and
randomly distributed at the surface. The skeleton was mainly composed of spongin fibers
(Fig. 1H) and a few short and thick oxeas of about 60–70 µm were observed but were not
specific to the sample (Fig. 1I) (Van Soest, 1978;Bergquist & Cook, 1978;Bergquist & Cook,
2002).
Antimicrobial activity
Antimicrobial assay
In order to evaluate the antibacterial activity of the three extract sponges in water or
ethanol, inhibition diameters were obtained by the disc diffusion method on three Gram+
bacterial strains: S. aureus, B. cereus and S. saprophyticus (Fig. 2A); and on two Gram-
bacterial strains: E. coli and P. aeruginosa (Fig. 2B).
Different positive controls were used to match bacteria sensitivity (T+). Ampicillin has
been used for S. saprophyticus,S. aureus and E. coli; chloramphenicol for B. cereus; and
fosfomycin for P. aeruginosa. Water only (T1-) or ethanol only (T2-) were used as negative
controls (Figs. 2A and 2B).
Large inhibition diameters were observed for the positive controls and no inhibition
diameters were obtained for negative controls as expected (Figs. 2A and 2B).
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 7/21
Table 2 Identification of sponge species collected.
Sample 1 Class: Demospongiae Sollas, 1885
Sub-class: Heteroscleromorpha
Cárdenas, Pérez & Boury-Esnault, 2012
Order: Agelasida Hartman, 1980
Familly: Agelasidae Verrill, 1907
Genus: Agelas Duchassaing & Michelotti, 1864
Agelas clathrodes Schmidt, 1870
Sample 2 Class: Demospongiae Sollas, 1885
Sub-class: Heterocleromorpha
Cárdenas, Pérez & Boury-Esnault, 2012
Order: Poecilosclerida Topsent, 1928
Familly: Desmacididae Schmidt, 1870
Genus: Desmaspamma Burton, 1934
Desmapsamma anchorata Carter, 1882
Sample 3 Class: Demospongiae Sollas, 1885
Sub-class: Verongimorpha Erpenbeck, Sutcliffe, De Cook,
Dietzel, Maldonado, Van Soest, Hooper & Wörheide, 2012
Ordre: Verongiida Bergquist, 1978
Verongula rigida Esper, 1794
For the sponge extracts, only the ethanolic extract of Agelas clathrodes showed inhibition
diameter for S. aureus and S. saprophyticus (Fig. 2A).
The inhibition diameter was then measured (Table 3) with an inhibition diameter
greater than nine mm around the disc indicating positive activity (Cita et al., 2017).
Table 3 shows the antimicrobial activity results obtained on the five bacterial strains.
Only the ethanolic extract of Agelas clathrodes shows high specific activity on the two
Gram + strains: S. aureus (inhibition diameter: 10.7 mm) and S. saprophyticus (inhibition
diameter: 9.5 mm).
MIC and MBC
Only Agelas clathrodes showed inhibition discs, it was therefore possible to measure MIC
and MBC values. The MIC determined for S. aureus was 15.62 µg/mL and was higher than
the 7.81 µg/mL obtained for S. saprophyticus (Table 4). This result was consistent with the
15.62 µg/mL MBC on S. saprophyticus and the 31.25 µg/mL on S. aureus. We could also
note that the MBC/MIC ratios were equal to 2, indicating a bacteriostatic effect for the
ethanolic extract of Agelas clathrodes.
Cytotoxic activity
Cytotoxic assay
A complete cytotoxicity evaluation of each sponge ethanolic extract was performed on four
cancer cell lines: MDA-MB-231 (breast cancer), RE259 (glioblastoma), MOLM-14 (acute
myeloid leukemia) and HL-60 acute (human promyelocytic leukemia). Figs. 3 and 4display
these cytotoxic dose response evaluations. Results for synthetic anticancer drugs used as
references (doxorubicin and Ara-C) are also indicated. These control experiments were
in agreement with previous internal evaluations, thus strengthening the sponge extracts
results and conclusions. Several extracts could be linked to strong to moderate cytotoxic
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 8/21
Figure 2 Antimicrobial screening and antibacterial activity of sponge species by the disc diffusion
method on five bacterial strains. (A) Three Gram+ bacterial strains: S. aureus, B. cereus and S. saprophyti-
cus. T+ (positive control): Ampicillin for S. aureus and S. saprophyticus; Chloramphenicol for B. cereus.
