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Citation: Varamogianni-Mamatsi, D.;
Nunes, M.J.; Marques, V.; Anastasiou,
T.I.; Kagiampaki, E.; Vernadou, E.;
Dailianis, T.; Kalogerakis, N.; Branco,
L.C.; Rodrigues, C.M.P.; et al.
Comparative Chemical Profiling and
Antimicrobial/Anticancer Evaluation
of Extracts from Farmed versus Wild
Agelas oroides and Sarcotragus foetidus
Sponges. Mar. Drugs 2023,21, 612.
https://doi.org/10.3390/
md21120612
Academic Editor: Daniela Giordano
Received: 9 October 2023
Revised: 8 November 2023
Accepted: 22 November 2023
Published: 26 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
marine drugs
Article
Comparative Chemical Profiling and Antimicrobial/Anticancer
Evaluation of Extracts from Farmed versus Wild Agelas oroides
and Sarcotragus foetidus Sponges
Despoina Varamogianni-Mamatsi 1,2,3,4,†, Maria João Nunes 5, † , Vanda Marques 6, Thekla I. Anastasiou 1,
Eirini Kagiampaki 1, Emmanouela Vernadou 1, Thanos Dailianis 1, Nicolas Kalogerakis 2, Luís C. Branco 5,
Cecília M. P. Rodrigues 6, Rita G. Sobral 3,4 , Susana P. Gaudêncio 3,4 ,* and Manolis Mandalakis 1, *
1Hellenic Centre for Marine Research, Institute of Marine Biology, Biotechnology and Aquaculture,
71500 Heraklion Crete, Greece; d.varamogianni@hcmr.gr (D.V.-M.); theanast@hcmr.gr (T.I.A.);
e.kagiampaki@hcmr.gr (E.K.); e.vernadou@hcmr.gr (E.V.); thanosd@hcmr.gr (T.D.)
2School of Chemical and Environmental Engineering, Technical University of Crete, 73100 Chania, Greece;
nicolas.kalogerakis@enveng.tuc.gr
3Associate Laboratory i4HB, Institute for Health and Bioeconomy, NOVA School of Science and Technology,
NOVA University of Lisbon, Campus Caparica, 2819-516 Caparica, Portugal; rgs@fct.unl.pt
4UCIBIO—Applied Molecular Biosciences Unit, Chemistry and Life Sciences Departments, NOVA School of
Science and Technology, NOVA University of Lisbon, Campus Caparica, 2819-516 Caparica, Portugal
5LAQV, REQUIMTE, Associated Laboratory for Green Chemistry, Chemistry Department, NOVA School of
Science and Technology, NOVA University of Lisbon, Campus Caparica, 2819-516 Caparica, Portugal;
mjm.nunes@fct.unl.pt (M.J.N.); l.branco@fct.unl.pt (L.C.B.)
6
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor
Gama Pinto, 1649-003 Lisboa, Portugal; vismsmarques@ff.ulisboa.pt (V.M.);
cmprodrigues@ff.ulisboa.pt (C.M.P.R.)
*Correspondence: s.gaudencio@fct.unl.pt (S.P.G.); mandalakis@hcmr.gr (M.M.); Tel.: +351-212948300 (S.P.G.);
+30-2810-337855 (M.M.); Fax: +351-212948550 (S.P.G.); +30-2810-337822 (M.M.)
†These authors contributed equally to this work.
Abstract:
Marine sponges are highly efficient in removing organic pollutants and their cultivation,
adjacent to fish farms, is increasingly considered as a strategy for improving seawater quality.
Moreover, these invertebrates produce a plethora of bioactive metabolites, which could translate into
an extra profit for the aquaculture sector. Here, we investigated the chemical profile and bioactivity
of two Mediterranean species (i.e., Agelas oroides and Sarcotragus foetidus) and we assessed whether
cultivated sponges differed substantially from their wild counterparts. Metabolomic analysis of crude
sponge extracts revealed species-specific chemical patterns, with A. oroides and S. foetidus dominated
by alkaloids and lipids, respectively. More importantly, farmed and wild explants of each species
demonstrated similar chemical fingerprints, with the majority of the metabolites showing modest
differences on a sponge mass-normalized basis. Furthermore, farmed sponge extracts presented
similar or slightly lower antibacterial activity against methicillin-resistant Staphylococcus aureus,
compared to the extracts resulting from wild sponges. Anticancer assays against human colorectal
carcinoma cells (HCT-116) revealed marginally active extracts from both wild and farmed S. foetidus
populations. Our study highlights that, besides mitigating organic pollution in fish aquaculture,
sponge farming can serve as a valuable resource of biomolecules, with promising potential in
pharmaceutical and biomedical applications.
Keywords:
porifera; demospongiae; marine sponge farming; chemical fingerprinting; marine natural
products; secondary metabolites; primary metabolites; MS/MS dereplication; bioactive compounds
supply; aquaculture and fish farming
Mar. Drugs 2023,21, 612. https://doi.org/10.3390/md21120612 https://www.mdpi.com/journal/marinedrugs
Mar. Drugs 2023,21, 612 2 of 27
1. Introduction
While aquaculture constitutes the fastest growing food production system [
1
], it can
exert pressure on adjacent marine habitats, mainly through the release of organic load
and other substances [
2
]. Among bioremediation candidates to reduce organic pollution,
marine sponges have attracted widespread interest [
3
–
6
], due to their innate filter-feeding
properties to remove particles (e.g., bacteria [
7
–
12
], phytoplankton [
13
–
16
]), and dissolved
organic [
17
–
19
]/inorganic substrates [
20
,
21
], as well as fish farm wastes [
22
–
24
] from sea-
water. Besides their high cleanup capacity, marine sponges also hold great biotechnological
potential, and their biomass can be turned into products of high added-value, serving as an
additional source of profit for aquaculture-related enterprises. In fact, to date, more than
5000 structurally characterized metabolites have been isolated from marine sponges, con-
tributing to about 30% of all marine natural products [
25
]. Although a variety of sponges
is currently known to produce an overwhelming array of secondary metabolites with
pharmaceutical potential [
26
], the problem of their supply still remains a typical limiting
factor for pre-clinical evaluation [27] and further drug development [28].
Over the years, various techniques have been proposed to overcome this bottleneck.
Synthesis of bioactive natural products or their related analogues (e.g., via chemical or
microbial processes) has always been the preferred method for drug manufacture in phar-
maceutical industry. Some of the few successful sponge-derived examples of this strategy
are the well-known drugs adenine arabinoside (Ara-A, Vidarabine
®
) and cytosine arabi-
noside (Ara-C, Cytarabine
®
). Both synthetic products constitute derivatives of sponge
nucleosides [
29
,
30
], and they have been clinically approved for use as antiviral and anti-
tumor drugs, respectively [
31
–
34
]. However, the development of synthetic strategies for
the production of other marine metabolites with greater structural complexity is often
challenging and economically unfeasible, even for the purpose of preclinical testing [
26
,
27
].
Wild harvesting of sponges that are prolific sources of bioactive compounds has com-
monly been suggested as a method to supply novel therapeutic agents. However, the
typically low naturally occurring concentrations of produced bioactive metabolites, com-
bined with valid concerns for the conservation of sponge diversity in marine ecosystems,
are the main reasons to consider this method unsuitable [
27
,
35
,
36
]. This also aligns with
the responsible research and innovation aspects that comply with environmental and so-
cietal values [
37
,
38
]. Nonetheless, a variety of novel marine drug-leads have proceeded
to preclinical and clinical trials using materials from wild harvesting. This is the case for
avarol, a novel sesquiterpenoid hydroquinone, which is uniquely found in the abundant
Mediterranean species Dysidea avara [
39
]. Being one of the most popular sponge-derived
bioactive compounds, avarol exhibited strong anti-HIV activity during its preliminary
testing [
40
], but it was later withdrawn from human clinical trials. Moreover, avarol was
patented as an anti-psoriasis agent [
41
], being used in paramedic medicine as one of the
ingredients of topical ointments against psoriasis.
Halichondrin B, a metabolite isolated from the sponge Lissodendoryx sp., was reported
by Hirata and Uemura (1986) [
27
] as a strong antitumor agent, with a special effect against
several melanoma types and leukemia. Although this compound has entered phase I of
clinical trials [
28
], it was produced at very low concentrations by harvested sponges. The
cytotoxic metabolite peloruside A, which is isolated from specimens of the sponge Mycale
hentscheli, is another highly promising antitumor agent, since it operates in a similar way
as the anticancer drug Taxol
®
, used to treat ovarian and breast cancers [
36
]. However,
similarly to halichondrin B, wild sponge populations were unable to provide high yields of
this compound to proceed in further drug development [42].
Other methods, such as cell lines, primmorphs, and ex situ culture, have extensively
been investigated as economically feasible approaches for obtaining sufficient quantities
of drug-lead sponge molecules, but they have shown a number of limitations (e.g., high
time and resources consumption, poor growth rates) [
43
–
45
]. By taking into account the
plant-like regeneration capability of sponges, and their resilience to overcome physical
damage [
46
], mariculture of these metazoans merges as a promising, cost-effective outlook
Mar. Drugs 2023,21, 612 3 of 27
for the sufficient and sustainable supply of biologically active metabolites [
47
]. In combi-
nation with their filter-feeding characteristics, setting up a sponge farm in proximity to
aquaculture operations is becoming highly appealing, since it can promote bioremediation
applications through profitability [48].
However, it is of great importance to reassure farmers that the cultivated sponge
fragments are able to reproduce the targeted bioactive metabolites of their wild coun-
terparts. In this context, we performed comparative chemical profiling of sponge crude
extracts, obtained from wild and farmed populations. The study focused on two widely
distributed Mediterranean species, namely Agelas oroides and Sarcotragus foetidus, which
have already been distinguished for their
in vitro
bioremediation efficiency against various
pollutants [
16
,
24
] and their production of metabolites with pharmaceutical and biotechno-
logical importance [
49
–
52
]. Chemical characterization of sponge extracts was performed by
using high-resolution analytical techniques, such as liquid chromatography coupled with
tandem mass spectrometry (LC–MS/MS), targeted to the bioactive metabolites reported in
the literature. For this purpose, a comprehensive metabolites/ions/MS fragments library,
including all the previously reported compounds for both species, was created. In addition,
the sponge extracts were assayed for their biological activities, with a particular focus on
their antibacterial and anticancer properties. Through our study, we aspire to demonstrate
the bioproduction potential of farmed marine sponges and pave the way for their inclusion
as prolific candidates in integrated aquaculture systems.
2. Results and Discussion
2.1. The Metabolomics Profile of Farmed and Wild Agelas oroides Sponges
The crude extracts obtained from wild and farmed A. oroides specimens were analyzed
by LC–MS/MS to determine their chemical composition with the aid of a comprehensive
metabolites/ions/MS fragments library (Table S1). Three chemical superclasses were identi-
fied in the A. oroides extracts—alkaloids, indoles, and lipids (Table 1)—with alkaloids being
the most predominant constituents in both types of sponge populations (farmed; 97.5%,
wild; 98.9%). The majority of the detected alkaloids comprised pyrrole classes, representing
96.2% and 98.3% of the total metabolite content in the farmed and wild A. oroides sponges,
respectively. Based on skeletal features of the produced pyrrole alkaloids, these were
further categorized into three chemical subclasses: (1) linear pyrrole alkaloids, (2) fused
cyclic pyrrole alkaloids, and (3) dimeric pyrrole alkaloids [
49
]. The subclass of terpenoid
alkaloids was detected in lower percentages among the farmed and wild sponges (1.3% and
0.6%, respectively). An intriguing class of indole metabolites with unexplored potential in
myriad research fields (e.g., chemistry, pharmacology, physiology, and medicine) [
53
] was
also present in the studied extracts, but in significantly low proportions (farmed; 0.03%,
wild; 0.02%).
Table 1.
Distribution of metabolite superclasses, classes, and subclasses in the extracts of wild and farmed
Agelas oroides sponges. Numbers in parentheses represent the standard deviation. ND—not detected.
Superclass Class Subclass Wild
(%)
Farmed
(%)
Alkaloids Pyrrole alkaloids
Linear pyrrole alkaloids 97.2 (1.4) 95.1 (0.9)
Fused cyclic
pyrrole alkaloids 0.9 (0.1) 0.7 (0.0)
Dimeric pyrrole alkaloids 0.2 (0.2) 0.4 (0.1)
Terpenoid alkaloids ND 0.6 (0.5) 1.3 (0.1)
Indoles ND ND <0.03 <0.03
Lipids
Fatty acyls ND 1.0 (0.9) 2.4 (0.8)
Glycerolipids ND <0.002 <0.003
Steroids ND <0.002 <0.001
Mar. Drugs 2023,21, 612 4 of 27
The lipids superclass was present in low abundance and accounted for 2.4% and 1.5%
of the total metabolite content in cultured and wild A. oroides extracts, respectively. MS
2
analysis indicated that the extracts with lipid molecules belong to three classes: (1) fatty
acyls, (2) glycerolipids, and (3) steroids. The fatty acyls compounds were detected in the
following content, (farmed sponges; 2.4%, wild sponges; 1.0%) and to a much lower extent,
the classes of glycerolipids and steroids (<0.01%).
The individual components detected in A. oroides extracts, along with their chemical
and mass spectrometric characteristics are summarized in Table S1. In total, 28 metabo-
lites were identified in wild A. oroides extracts, with only three of these compounds being
completely absent in the farmed sponge samples (i.e., ageliferin [
54
], oxysceptrin [
55
],
and trichodermanone C [
56
]). However, it should be highlighted that these specific com-
pounds were detected in only one replicate of the wild sponges, at significantly low levels
(i.e., <0.01%).
The content of each metabolite, expressed by its relative abundance (%) in the tested
samples, is presented in Table S2. Based on these results, the linear pyrrole–imidazole
alkaloid oroidin, which was identified using electrospray ionization in positive ion mode
(ESI+), was the most abundant metabolite found in both farmed and wild A. oroides popula-
tions (70.3% and 80.4%, respectively). Oroidin constitutes the characteristic metabolite of
A. oroides sponges [
57
–
59
]. In fact, it was the first metabolite isolated from this species [
60
]
and possesses broad-spectrum biological activities (e.g., antibacterial, antifouling, anti-
malarial properties and anti-predatory defenses against reef fish) [49].
Apart from oroidin, abundance differences between farmed and wild sponges were
observed in other detected metabolites. Regarding cultivated sponges, the following most
abundant metabolites were the closely oroidin-related molecules keramadine [
61
] (8.0%)
and dispacamide B [
62
] (7.8%). Their respective levels in wild extracts were determined to
be as high as 3.6% and 2.9%, respectively. Conversely, the second major metabolite of wild
sponges (i.e., 6.8%) was the oroidin hydrolysis product 4,5-dibromopyrrole-2-carboxylic
acid [
63
]. This metabolite was detected at similar abundance levels in the extracts of
farmed A. oroides specimens (6.6%) and yet was the fourth major metabolite of these
extracts. Additionally, small amounts of the linear pyrrole alkaloids dispacamide A [
62
]
and hymenidin [
64
] (farmed; 1.6–1.0%, wild; 2.1–1.4%, respectively) were determined in
both sponge populations.
The differences observed in the ranking of metabolites between wild and cultivated
specimens are partially in contrast with the findings of Rodriguez et al. (1994) [
65
]. In
the reported study, it was found that Acanthella cavernosa sponges, collected from natural
habitats and populations held in controlled aquaculture systems, shared common major
constituents, but with different contents in their respective chemical profiles. In our case,
oroidin was the common dominant metabolite among studied extracts, but the chemical
composition thereafter seemed to be population-specific.
In addition to linear pyrrole alkaloids, which determined the overall metabolite compo-
sition of both farmed (97.2%) and wild (95.1%) extracts, compounds belonging to other alka-
loid subclasses were also present. Dibromophakellin, an analogue derived through oroidin
cyclization/oxidation processes [
49
,
66
], previously described from the sponge Phakellia
flabellata [
67
], was the most abundant fused cyclic pyrrole alkaloid; however, it was detected
at a low percentage (i.e., <1.0%) in both types of extracts. This was followed by its relative
congeners longamide B methyl ester [68], longamide B [69], monobromoisophakellin [70],
and 3-debromohanishin [
71
]. Overall, the percentage of fused cyclic pyrrole alkaloids
reached up to 0.7% and 0.9% for cultured and wild A. oroides specimens.
Dimeric pyrrole alkaloids, formed by oxidation, cyclization, and dimerization reactions
of simple monomers (i.e., oroidin, clathrodin, and hymenidin) [
72
] was the least abundant
alkaloids group within the extracts (farmed; 0.4% and wild; 0.2%). Nakamuric acid [
73
]
and debromosceptrin acetate [
74
] represented the majority of such metabolites, while
bromoageliferin [54] and sceptrin [75] were present in very low percentages (<0.01%).
Mar. Drugs 2023,21, 612 5 of 27
Terpenoid alkaloids, which involve a nitrogen-based functional group (in the form of
an amine or ammonia, etc.) attached to preformed terpenoid moieties [
49
,
76
], were also
present at low levels in A. oroides extracts (up to 1.1% for farmed and 0.5% for wild sponges).
This was the case for metabolites belonging to the families of agelasines (e.g., agelasine [
77
],
agelasine A [78] and E [79]), as well as agelasidines (e.g., agelasidine A [80]).