(B) Two Gram- bacterial strains: E. coli and P. aeruginosa. T+ (positive control): Ampicillin = for E. coli;
fosfomycin for P. aeruginosa. (A) and (B) T1- (negative control 1): H2O only. T2- (negative control 2):
EtOH only. S1 H2O: A. clatrodes aqueous extract/S1 EtOH: A. clatrodes ethanolic extract. S2 H2O: D. an-
chorata aqueous extract/ S2 EtOH: D. anchorata ethanolic extract. S3 H2O: V. rigida aqueous extract/S3
EtOH: V. rigida ethanolic extract. For each panel, visible inhibition discs are marked with yellow arrows.
Full-size DOI: 10.7717/peerj.13955/fig-2
activity with cell viability percentages reduced in a dose response manner. All the IC50
extracted from the dose response analysis were kept independent to illustrate the good
homogeneity observed across different evaluation experiments and for several extraction
campaigns. Table 5 lists all these measured IC50 (per experiment and per extract) as well
as the 95% confidence interval range (CIR) generated during the fitting. Overall, the Agelas
clathrodes extracts exhibited a strong cytotoxicity on all evaluated cell lines. The Verongula
rigida extracts also exhibited strong to moderate cytotoxicity on three cell lines, while the
Desmapsamma anchorata extracts were mostly inactive. A detailed individual analysis is
presented below for each cell line.
Antiproliferative activity on the human breast cancer line (MDA-MB-231)
The results reported in Table 5, show that the ethanolic extracts of the two species: A.
clathrodes and V. rigida were active on cell viability for this cell line. On the other hand,
the D. anchorata extracts were inactive. Indeed, the MDA-MB-231 cells were more sensible
to A. clathrodes with IC50 values in the µg/mL range (3.42 to 7.91 µg/mL). The V. rigida
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 9/21
Table 3 Inhibition diameter of aqueous and ethanolic extracts of the different sponge species on the five strains (mm).
Extracts sponges
and controls
Extract
solvent
Strains
Gram + Gram -
S. aureus B. cereus S. saprophyticus E. coli P. aeruginosa
Ampicillin (T+) 46 ±1 – 40 ±1 26 ±0 –
Chloramphenicol
(T+)
– 25 ±0 – – –
Fosfomycin (T+) – – – – 17.5 ±0.71
(H2O) T1- A R R R R R
(EtOH) T2- E R R R R R
A R R R R R
A. clathrodes E 10.66 ±0.58 R 9.5 ±0.5 R R
A R R R R R
D. anchorata E R R R R R
A R R R R R
V. rigida E R R R R R
Notes.
A, aqueous extract; E, ethanolic extract; R, resistant; -, not used for this strain.
Table 4 MIC and MBC for the ethanolic extract (E) of Agelas clathrodes.
Strain MIC
(µg/mL)
MBC
(µg/mL)
MBC/
MIC ratio
S. saprophyticus 7.81 15.62 2
S. aureus 15.62 31.25 2
extracts were also active but with 1 log less potency, exhibiting IC50 values ranging from
18.62 to 64.41 µg/mL.
Antiproliferative activity on the glioblastoma cell line (RES259)
The activity of the extracts on the RES259 glioblastoma cell line was similar to the results
observed for the breast cancer cell line. Strong cytotoxicity properties were observed for
A. clathrodes and V. rigida with IC50 values lower than 10 µg/mL as shown in Table 5.A.
clathrodes is once again the most active extract with IC50 values in the µg/mL range (1.45 to
6.16 µg/mL) followed by V. rigida with again 1 log weaker potency (16.33 to 25.89 µg/mL).
In contrast, D. anchorata did not exhibit significant antiproliferative properties.
Antiproliferative activity on the leukemia cell lines (MOLM-14 and HL-60)
The MOLM-14 leukemia cell line was the most sensitive to all sponge extracts, closely
followed by HL-60. Indeed, the species A. clathrodes exhibited similar antiproliferative
properties on both cell lines in the µg/mL range (0.42 to 1.57 µg/mL on MOLM-14 and
1.82 to 2.5 µg/mL on HL-60 as seen in Table 5). While V. rigida was active on both cell
lines with the same activity range, a slight preference was measured for MOLM-14 over
HL-60 (3.18 to 11.94 µg/mL for MOLM-14 versus 11.03 to 36.19 µg/mL for HL-60). While
one experiment (#3, X2) could generate a dose response curve for D. anchorata with an
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 10/21
Figure 3 Cytotoxic evaluation of the sponge extracts on the for MDA-MB-231 and RES259 cell lines.
For each sample, three dose response experiments (1st blue; 2nd red; 3rd green) were performed as tripli-
cates. Doxorubicin and Ara-C and were evaluated as positive controls for MDA-MB-231 and RES259 re-
spectively, in two independent experiments.
Full-size DOI: 10.7717/peerj.13955/fig-3
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 11/21
Figure 4 Cytotoxic evaluation of the sponge extracts on the MOLM-14 and HL60 cell lines. For each
sample, two or three dose response experiments (1st blue; 2nd red; 3rd green) were performed as tripli-
cates. Ara-C was evaluated as positive control in two independent experiments for MOLM14 and one ex-
periment for HL60.
Full-size DOI: 10.7717/peerj.13955/fig-4
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 12/21
IC50 below 100 µg/mL (47.72 µg/mL), this result could not be confirmed for the two other
evaluations confirming the overall lack of activity of this species extracts during all the
cytotoxicity evaluation and on all cell lines.
DISCUSSION
Antimicrobial activity
The results obtained highlight the effects of Agelas clathrodes whose ethanolic extract is the
only sample presenting a specific activity on three strains of staphylococcus.
In similar tests carried out with the Agelas sventres species, such strain-specific activity
on staphylococcus had not been observed. In fact, an activity had been observed on E. coli
(CIP 54.127) for methanolic extracts and in n-hexane. Similar results were obtained on S.
aureus (CIP 67.8) with chloroform and hexane extracts, and finally on C. albicans (ATCC
10231) with chloroform extracts (Galeano & Martínez, 2007). The specificity observed
with A. clathrodes probably indicates a difference in active biomolecule composition due to
either the species or the nature of the solvent. Indeed, with the aqueous extract having no
effect, we can conclude that the nature of the solvent plays a crucial part in the extraction
of active molecules. A test performed using a crude methanolic extract of Agelas sp. showed
a significant effect on the same S. aureus strain (CIP 67.8), but again these were not
specific to staphylococcus (Balansa et al., 2020). Thus, the alcohol extracts appear to have
a greater effect on staphylococci than on other strains. Alcoholic solvents would probably
allow a better extraction of alkaloid type biomolecules. Indeed, the alkaloids present in
marine sponges are known for their antibacterial properties, particularly on S. aureus. For
example, bromo-pyrrole alkaloids extracted from the species Agelas dispar, are known for
their moderate antimicrobial activity on Gram +: B. subtilis and S. aureus (Chairman, AR
& Ramesh, 2012). This suggests the potential presence of similar biomolecules in the A.
clathrodes extract or a biomolecule with similar effect. It could also be a combination of
biomolecules. The lack of effect on these same strains with the aqueous extract confirms
the necessity of an alcoholic solvent for this type of biomolecules. Similarly, in Agelas
dilatata, pyrrole-imidazoles were tested on two pathogenic strains of P. aeruginosa (CIP
A22 and PAO1) and showed a moderate to strong activity. More specifically, oroidin 1
(pryrrole-imidazole which was first isolated in Agelas oroides in 1971) has also showed a
moderate activity against laboratory strains of P. aeruginosa (PA01 and PA14) (Melander
et al., 2016). Bromoageliferin is an isolated molecule that showed significant activity,
specifically on the P. aeruginosa strain CIP A22 (Pech-Puch et al., 2020,Pech-Puch et al.,
2020b). This could indicate the absence of this molecule in A. clathrodes.