The lipid content of both farmed and wild A. oroides was mainly characterized by
fatty acyls, which were dominated by the compound 10-methyl-9(Z)-octadecenoic acid [
81
],
accounting for 2.4% and 1.0% of the total metabolite composition in the cultured and
wild sponge extracts, respectively. Interestingly, this unsaturated fatty acid has previously
only been recorded in extracts of the marine fungus Microsphaeropsis olivacea, which was
isolated by Yu et al. (1996) from a sponge collected in Florida. Along with Tasdemir et al.
(2007) [
82
], who previously reported the presence of the isomer 11-methyloctadecanoic
acid in A. oroides, we are the first to discover the existence of 10-methyl-9(Z)-octadecenoic
acid in this sponge species. Its glyceride [
81
] was also detected, but at trace concentrations
in both types of extracts (i.e., <0.003%). Detected steroids were mainly members of the
ecdysteroids class, such as 20-hydroxyecdysone-22-acetate [
83
],
β
-ecdysterone [
69
] and
ponasterone A [83], which collectively accounted for <0.01% of the sponge extracts.
5,6-dihydroxyindole was the only representative of the indole family. However, it
was present in very low concentrations for both farmed (0.03%) and wild (0.02%) sponge
extracts. This metabolite is an intermediate of the melanin biosynthetic pathway, which has
previously demonstrated antibacterial activity against Gram-negative (e.g., Escherichia coli)
and Gram-positive bacterial pathogens (e.g., Staphylococcus aureus), as well as antifungal
activity [84].
The metabolic profiling of the crude sponge extracts was further investigated by
an unsupervised principal component analysis (PCA), to evaluate the similarities and
differences between the extracts of the two populations and access clustering trends, as
well as identifying outliers. The results obtained from the PCA (Figure 1) indicated the
high spatial distribution of the extracts produced by wild A. oroides fragments, which
are highlighted in green. Extracts derived from farmed specimens (highlighted in red)
are closely clustered. Based on these findings, it can be assumed that A. oroides sponges
are likely to provide extracts with a similar chemical profile when subjected to farm
conditions, whereas the composition of the wild individuals can be more diverse. This
result presumably reflects the more homogenous environment of the farm conditions, in
contrast to the complexity of a natural habitat, where the biodiversity assemblages and
interactions between their organisms at micro and macro levels are more complex and
intricate, thus inducing diversification to the sponges’ chemical phenotype. However,
according to the PCA scores plot (Figure 1a), the extracts derived from the farmed sponges
are closely similar to one of the wild specimens, supporting a consistency of the core
chemical profile of A. oroides, disregarding the prevalent drivers in the natural versus
aquaculture environments.
In a similar study, Page et al. (2005) [
85
] investigated the biosynthesis of metabolites
from sponges Mycale hentscheli, collected from aquaculture and natural habitats in New
Zealand. Their results showed that the levels of the cytotoxic compounds mycalamide
A, pateamine, and peloruside A varied not only within wild specimens, but also among
farmed explants. Consequently, differences in the chemical profile were evident at an
individual-specific level, rather than among different populations. Similar findings were
reported for the bioactive metabolite amphitoxin, produced by cultured and natural species
of the Indonesian reef-dwelling sponge Callyspongia biru [86].
Studies have shown that the variability of secondary metabolite production in sponges
can be pronounced at the intraspecific level, among populations or even among individ-
uals [
42
,
87
,
88
]. A variety of biological traits (e.g., sponge shape [
89
] and size [
90
]) or
environmental factors (e.g., response to predation [
91
], pollutants [
92
], light [
93
,
94
], and
temperature [
42
]) can induce these marine organisms to modify their levels of secondary
metabolites. Considering the fluctuation of the aforementioned parameters in natural habi-
Mar. Drugs 2023,21, 612 6 of 27
tats, compared to the less complex artificial environment of a fish farm, a higher diversity
of secondary metabolites is expected for wild versus farmed sponge specimens.
Mar. Drugs 2023, 21, x 6 of 28
Figure 1. Principal component analysis (a) scores plot and (b) loadings plot of crude A. oroides ex-
tracts belonging to wild and farmed sponges. Color circles represent the origin of the analyzed
sponge extract (i.e., green for wild or red for farmed) and triangles represent the dierent chemical
subclasses of the identied metabolites, according to the legend.
In a similar study, Page et al. (2005) [85] investigated the biosynthesis of metabolites
from sponges Mycale hentscheli, collected from aquaculture and natural habitats in New
Wild #1
Wild #2
Wild #3
Farmed #1
Farmed #2
Farmed #3
-10
-8
-6
-4
-2
0
2
4
6
8
10
-10 -8 -6 -4 -2 0246810
PC2 (17.8 %)
PC1 (52.5 %)
Observations (axes F1 and F2: 70.30 %)
(a)
3-debromohanishin
4,5-dibromopyrrole-2-carboxylic acid
Bromoageliferin
Debromosceptrin
acetate Dibromophakellin
Dispacamide A
Dispacamide B
Hymenidin
Keramadine
Longamide B
Longamide B methyl
ester
Monobromoisophakellin
Nakamuric acid Oroidin
Agelasidine A
Agelasine
Agelasine A
4,6-Dihydroxyindole
10-methyl-9(Z)-
octadecenoic acid
2,3-dihydroxypropyl(Z)-10-methyloctadec-
9-enoate
20-hydroxyecdysone-22-acetate
β-ecdysterone
-10
-8
-6
-4
-2
0
2
4
6
8
10
-10 -8 -6 -4 -2 0246810
PC2 (17.8 %)
PC1 (52.5 %)
Biplot (axes F1 and F2: 70.30 %)
(b)
Farmed sponge extract
Wild sponge extract
Linear pyrrole alkaloids
Fused cyclic pyrrole alkaloids
Dimeric pyrrole alkaloids
Terpenoid alkaloids
Indoles
Fatty acyls
Glycerolipids
Steroids
Figure 1.
Principal component analysis (
a
) scores plot and (
b
) loadings plot of crude A. oroides extracts
belonging to wild and farmed sponges. Color circles represent the origin of the analyzed sponge
extract (i.e., green for wild or red for farmed) and triangles represent the different chemical subclasses
of the identified metabolites, according to the legend.
Mar. Drugs 2023,21, 612 7 of 27
Overall, the first two principal components (PC1 and PC2) explained 52.5% and 17.8%
of the total variance present in the dataset (Figure 1). Interestingly, the loadings plot
(Figure 1b) shows that the metabolites highly enriched in carbon content (i.e., C
22
–C
29
),
such as dimeric pyrrole and terpenoid alkaloids, as well as steroids, are grouped together
in the bottom left quadrant of the panel. As was expected, the linear pyrrole alkaloids
oroidin and 4,5-dibromoprrole-2-carboxylic acid, which constitute the major constituents
of the wild specimens, are scattered closely to the wild extracts. The same stands for the
linear pyrrole alkaloids keramadine and dispacamide B with respect to farmed sponges.
However, a series of t-tests (Figure 2) revealed that significant differences in the metabo-
lite content were observed only for the bromopyrrole alkaloids oroidin (p= 0.025) and
dibromophakellin (p= 0.025), which were richer in wild A. oroides sponges. Furthermore,
compounds of the same class, including ageliferin and oxysceptrin, were completely absent
in farmed A. oroides sponges. Previous studies have highlighted the role of oroidin-like
brominated pyrrole alkaloids as chemical defenders against fish predation for the genus
Agelas [
63
,
95
] and enhanced bromine content with increased feeding deterrent potency [
96
].
Given that sponges occurring in natural habitats are prone to face increased antagonistic
interactions compared to their farmed counterparts, a higher content of bromopyrrole
alkaloids can thus be expected in wild specimens.
Mar.Drugs2023,21,x 7of28
Zealand.TheirresultsshowedthatthelevelsofthecytotoxiccompoundsmycalamideA,
pateamine,andpelorusideAvariednotonlywithinwildspecimens,butalsoamong
farmedexplants.Consequently,differencesinthechemicalprofilewereevidentatanin-
dividual-specificlevel,ratherthanamongdifferentpopulations.Similarfindingswerere-
portedforthebioactivemetaboliteamphitoxin,producedbyculturedandnaturalspecies
oftheIndonesianreef-dwellingspongeCallyspongiabiru[86].
Studieshaveshownthatthevariabilityofsecondarymetaboliteproductionin
spongescanbepronouncedattheintraspecificlevel,amongpopulationsorevenamong
individuals[42,87,88].Avarietyofbiologicaltraits(e.g.,spongeshape[89]andsize[90])
orenvironmentalfactors(e.g.,responsetopredation[91],pollutants[92],light[93,94],and
temperature[42])caninducethesemarineorganismstomodifytheirlevelsofsecondary
metabolites.Consideringthefluctuationoftheaforementionedparametersinnaturalhab-
itats,comparedtothelesscomplexartificialenvironmentofafishfarm,ahigherdiversity
ofsecondarymetabolitesisexpectedforwildversusfarmedspongespecimens.
Overall,thefirsttwoprincipalcomponents(PC1andPC2)explained52.5%and
17.8%ofthetotalvariancepresentinthedataset(Figure1).Interestingly,theloadingsplot
(Figure1b)showsthatthemetaboliteshighlyenrichedincarboncontent(i.e.,C
22
–C
29
),
suchasdimericpyrroleandterpenoidalkaloids,aswellassteroids,aregroupedtogether
intheboomleftquadrantofthepanel.Aswasexpected,thelinearpyrrolealkaloids
oroidinand4,5-dibromoprrole-2-carboxylicacid,whichconstitutethemajorconstituents
ofthewildspecimens,arescaeredcloselytothewildextracts.Thesamestandsforthe
linearpyrrolealkaloidskeramadineanddispacamideBwithrespecttofarmedsponges.
However,aseriesoft-tests(Figure2)revealedthatsignificantdifferencesintheme-
tabolitecontentwereobservedonlyforthebromopyrrolealkaloidsoroidin(p=0.025)and
dibromophakellin(p=0.025),whichwerericherinwildA.oroidessponges.Furthermore,
compoundsofthesameclass,includingageliferinandoxysceptrin,werecompletelyab-
sentinfarmedA.oroidessponges.Previousstudieshavehighlightedtheroleoforoidin-
likebrominatedpyrrolealkaloidsaschemicaldefendersagainstfishpredationforthege-
nusAgelas[63,95]andenhancedbrominecontentwithincreasedfeedingdeterrentpo-
tency[96].Giventhatspongesoccurringinnaturalhabitatsarepronetofaceincreased
antagonisticinteractionscomparedtotheirfarmedcounterparts,ahighercontentofbro-
mopyrrolealkaloidscanthusbeexpectedinwildspecimens.
Figure2.Boxplotofrelativeabundancesformetabolitesoroidin,dibromophakellin,anddispaca-
mideB,whichpresentedsignificantabundancedifferences(t-test,p<0.05)betweentheextracts
contentoffarmed(red)andwild(green)A.oroidessponges.Datawerenormalizedtothetotalspec-
tralareaandpresentedintheauto-scalemodeoftheMetaboAnalyst5.0software.Boxesrangefrom
the25%to75%percentile;whiskersindicatethe5%and95%percentiles;blackdotsshowtheindi-
vidualdatapoints;thehorizontallineandtheyellowdiamondwithineachboxindicatethemedian
andmeanvalue,respectively.
Figure 2.
Boxplot of relative abundances for metabolites oroidin, dibromophakellin, and dispacamide
B, which presented significant abundance differences (t-test, p< 0.05) between the extracts content of
farmed (red) and wild (green) A. oroides sponges. Data were normalized to the total spectral area and
presented in the auto-scale mode of the MetaboAnalyst 5.0 software. Boxes range from the 25% to
75% percentile; whiskers indicate the 5% and 95% percentiles; black dots show the individual data
points; the horizontal line and the yellow diamond within each box indicate the median and mean
value, respectively.
Interestingly, farmed A. oroides sponges exhibited a significantly higher content in
the pyrrole alkaloid dispacamide B (p= 0.031), which was also included in the top three
abundant metabolites of the respective extracts (Figure 2). However, the ecological role of
this alkaloid has not yet been well established. It is likely to be related to other biological
functions of this species, such as growth or reproduction, but a lack of evidence means its
higher biosynthesis in the farmed population cannot be explained. Nevertheless, oroidin,
dibromophakellin, and dispacamide B could serve as chemotaxonomic markers for distin-
guishing farmed from wild A. oroides specimens, but further investigation is required to
support this statement. Overall, the distribution of the various metabolite classes between
the two sponge populations remained constant, but the composition of the three above
mentioned compounds varied according to the population.
Apart from assessing differences in the metabolite content, we further aimed to com-
pare production levels of the respective bioactive compounds in the two sponge populations.
For the quantification of the detected metabolites, we introduced the term of the weight-
Mar. Drugs 2023,21, 612 8 of 27
normalized intensity, that considers the dry weight of each sponge fragment subjected to
extraction (expressed as intensity units per gram of sponge dry weight). The calculated
values are presented in Table S3. Out of the 25 metabolites shared between farmed and
wild sponges, only two showed significant variation in their production levels. These were
the terpenoid alkaloids agelasine (t-test, p= 0.012) and agelasine A (t-test, p= 0.019), which
were increasingly produced by farmed explants (Figure 3).
Mar.Drugs2023,21,x 8of28
Interestingly,farmedA.oroidesspongesexhibitedasignificantlyhighercontentin
thepyrrolealkaloiddispacamideB(p=0.031),whichwasalsoincludedinthetopthree
abundantmetabolitesoftherespectiveextracts(Figure2).However,theecologicalroleof
thisalkaloidhasnotyetbeenwellestablished.Itislikelytoberelatedtootherbiological
functionsofthisspecies,suchasgrowthorreproduction,butalackofevidencemeansits
higherbiosynthesisinthefarmedpopulationcannotbeexplained.Nevertheless,oroidin,
dibromophakellin,anddispacamideBcouldserveaschemotaxonomicmarkersfordis-
tinguishingfarmedfromwildA.oroidesspecimens,butfurtherinvestigationisrequired
tosupportthisstatement.Overall,thedistributionofthevariousmetaboliteclassesbe-
tweenthetwospongepopulationsremainedconstant,butthecompositionofthethree
abovementionedcompoundsvariedaccordingtothepopulation.
Apartfromassessingdifferencesinthemetabolitecontent,wefurtheraimedtocom-
pareproductionlevelsoftherespectivebioactivecompoundsinthetwospongepopula-
tions.Forthequantificationofthedetectedmetabolites,weintroducedthetermofthe
weight-normalizedintensity,thatconsidersthedryweightofeachspongefragmentsub-
jectedtoextraction(expressedasintensityunitspergramofspongedryweight).Thecal-
culatedvaluesarepresentedinTabl eS3.Outofthe25metabolitessharedbetweenfarmed
andwildsponges,onlytwoshowedsignificantvariationintheirproductionlevels.These
weretheterpenoidalkaloidsagelasine(t-test,p=0.012)andagelasineA(t-test,p=0.019),
whichwereincreasinglyproducedbyfarmedexplants(Figure3).
Figure3.Boxplotofweight-normalizedintensitiesformetabolitesagelasineandagelasineA,which
presentedsignificantdifferencesintheirproduction(t-test,p<0.05)betweentheextractsoffarmed
(red)andwild(green)A.oroidessponges.Dataarepresentedintheauto-scalemodeoftheMetabo-
Analyst5.0software.Boxesrangefromthe25%to75%percentile;whiskersindicatethe5%and95%
percentiles;blackdotsshowtheindividualdatapoints;thehorizontallineandtheyellowdiamond
withineachboxindicatethemedianandmeanvalue,respectively.
Ahandfulofstudieshavesimilarlyindicatedthatfarmingcanpromotetheproduc-
tionofspecificbioactivemetabolitesinsponges.Byfollowinga9-monthstrategyofhar-
vestingexplantsfromculturesoftheNewZealanddemospongesLatrunculiawellingtonen-
sisandPolymastiacroceus,Duckworthetal.(2003)[47]measuredsimilarorhigherbioac-
tivityinfarmedspecimensthanthatexhibitedbynaturalpopulations.However,itshould
benotedthattheoverallmetaboliteproductionwasestimatedintermsofamurineleu-
kemiabioassay,andnotbyusingmetabolomicsapproaches.
Furthermore,anarrayofexsitutrialsrevealedthatfarmedspongeexplantsare
greaterproducersthantheirwildcounterpartswithrespecttospecificbiomolecules.This
wasthecasefortheMediterraneanspongeAplysinaaerophoba,anditsbrominatedisoxa-
zolinealkaloidsaplysinamisin-1,aerophobin-2,andisofistularin-3,aswellasthebiotrans-
formationproductaeroplysinin-1,whenexplantsofthespecificspecieswerekeptfor9
monthsincontrolledaquariumsystems[97].Similartothepreviouswork,Duckworthet
Figure 3.
Boxplot of weight-normalized intensities for metabolites agelasine and agelasine A, which
presented significant differences in their production (t-test, p< 0.05) between the extracts of farmed
(red) and wild (green) A. oroides sponges. Data are presented in the auto-scale mode of the Metabo-
Analyst 5.0 software. Boxes range from the 25% to 75% percentile; whiskers indicate the 5% and 95%
percentiles; black dots show the individual data points; the horizontal line and the yellow diamond
within each box indicate the median and mean value, respectively.
A handful of studies have similarly indicated that farming can promote the production
of specific bioactive metabolites in sponges. By following a 9-month strategy of harvesting
explants from cultures of the New Zealand demosponges Latrunculia wellingtonensis and
Polymastia croceus, Duckworth et al. (2003) [
47
] measured similar or higher bioactivity
in farmed specimens than that exhibited by natural populations. However, it should be
noted that the overall metabolite production was estimated in terms of a murine leukemia
bioassay, and not by using metabolomics approaches.