Concerning the species V. rigida, this is the first study to analyze the antibacterial activity
of this extract on non-marine bacterial strains (Newbold et al., 1999). Indeed, this species is
known for its antibacterial effect on some sponge pathogenic strains, in particular Bacillus
sp. and Vibrio alginolyticus, but no work has been done on the evaluation of their possible
activity on non-marine strains. In addition, antiparasitic (Putra, Hadi & Murniasih, 2016a;
Bianco et al., 2015) or antidepressant (Zhang, 2020) effects have also been described. Also,
based on our results, we find that this species does not possess activity on non-marine
bacterial strains.
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 13/21
Table 5 Cytotoxic evaluation results. Half maximal inhibitory concentration (IC50 ) and confidence interval range (CRI) measured for the ethanolic extracts (E) on dif-
ferent cancer cell lines (MDA-MB231 (breast cancer), RES259 (glioblastoma), MOLM-14 (leukemia) and HL-60 (leukemia)). For each sponge sample, two extractions
were evaluated (X1 and X2) for a total of 3 experiments labelled as #1, #2 or #3. All values are listed as µg/ml concentrations and ‘ND’ is specified for non-determined val-
ues and ‘NC’ for not converged values during the fitting process. Doxorubicin and Ara-C are used as positive control anticancer agents.
MDA-MB-231 RES259 MOLM-14 HL-60
IC50** 95% CIR** IC50 ** 95% CIR*** IC50** 95% CIR*** IC50 ** 95% CIR***
#1 0.20 0.14 to 0.29 ND -ND -ND -
Doxorubicin #2 0.18 0.13 to 0.23 ND -ND -ND -
#1 ND -0.07*0.05 to 0.08 0.085*0.06 to 0.11 ND -
Ara-C #2 ND -0.08 0.07 to 0.10 0.222 0.15 to 0.25 0.12 0.10 to 0.15
#1 (X1) 3.42 2.38 to 4.91 1.45 0.91 to 2.07 0.41 0.34 to 0.51 ND -
#2 (X1) 7.91 2.02 to 30.99 3.52 2.64 to 4.66 1.57 1.30 to 1.85 1.82 1.58 to 2.18
Agelas
clathrodes #3 (X2) 7.71 NC 6.16 NC 1.21 1.06 to 1.37 2.5 2.20 to 2.85
#1 (X1) >100 ->100*->100*-ND -
#2 (X1) >100 ->100 ->100 ->100 -
Desmapsamma
anchorata #3 (X2) >100 -97.15 NC 47.72 40.04 to 56.95 >100 -
#1 (X1) 18.62 16.77 to 20.67 16.33 NC 3.83 2.74 to 5.20 ND -
#2 (X1) 30.68 26.06 to 36.11 11.83 NC 3.18 2.18 to 4.56 11.03 8.25 to 14.50
Verongula
rigida
#3 (X2) 64.41 54.41 to 76.25 25.89 NC 11.94 6.40 to 20.65 36.19 NC
Notes.
*Duplicate instead of triplicate.
**Half maximal inhibitory concentration (µg/mL).
***CIR: confidence interval range (µg/mL).
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 14/21
Regarding D. anchorata species, we confirmed its lack of activity against Gram- bacteria,
including E. coli and P. aeruginosa (Bianco et al., 2015). Indeed, it is becoming increasingly
difficult to find antibacterial molecules effective against Gram- bacteria, as confirmed by
our results. Moreover, antimicrobial activity tests performed with D. anchorata extracts
revealed no effect on S. aureus, a Gram+. This study confirmed the inactivity of the extract
on the strains selected for this study but it may be active on other bacterial strains not used
here.