Furthermore, an array of ex situ trials revealed that farmed sponge explants are greater
producers than their wild counterparts with respect to specific biomolecules. This was the
case for the Mediterranean sponge Aplysina aerophoba, and its brominated isoxazoline alka-
loids aplysinamisin-1, aerophobin-2, and isofistularin-3, as well as the biotransformation
product aeroplysinin-1, when explants of the specific species were kept for 9 months in
controlled aquarium systems [
97
]. Similar to the previous work, Duckworth et al. (2003) re-
ported higher levels of the antitumor compound stevensine in cultures of Axinella corrugata,
by following a multicomponent diet [
98
]. Contrarily, Munro et al. (1999) [
28
] found that the
overall halichondrin content of the cultured Lissodendoryx sponge was not as high as that of
the wild specimens, reaching the opposite conclusion. No effect of farming on metabolite
production was indicated by Carballo et al. (2010) [
27
] and Ternon et al. (2017) [
99
], who
conducted sponge mariculture of the species Mycale cecilia and Crambe crambe, respectively.
2.2. The Metabolomics Profile of Farmed and Wild Sarcotragus foetidus Sponges
The LC–MS/MS analysis of S. foetidus crude extracts revealed that, in total, 31 metabo-
lites were shared between farmed and wild sponges, with all the compounds being present
in at least one biological replicate of each population (Tables S4 and S5). Detection was
supported by building a comprehensive metabolites/ions/MS fragments library (Table S4).
The detected S. foetidus metabolites were classified into five superclasses, namely ben-
zenoids, dipeptides, indoles, lipids, and polyketides (Table 2). This species was revealed
to be an efficient lipid producer, by presenting extracts with an average lipid content as
Mar. Drugs 2023,21, 612 9 of 27
high as 95.5% and 94.3% among wild and farmed specimens, respectively. The detected
lipids classes were: (1) fatty acyls, (2) glycerolipids, (3) prenol lipids, and (4) steroids. This
slightly higher predominance of lipids observed in the extracts of farmed sponges was
evaluated as statistically significant (t-test, p= 0.002), and is attributed to the higher levels
of prenol lipids (t-test, p= 0.011) in the respective extracts when compared to their wild
counterparts. However, this specific class held only a small share in the overall chemical
composition of both sponge populations (farmed; 3.1% and wild; 1.5%). Lipids were mainly
dominated by the fatty acyls class, which accounted for more than 55% of the overall
metabolite abundance, followed by steroids, averaging at approximately 28.7% and 30.1%
within farmed and wild sponges, respectively. However, this steroid dominance in wild
individuals was regarded as marginally significant (t-test, p= 0.037). Glycerolipids were
present at similar abundance levels within the sponge populations, collectively accounting
for 7.4% and 7.2% of the farmed and wild sponge extracts.
Table 2.
Distribution of metabolite superclasses and classes in the extracts of wild and farmed Sarcotragus
foetidus sponges. Numbers in parentheses represent the standard deviation. ND—not detected.
Superclass Class Wild
(%)
Farmed
(%)
Benzenoids
Anthracenes <0.01 <0.01
Benzene and substituted derivatives 4.2 (0.3) 3.2 (0.4)
Benzopyrans 0.5 (0.2) 0.4 (0.0)
Phenols <0.01 <0.01
Dipeptides ND <0.02 <0.02
Indoles ND 0.1 (0.0) 0.1 (0.1)
Lipids
Fatty acyls 55.5 (1.2) 56.2 (0.5)
Glycerolipids 7.2 (0.4) 7.4 (0.3)
Prenol lipids 1.5 (0.4) 3.1 (0.5)
Steroids 30.1 (0.7) 28.7 (0.4)
Polyketides ND 0.8 (0.1) 0.8 (0.0)
Benzenoids were the second major compound superclass, and, yet, were only de-
tected at small percentages in both types of extracts (i.e., <4.2%). It comprised four classes,
(1) anthracenes, (2) benzene and substituted derivatives, (3) benzopyrans, and (4) phe-
nols. Interestingly, benzenoid moieties were more pronounced in wild specimens (t-test,
p= 0.007), due to their higher content in benzenes and substituted derivatives (t-test,
p= 0.016). Moreover, metabolites belonging to the class of benzopyrans were identified at
similarly low levels within the farmed and wild sponge populations (i.e., 0.4% and 0.5%,
respectively). Furthermore, other compound classes contributed to the benzenoid profile
of farmed and wild S. foetidus extracts, such as anthracenes and phenols, but these were
present as trace elements (i.e., <0.01%).
The chemical profile of S. foetidus exhibited superclasses of polyketides and indoles
that were found in all extracts of this keratose demosponge. The respective contents
were similar for sponges of farmed and wild populations. More specifically, polyketides
accounted for 0.8% of both farmed and wild sponge extracts. Indoles were present at
percentages lower than 0.1% in both S. foetidus populations. Additionally, dipeptides were
the least abundant metabolite superclass among the sponges’ populations (i.e., <0.02%).
The distribution of metabolites within the extracts of S. foetidus was determined us-
ing the MS
2
peak intensity data, and the calculated abundance values are presented in
Table S5. Fatty acyls of farmed and wild sponge extracts were entirely represented by the
primary metabolite N-hexadecanoyl-L-homoserine lactone [
100
], which has been previ-
ously detected in wild specimens of this species through in situ chemical extraction [
101
]
and its existence has been associated with the quorum-sensing activities of its host micro-
biome [
102
]. The steroid 24-methylcholesta-5,7,22-trien-3
β
-ol (i.e., ergosterol) [
103
] was the
Mar. Drugs 2023,21, 612 10 of 27
second most abundant metabolite, and it accounted for the majority of steroids detected in
S. foetidus extracts. Its content was determined to be as high as 28.6% and 30.0% in sponges
derived from farmed and wild sponges, respectively.
Monovaccenin [
104
] and 1-O-(2,3,4,5-tetrahydroxycyclopentyl)-3-O-(10-methylhexadecyl)
glycerol (1-O-(2,3,4,5-4OHCyp-3-O-10-MeHG)) [
105
] were the major compounds of the
glycerolipids family, and their respective average percentages were 4.6% and 2.5% within
the analyzed extracts. However, the prenol lipid 4-hydroxy-3-tetraprenylbenzoic acid [
103
]
was detected at similar levels as 1-O-(2,3,4,5-4OHCyp-3-O-10-MeHG), but only in farmed
specimens (i.e., 2.6%). Wild extracts exhibited approximately two times lower percentages
of this compound (i.e., 1.3%). The opposite trend was observed for the benzenoid molecule
toluate (i.e., 2.1% and 1.5% in wild and farmed sponges, respectively), while the second
major benzenoid 8-O-4’-dehydrodiferulic acid [
106
] accounted for 1.3% and 1.6% of the
total metabolite content of farmed and wild sponge populations.
Other compounds belonging to benzenoids (i.e., 3-phenylpropane-1,2-diol [
106
], 3-
isochromanone [
107
], and 7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone [
108
]), indoles
(i.e., indole-3-methylethanoate [
109
]), lipids (i.e., 1-O-(2,3,4,5-tetrahydroxycyclopentyl)-
3-O-(11-hexadecenyl)glycerol, 1-O-(2,3,4,5)-4OHCyp-3-O-HG [
105
], and 1,4-dihydroxy-2-
tetraprenylbenzoic acid [
103
]) and polyketides (i.e., 3,8-dihydroxy-6-methoxy-8-
methylxanthone [
110
], chrysophanol [
111
], and griseofulvin [
112
]) exhibited minor abun-
dances (i.e., 0.1-0.4%) in relation to the total metabolite content of S. foetidus. Anthracenes,
represented by emodin [
113
] and endocrocin [
114
], and dipeptides comprised 3-nitropropionic
acid [
115
], along with some benzene derivatives (i.e., 3,4-dimethoxybenzoic acid [
116
], 4-
hydroxyphenylacetic acid [
117
], tyrosol [
118
]), prenol lipids (i.e., 7E,12E,20Z-variabilin [
119
]
and (+)-12,15-dihydroxycurcuphenol [
120
]), steroids (i.e., 24-methylcholest-7-en-3
β
-ol, 24-
methylcholesta-5,7-dien-3
β
-ol, 24-methylcholesta-7,22-dien-3
β
-ol, cholest-7-en-3
β
-yl acetate,
and cholesta-5,7-dien-3
β
-ol [
121
]), and polyketides (i.e., dechlorogriseofulvin [
112
], and nor-
lichexanthone [122]) were detected as trace amounts within the tested extracts (i.e., <0.1%).
The compositional differences between the extracts derived from farmed and wild
S. foetidus sponges were further evaluated by PCA analysis. Similar to the case of A.
oroides, data points representing the chemical profiles of wild sponges showed a broader
distribution, while a clear clustering was observed for the samples obtained from farmed
explants (Figure 4a). The first two principal components (PC1 and PC2) explained 41.6%
and 22.8% of the total variance present in the dataset.
Several members of lipids superclass, belonging to the classes of glycerolipids (i.e., 1-O-
(2,3,4,5)-4OHCyp-3-O-HG and monovaccenin), prenol lipids (i.e., (+)-12,15-dihydroxycurcuphenol,
1,4-dihydroxy-2-tetraprenylbenzoic acid, and 4-hydroxy-3-tetraprenylbenzoic acid), and steroids
(i.e.,
∆7
-cholesterol and 22,23-dihydroergosterol), together with the majority of detected
polyketides (i.e., griseoxanthone C and norlichexanthone), are visually clustered in the
bottom left side of the PCA loadings plot (Figure 4b), corresponding to the extracts derived
from farmed S. foetidus sponges in the scores plot (Figure 4a). This supports the notion that
these specific compounds might be the key elements accounting for the variation presented
in the farmed and wild sponges’ chemical profiles. To shed light onto this pattern, we
tested the differences in the % abundance of each metabolite between farmed and wild
sponges. The results showed that, indeed, some of the abovementioned compounds
were significantly higher in abundance in farmed S. foetidus explants, namely the prenol
lipids (+)-12,15-dihydroxycurcuphenol (p= 0.006) and 4-hydroxy-3-tetraprenylbenzoic acid
(p= 0.018) and the polyketide norlichexanthone (p= 0.029) (Figure 5). Furthermore, wild
S. foetidus specimens were enriched in ergosterol (p= 0.039), which was illustrated in the
upper right side of PCA biplot, close to one of the wild samples.
Mar. Drugs 2023,21, 612 11 of 27
Mar. Drugs 2023, 21, x 11 of 28
Figure 4. Principal component analysis (a) scores plot and (b) loadings plot of crude S. foetidus ex-
tracts belonging to wild and farmed sponges. Color circles represent the origin of the analyzed
sponge extract (i.e., green for wild or red for farmed) and triangles represent the dierent chemical
classes of the identied metabolites, according to the legend: 1-O-(2,3,4,5)-4OHCyp-3-O-HG, 1-O-
(2,3,4,5-tetrahydroxycyclopentyl)-3-O-(11-hexadecenyl)glycerol, 1-O-(2,3,4,5-4OHCyp-3-O-10-
MeHG, 1-O-(2,3,4,5-tetrahydroxycyclopentyl)-3-O-(10-methylhexadecyl)glycerol); aloesol, 7-hy-
droxy-2-(2-hydroxypropyl)-5-methylchromone; C16-HSL, N-hexadecanoyl-L-homoserine lactone;
Griseoxanthone C, 3,8-dihydroxy-6-methoxy-8-methylxanthone; Δ7-Cholesterol, Cholesta-5,7-dien-
3β-ol; 22,23-Dihydroergosterol, 24-methylcholesta-5,7-dien-3β-ol; stellasterol, 24-methylcholesta-
7,22-dien-3β-ol; ergosterol, 24-methylcholesta-5,7,22-trien-3β-ol.
Wild #1
Wild #2
Wild #3
Farmed #1
Farmed #2
Farmed #3
-8
-6
-4
-2
0
2
4
6
8
-8 -6 -4 -2 0 2 4 6 8
PC2 (22.8 %)
PC1 (41.6 %)
Observations (axes F1 and F2: 64.49 %)
(a)
Endocrocin
3-Phenylpropane-1,2-diol
3,4-Dimethoxybenzoic acid
8-O-4'-Dehydrodiferulic
acid
Toluate
3-Isochromanone
Aloesol
4-Hydroxyphenylacetic acid
Tyrosol
3-Nitropropionic acid
Indole-3-
methylethanoate C16-HSL
1-O-(2,3,4,5-4OH-Cyp)-3-O-(10-MeHG)
1-O-(2,3,4,5-4OH-Cyp)-3-O-HG
Monovaccenin
Variabilin
4-Hydroxy-3-tetraprenylbenzoic acid
1,4-Dihydroxy-2-tetraprenylbenzene
(+)-12,15-Dihydroxycurcuphenol
24-Methylcholest-7-en-3β-ol
22,23-Dihydroergosterol
Stellasterol
Ergosterol
Δ7-Cholesterol
Griseoxanthone C
Chrysophanol
Griseofulvin
Norlichexanthone
-8
-6
-4
-2
0
2
4
6
8
-8 -6 -4 -2 0 2 4 6 8
PC2 (22.8 %)
PC1 (41.6 %)
Biplot (axes PC 1 and PC 2: 64.4 %)
(b)
Farmed sponge extract
Wild sponge extract
Anthracenes
Benzenes & substituted derivatives
Benzopyrans
Phenols
Dipeptides
Indoles
Fatty acyls
Glycerolipids
Prenol lipids
Steroids
Polyketides
Figure 4.
Principal component analysis (
a
) scores plot and (
b
) loadings plot of crude S. foetidus
extracts belonging to wild and farmed sponges. Color circles represent the origin of the analyzed
sponge extract (i.e., green for wild or red for farmed) and triangles represent the different chemical
classes of the identified metabolites, according to the legend: 1-O-(2,3,4,5)-4OHCyp-3-O-HG, 1-O-
(2,3,4,5-tetrahydroxycyclopentyl)-3-O-(11-hexadecenyl)glycerol, 1-O-(2,3,4,5-4OHCyp-3-O-10-MeHG,
1-O-(2,3,4,5-tetrahydroxycyclopentyl)-3-O-(10-methylhexadecyl)glycerol); aloesol, 7-hydroxy-2-(2-
hydroxypropyl)-5-methylchromone; C16-HSL, N-hexadecanoyl-L-homoserine lactone; Griseoxan-
thone C, 3,8-dihydroxy-6-methoxy-8-methylxanthone;
∆7
-Cholesterol, Cholesta-5,7-dien-3
β
-ol; 22,23-
Dihydroergosterol, 24-methylcholesta-5,7-dien-3
β
-ol; stellasterol, 24-methylcholesta-7,22-dien-3
β
-ol;
ergosterol, 24-methylcholesta-5,7,22-trien-3β-ol.
Mar. Drugs 2023,21, 612 12 of 27
Mar.Drugs2023,21,x 12of28
Severalmembersoflipidssuperclass,belongingtotheclassesofglycerolipids(i.e.,
1-O-(2,3,4,5)-4OHCyp-3-O-HGandmonovaccenin),prenollipids(i.e.,(+)-12,15-dihy-
droxycurcuphenol,1,4-dihydroxy-2-tetraprenylbenzoicacid,and4-hydroxy-3-
tetraprenylbenzoicacid),andsteroids(i.e.,Δ
7
-cholesteroland22,23-dihydroergosterol),
togetherwiththemajorityofdetectedpolyketides(i.e.,griseoxanthoneCandnorli-
chexanthone),arevisuallyclusteredintheboomleftsideofthePCAloadingsplot(Fig-
ure4b),correspondingtotheextractsderivedfromfarmedS.foetidusspongesinthescores
plot(Figure4a).Thissupportsthenotionthatthesespecificcompoundsmightbethekey
elementsaccountingforthevariationpresentedinthefarmedandwildsponges’chemical
profiles.Toshedlightontothispaern,wetestedthedifferencesinthe%abundanceof
eachmetabolitebetweenfarmedandwildsponges.Theresultsshowedthat,indeed,some
oftheabovementionedcompoundsweresignificantlyhigherinabundanceinfarmedS.
foetidusexplants,namelytheprenollipids(+)-12,15-dihydroxycurcuphenol(p=0.006)and
4-hydroxy-3-tetraprenylbenzoicacid(p=0.018)andthepolyketidenorlichexanthone(p=
0.029)(Figure5).Furthermore,wildS.foetidusspecimenswereenrichedinergosterol(p=
0.039),whichwasillustratedintheupperrightsideofPCAbiplot,closetooneofthewild
samples.
Figure5.Boxplotofrelativeabundancesformetabolites(+)-12,15dihydroxycurcuphenol,4-hy-
droxy-3-tetraprenylbenzoicacid,ergosterol,andnorlichexanthone,whichpresentedsignificant
abundancedifferences(t-test,p<0.05)betweentheextractscontentoffarmed(red)andwild(green)
S.foetidussponges.Datawerenormalizedtothetotalspectralareaandpresentedintheauto-scale
modeoftheMetaboAnalyst5.0software.Boxesrangefromthe25%to75%percentile;whiskers
indicatethe5%and95%percentiles;blackdotsshowtheindividualdatapoints;thehorizontalline
andtheyellowdiamondwithineachboxindicatethemedianandmeanvalue,respectively.
Figure 5.