The determination of MIC and MBC could only be performed on A. clathrodes
showing bioactivity on the selected bacterial strains. The extracts were more potent
for S. saprophyticus than S. aureus. Given the quality of the extractions performed and the
quantity of extracts obtained after drying, a higher MIC for S. aureus does not mean an
absence of active biomolecules but maybe a lower concentration. Indeed, a higher MIC
has already been obtained for an alcoholic extract on E. coli for the S. massa sponge (Putra,
Hadi & Murniasih, 2016a). Similarly for the aqueous extract, the number of biomolecules
may be too low to observe a significant effect on the bacteria.
The bacteriostatic effect of this extract may, again, indicate the presence of biomolecules
different from those known to exist in the other studied Agelas genus species.
Moreover, Agelas genus seems to have a specific type of alkaloid biomolecules or a
specific combination of them, hence the absence of effect for the species D. anchorata and
V. rigida.
Cytotoxic activity
The cytotoxicity evaluation highlighted that the Agelas clathrodes species has a strong
cytotoxic activity in addition to antimicrobial properties as previously demonstrated.
Agelas clathrodes species is also the one with the highest activity on all cell lines followed
by Verongula rigida. Their order of activity for both these sponges is the same, namely, a
stronger activity on the leukemia cell lines (MOLM-14 and HL-60) followed by the activity
on the RES259 glioblastoma cells and finally on the MDA-MB231 breast cancer lineage.
The sponges of the Agelas genus are known for their cytotoxic effect on many cancerous
cells associated with several extracted molecules such as agelasins, agelasidins and agelines.
The Agelasphins extracted from the A. mauritianus species exhibit, notably, antitumor and
immunostimulatory effects (Natori et al., 1994). Agelasine B extracted from A. clathrodes
species has demonstrated effects on the MCF-7 line of human breast cancer cells (Dipolo
& Suarez, 2012). Moderate effects of A. clathrodes extracts were also observed on the
MDA-MB-435 cell line (Custódio & RJ, 2007).
In our study, we observed similar effects on a different breast cancer cell line.
Nevertheless, working with a crude extract, we could not establish here if it was an
effect of agelasine B or if it was due to a different molecule or set of molecules. Indeed,
other tests conducted on several species of the Agelas genus (A. citrina, A. clathrodes, A.
dilatata and A. sceptrum) on the same MCF-7 cell line showed more negligible effects,
except for A. citrina which stood out with a percentage of inhibition of 100% at 30 and
15 µg/mL (Pech-Puch et al., 2020,Pech-Puch et al., 2020b). Similar results for extracts of
the Agelas clathrodes species were observed in the MDA-MB-435 cancer cell line, as well as
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 15/21
for Agelas sp. in the same study (Custódio & RJ, 2007). Furthermore, a moderate activity
had been found for A. clathrodes in the HL-60 cell line (IC50 : 48.51 µg/mL) as well as in
a glioblastoma cell line SF-295 (IC50: 62.36 µg/mL) (Custódio & RJ, 2007). The stronger
activity in our evaluation campaign could be explained by a synergy of active biomolecules.
The ethanolic crude extract of Verongula rigida showed a high cytotoxic response in
this study. This activity is specific to leukemic (MOLM-14 and HL-60), glioblastoma (RES
259) and breast cancer (MDA-MB231) cell lines. Although no specific studies have been
performed on these cells, the published results of (Galeano et al., 2011) on the cytotoxicity
of V. rigida on U937 cells of the human myeloid linage, could sustain our observations.
Indeed, Galeano showed that this property would be due to aeroplysinin-1, a protein
tyrosine kinase inhibitor found in V. rigida extracts. These active biomolecules will have to
be purified from the extracts and evaluated to confirm our preliminary results.