Boxplot of relative abundances for metabolites (+)-12,15 dihydroxycurcuphenol, 4-hydroxy-
3-tetraprenylbenzoic acid, ergosterol, and norlichexanthone, which presented significant abundance
differences (t-test, p< 0.05) between the extracts content of farmed (red) and wild (green) S. foetidus
sponges. Data were normalized to the total spectral area and presented in the auto-scale mode of the
MetaboAnalyst 5.0 software. Boxes range from the 25% to 75% percentile; whiskers indicate the 5%
and 95% percentiles; black dots show the individual data points; the horizontal line and the yellow
diamond within each box indicate the median and mean value, respectively.
All the lipids that varied in abundance between farmed and wild S. foetidus pop-
ulations have been associated with sponge defense mechanisms. More specifically, (+)-
12,15-dihydroxycurcuphenol constitutes an intermediate metabolite in the synthesis of
abscisic acid, which is assumed to be produced by sponges as a response to heat stress [
120
].
Additionally, the compound 4-hydroxy-3-tetraprenylbenzoic acid has been shown to pos-
sess strong pungent activity [
123
], potentially serving as a defensive compound by the
sponges to deter predators. Norlichexanthone is a marine-derived fungi metabolite [
122
],
which has been previously reported in the fungal extracts of the Sarcotragus muscarum
symbiont Arthrinium sp. [
106
]. Ahluwalia et al. (2015) [
124
] included this metabolite in
high oxidation state compounds, which have been previously reported to exhibit great
antimicrobial activity, while boosting symbionts’ competitive efficiency and host resistance
to other pathogens [
125
]. However, all these defensive lipid chemicals were found to be
more abundant in farmed populations of S. foetidus, which is quite contradictory, given the
lesser environmental pressures they face, compared to their wild counterparts. However, it
should be mentioned that extracts provided by farmed S. foetidus specimens are less rich
in ergosterol. This specific compound is the prime sterol in plasma membranes of fungal
cells, and like many steroids, has been characterized as a bioactive compound, due to its
Mar. Drugs 2023,21, 612 13 of 27
antibacterial and anti-inflammatory activities [
126
]. Higher levels of this fungi steroid in
wild sponge extracts indicate a higher pronounced presence of symbionts in the respective
specimens and their potential involvement in the sponge-associated defense mechanisms.
Taking this into account, we can perceive why the metabolism of farmed S. foetidus sponges
involved alternative defense pathways, enriched with other minor metabolites, such as
prenol lipids.
In terms of production levels, the weight-normalized intensity values calculated
for each metabolite of S. foetidus sponges are presented in Table S6. Only the prenol
lipid (+)-12,15-dihydroxycurcuphenol was found to vary significantly between farmed
and wild S. foetidus sponges (p= 0.006) (Figure 6). In agreement with the previously
reported one-way ANOVA results (i.e., assessing differences in % metabolite abundance
among extracts), the production of (+)-12,15-dihydroxycurcuphenol was more intensive in
farmed specimens. Consequently, rearing in proximity to a fish farm facility may affect the
production efficiency of specific bioactive compounds in S. foetidus individuals, but this is
an exception to a broader uniformity in metabolite production levels.
Mar.Drugs2023,21,x 13of28
AllthelipidsthatvariedinabundancebetweenfarmedandwildS.foetiduspopula-
tionshavebeenassociatedwithspongedefensemechanisms.Morespecifically,(+)-12,15-
dihydroxycurcuphenolconstitutesanintermediatemetaboliteinthesynthesisofabscisic
acid,whichisassumedtobeproducedbyspongesasaresponsetoheatstress[120].Ad-
ditionally,thecompound4-hydroxy-3-tetraprenylbenzoicacidhasbeenshowntopossess
strongpungentactivity[123],potentiallyservingasadefensivecompoundbythesponges
todeterpredators.Norlichexanthoneisamarine-derivedfungimetabolite[122],which
hasbeenpreviouslyreportedinthefungalextractsoftheSarcotragusmuscarumsymbiont
Arthriniumsp.[106].Ahluwaliaetal.(2015)[124]includedthismetaboliteinhighoxida-
tionstatecompounds,whichhavebeenpreviouslyreportedtoexhibitgreatantimicrobial
activity,whileboostingsymbionts’competitiveefficiencyandhostresistancetoother
pathogens[125].However,allthesedefensivelipidchemicalswerefoundtobemore
abundantinfarmedpopulationsofS.foetidus,whichisquitecontradictory,giventhe
lesserenvironmentalpressurestheyface,comparedtotheirwildcounterparts.However,
itshouldbementionedthatextractsprovidedbyfarmedS.foetidusspecimensarelessrich
inergosterol.Thisspecificcompoundistheprimesterolinplasmamembranesoffungal
cells,andlikemanysteroids,hasbeencharacterizedasabioactivecompound,duetoits
antibacterialandanti-inflammatoryactivities[126].Higherlevelsofthisfungisteroidin
wildspongeextractsindicateahigherpronouncedpresenceofsymbiontsintherespective
specimensandtheirpotentialinvolvementinthesponge-associateddefensemechanisms.
Takingthisintoaccount,wecanperceivewhythemetabolismoffarmedS.foetidus
spongesinvolvedalternativedefensepathways,enrichedwithotherminormetabolites,
suchasprenollipids.
Intermsofproductionlevels,theweight-normalizedintensityvaluescalculatedfor
eachmetaboliteofS.foetidusspongesarepresentedinTab leS6.Onlytheprenollipid(+)-
12,15-dihydroxycurcuphenolwasfoundtovarysignificantlybetweenfarmedandwildS.
foetidussponges(p=0.006)(Figure6).Inagreementwiththepreviouslyreportedone-way
ANOVAresults(i.e.,assessingdifferencesin%metaboliteabundanceamongextracts),
theproductionof(+)-12,15-dihydroxycurcuphenolwasmoreintensiveinfarmedspeci-
mens.Consequently,rearinginproximitytoafishfarmfacilitymayaffecttheproduction
efficiencyofspecificbioactivecompoundsinS.foetidusindividuals,butthisisanexcep-
tiontoabroaderuniformityinmetaboliteproductionlevels.
Figure6.Boxplotofweight-normalizedintensitiesforthemetabolite(+)-12,15dihydroxycurcuphe-
nol,whichpresentedsignificantdifferencesinitsproduction(t-test,p<0.05)betweentheextracts
offarmed(red)andwild(green)S.foetidussponges.Dataarepresentedintheauto-scalemodeof
theMetaboAnalyst5.0software.Boxesrangefromthe25%to75%percentile;whiskersindicatethe
5%and95%percentiles;blackdotsshowtheindividualdatapoints;thehorizontallineandthe
yellowdiamondwithineachboxindicatethemedianandmeanvalue,respectively.
Figure 6.
Boxplot of weight-normalized intensities for the metabolite (+)-12,15 dihydroxycurcuphenol,
which presented significant differences in its production (t-test, p< 0.05) between the extracts of
farmed (red) and wild (green) S. foetidus sponges. Data are presented in the auto-scale mode of the
MetaboAnalyst 5.0 software. Boxes range from the 25% to 75% percentile; whiskers indicate the 5%
and 95% percentiles; black dots show the individual data points; the horizontal line and the yellow
diamond within each box indicate the median and mean value, respectively.
2.3. Evaluation of the Sponges’ Biotechnological Potential
In addition to comparing the chemical profiles of cultivated and wild sponges, we
further examined the biological activities of A. oroides and S. foetidus extracts to evaluate
whether farming can affect those properties. Antimicrobial assays were performed against
two human pathogens: methicillin-resistant Staphylococcus aureus (MRSA, COL), represent-
ing Gram-positive bacteria, and Escherichia coli (ATCC 25922), representing Gram-negative
bacteria. In addition, the anticancer activities and general toxicities of the extracts were
assessed against the human colorectal carcinoma cell line HCT-116 (ECACC 91091005).
2.3.1. Antimicrobial Activity Evaluation
To assess the antibacterial potential of A. oroides and S. foetidus marine sponges and
compare the bioactivities of wild versus farmed populations, a total of 12 extracts were
tested against two strains of Gram-positive and -negative bacteria, representative of clini-
cally relevant human pathogens, as mentioned above: Staphylococcus aureus (MRSA strain
COL) and Escherichia coli (strain ATCC 25922). The studied samples consisted of the crude
extracts of three explants collected from both farmed and wild populations of targeted
Mar. Drugs 2023,21, 612 14 of 27
sponge species A. oroides and S. foetidus, analyzed in triplicate, using the microdilution
assay to determine the minimum inhibitory concentration (MIC) (Table 3). In total, eight
crude sponge extracts revealed antibacterial properties, with all showing marginal potency.
More specifically, seven extracts were found to be active against the S. aureus strain, while
only one was proven to be effective against E. coli.
Table 3.
Antimicrobial activity of A. oroides and S. foetidus crude extracts against methicillin-resistant
Staphylococcus aureus (MRSA, strain COL) and Escherichia coli (strain ATCC 25922). MIC values are
expressed in
µ
g/mL. NA—not active at the tested concentrations. Vancomycin and tetracycline were
used as positive controls for S. aureus and E. coli growth, respectively. Inoculated medium without
any extract addition was used as negative control.