As for the antimicrobial and cytotoxic activities of crude ethanolic extract of
Desmapsamma anchorata, we validated a lack of activity on our cell lines for the first
time, but this was also shown on other cell lines in previous studies (Lhullier et al., 2019;
Marques et al., 2016). This was presumably due to the presence of relatively low amount of
active biomolecules.
CONCLUSION
Taken together, the results of this study suggest that crude extracts of marine sponges from
Martinique have a potential antimicrobial and cytotoxic effect. Two sponge species stood
out: Agelas clathrodes for both antimicrobial and antitumor properties, and Verongula
rigida for its antitumor properties. The existence of such active compounds in this species
extracts would respond to a need for natural antibacterial and antitumor molecules, with
limited side effects or drug resistances. Hypotheses have also emerged concerning the
biomolecular composition of Agelas clathrodes and Verongula rigida such as the presence
of alkaloids and aeroplysin-1, or biomolecules with similar effects. Moreover, having
identified these species in Martinique constitutes a key element for the valorization of the
marine biodiversity of the island within the Caribbean area. As this study is a preliminary
work never done before in the French West Indies, especially in Martinique, it would be
interesting to widen the spectrum of study to other species of the region and to carry out
complementary chemical analyses in order to isolate and confirm the bioactive candidate
or to identify other biomolecules of interest.
ACKNOWLEDGEMENTS
The authors thank Dr. Ferry for his involvement in the sampling and identification
of sponges. They also thank Dr. Stéphanie Morin, for their help in the realization
of antibiograms. The authors also thank Dr. Thomas Miller for helping rewriting the
manuscript. This work was part of the PO FEDER project No. MQ0023978.
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 16/21
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This work was supported by the ‘‘Collectivité Térritoriale de Martinique’’ (CTM), ‘‘Société
Anonyme de la Raffinerie des Antilles’’ (SARA) and ‘‘Office De l’Eau’’ (ODE). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Collectivité Térritoriale de Martinique (CTM).
Société Anonyme de la Raffinerie des Antilles (SARA).
Office De l’Eau (ODE).
Competing Interests
The authors declare there are no competing interests.
Author Contributions
•Julie Piron performed the experiments, analyzed the data, prepared figures and/or tables,
authored or reviewed drafts of the article, and approved the final draft.
•Stephane Betzi conceived and designed the experiments, analyzed the data, prepared
figures and/or tables, authored or reviewed drafts of the article, and approved the final
draft.
•Jessica Pastour performed the experiments, analyzed the data, prepared figures and/or
tables, and approved the final draft.
•Audrey Restouin performed the experiments, prepared figures and/or tables, and
approved the final draft.
•Rémy Castellano conceived and designed the experiments, prepared figures and/or
tables, and approved the final draft.
•Yves Collette conceived and designed the experiments, prepared figures and/or tables,
and approved the final draft.
•Niklas Tysklind conceived and designed the experiments, authored or reviewed drafts
of the article, and approved the final draft.
•Juliette Smith-Ravin conceived and designed the experiments, analyzed the data,
authored or reviewed drafts of the article, and approved the final draft.
•Fabienne Priam conceived and designed the experiments, analyzed the data, prepared
figures and/or tables, authored or reviewed drafts of the article, and approved the final
draft.
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving
body and any reference numbers):
The sponge samples were taken in Fins Mask Snorkel (FMS) in 2017 and were authorized
in FMS before the 2019 recreational fishing decree. We had also obtained the verbal
Piron et al. (2022), PeerJ, DOI 10.7717/peerj.13955 17/21
agreement of Mrs. Sabrina MUNIER, in charge of marine and coastal biodiversity, marine
environment referent - DEAL, Martinique.
Data Availability
The following information was supplied regarding data availability:
The raw data for antibacterial assay and cytotoxicity assay are available in the
Supplementary Files.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.13955#supplemental-information.
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