Species Type of Sponge
Population
Sponge
Replicate
MIC Values
(µg/mL) for S.
aureus MRSA COL
MIC Values
(µg/mL) for E.
coli ATCC 25922
Agelas oroides
Wild
#1 NA NA
#2 NA NA
#3 250 NA
Farmed
#1 NA NA
#2 250 NA
#3 250 250
Sarcotragus
foetidus
Wild
#1 125 NA
#2 125 NA
#3 250 NA
Farmed
#1 NA NA
#2 250 NA
#3 NA NA
Positive control 1.9 3.9
Regarding the species A. oroides, one out of the three wild specimens (33%) provided
extracts with inhibitory activities against the Gram-positive methicillin-resistant bacterium
S. aureus, while the same stood for two out of the three studied farmed samples (67%).
However, both types of A. oroides extracts (i.e., farmed vs. wild) exhibited activity against
MRSA, albeit only for the highest tested concentration (i.e., 250 µg/mL).
Interestingly, one farmed A. oroides extract (i.e., #3), besides revealing S. aureus growth
inhibition, also showed activity against E. coli (MIC; 250
µ
g/mL). None of the wild sponge
extracts belonging to A. oroides demonstrated inhibitory effects against E. coli. The same
results were observed for farmed A. oroides extracts, expect for replicate #3, which targeted
both Gram-positive and -negative bacteria, suggesting a mechanism not related to the
cell wall. Indeed, among all the tested biological samples, the farmed A. oroides replicate
#3 extract contained the highest levels of the alkaloids dispacamide B, keramadine, and
agelasidine A, as well as the indole compound 4,6-dihydroxyindole and the glycerolipid 2,3-
dihydroxypropyl(Z)-10-methyloctadec-9-enoate (Table S2). Of these metabolites, kerama-
dine and agelasidine A have been previously reported to display partial growth inhibitory
effects against E. coli strains, with MIC values in the range of 32–100
µ
g/mL [
127
,
128
],
while 4,6-dihydroxyindole was found to have strong antibacterial activity against both
Gram-positive and -negative bacteria, including E. coli [
84
]. These compounds might be the
key metabolites equipping A. oroides sponges with broad-spectrum antibacterial properties,
by acting either individually or synergistically in the respective extracts.
In contrast, extracts of wild S. foetidus sponges had a stronger effect on S. aureus
growth, with one of these being active at the highest tested concentration and the rest
of the samples reaching lower MIC values of 125
µ
g/mL. However, it is worth noticing
that this value represented the highest antimicrobial activity exhibited by all the studied
extracts. Concerning the cultivated S. foetidus fragments, only one out of three (33%)
generated extracts that marginally inhibited the growth of the Gram-positive bacterium
Mar. Drugs 2023,21, 612 15 of 27
(MIC; 250
µ
g/mL), but did not reach as low values as their wild counterparts. On the other
hand, all the S. foetidus extracts did not show any effect on E. coli growth.
Based on these observations, we can assume a higher presence of anti-S. aureus com-
pounds in the extracts of wild S. foetidus populations, which target the cell wall structure,
because only Gram-positive bacteria are inhibited. More specifically, wild extracts #1 and
#2, which stood out for their inhibitory properties, demonstrated the highest contents of
the natural products toluate (i.e., >2.0%) and ergosterol (i.e., >29.9%) among all the tested
samples. Toluate, or p-toluic acid, is a primary metabolite involved in the natural degrada-
tion of p-xylene [
129
] and can be found in a myriad of organisms [
130
,
131
], including the
sponge S. foetidus [
101
]. To the best of our knowledge, there are no reports examining its
antimicrobial effects, except for those focused on its congener, benzoic acid. This compound
is a food preservative, and its MIC values against various S. aureus strains have been demon-
strated in several previous studies [
132
,
133
]. On the other hand, ergosterol, a derivative
of cholesterol, plays an important role in the function of eukaryotic cell membranes by
maintaining their permeability. However, it is absent in the cytoplasmic membrane of
bacteria [
134
]. Sullivan et al. (2020) reported that ergosterol causes detrimental effects on
bacterial cells [
135
]. This could be a possible explanation for the enhanced antimicrobial
activity observed in the majority of crude extracts collected from wild S. foetidus specimens.
However, there are no data in the literature describing ergosterol-driven effects on bacteria.
Although the reported MIC values of the present study are relatively high, it should be
stressed that they concern the activity of crude sponge extracts, which have not been priorly
subjected to any fractionation. In agreement with the results obtained from Govinden-
Soulange et al. (2014) [
136
] for crude and fractionated extracts of the Mauritian sponges
Biemna tubulosa and Stylissa sp., MIC values of our crude samples were rarely found to
be higher than 100
µ
g/mL. This is attributed to the chemical complexity of the specific
extracts, which constitute mixtures of both active and non-active compounds.
2.3.2. Anticancer Activity Evaluation
The anticancer properties of crude sponge extracts, derived from farmed and wild pop-
ulations of A. oroides and S. foetidus species, were investigated against the human colorectal
carcinoma cell line HCT-116, by employing the MTS assay. Figures 7and 8illustrate the
variation of MTS cell metabolism as a function of the applied extract concentration, with re-
spect to A. oroides and S. foetidus sponges. The cytotoxicity effects were evaluated according
to half-maximal (50%) inhibitory concentration (IC
50
) values, which were obtained from
the curve slopes of the abovementioned graphs and are presented in Table 4. As shown
in Figure 7, none of the extracts belonging to wild and farmed A. oroides explants exerted
anticancer activity against HTC-116 cells, except for one extract of a farmed specimen
(#3, Figure 7f) that showed approximately 25% inhibition at the highest test concentration
(i.e., 125
µ
g/mL). However, this inhibition rate was considered to be relatively low, hence,
the IC50 values were not determined.
All S. foetidus sponges provided extracts with moderate anticancer activity, whether
they derived from wild or farmed populations. More specifically, wild explants exhibited
IC
50
values in the range of 70.8–71.5
µ
g/mL, while their cultivated counterparts showed a
more profound variation, with values starting from 80.5
µ
g/mL and reaching 41.2
µ
g/mL.
However, the average cytotoxicity against HTC-116 cells, which was derived from the IC
50
values of the three tested sponge extracts, was evaluated as statistically similar between the
two populations (t-test, p= 0.7). This was found to be as high as 70.9
±
0.5
µ
g/mL for wild
sponges and 65.3 ±21.1 µg/mL for the farmed ones.
Based on these observations, it can be assumed that S. foetidus extracts contain metabo-
lites that are more active against HCT-116 cells than those detected in A. oroides. However,
previous studies have indicated the cytotoxicity of Agelas species against various carcinoma
cells, but the associated results concern the activity of isolated metabolites, like oroidin [
137
]
and sceptrin derivatives [
138
], and not the whole sponge extract. Although these metabo-
lites were present in our tested Agelas extracts, it is likely that the levels at which they
Mar. Drugs 2023,21, 612 16 of 27
were produced, remained relatively low to exhibit bioactivity, or other components might
counteract their inhibitory properties. Nevertheless, the highest percentage of HCT-116 cell
inhibition, observed for one of our farmed A. oroides specimens, was within the range re-
ported by Ang et al. (2023) for various crude extracts of Agelas species against the HCT-116
carcinoma cell line [
139
]. However, it should be mentioned that the latter study revealed
the lowest percent of cell viability (i.e., 75.8%) at 30
µ
g/mL, which was four times lower
than our “active” concentration (i.e., 125 µg/mL) for crude Agelas extracts.
Mar. Drugs 2023, 21, x 16 of 28
The anticancer properties of crude sponge extracts, derived from farmed and wild
populations of A. oroides and S. foetidus species, were investigated against the human col-
orectal carcinoma cell line HCT-116, by employing the MTS assay. Figures 7 and 8 illus-
trate the variation of MTS cell metabolism as a function of the applied extract concentra-
tion, with respect to A. oroides and S. foetidus sponges. The cytotoxicity eects were evalu-
ated according to half-maximal (50%) inhibitory concentration (IC50) values, which were
obtained from the curve slopes of the abovementioned graphs and are presented in Table
4. As shown in Figure 7, none of the extracts belonging to wild and farmed A. oroides ex-
plants exerted anticancer activity against HTC-116 cells, except for one extract of a farmed
specimen (#3, Figure 7f) that showed approximately 25% inhibition at the highest test con-
centration (i.e., 125 μg/mL). However, this inhibition rate was considered to be relatively
low, hence, the IC50 values were not determined.
Figure 7. HCT-116 cells viability determined by the MTS assay after a 72-h treatment with extracts
derived from wild (a–c) and farmed (d–f) A. oroides specimens at dierent concentrations (presented
in a logarithmic scale). All data points are expressed as mean ± standard error of the mean from at
least three independent experiments. DMSO was used as vehicle control, and 10 μM 5-Fu as positive
control.
All S. foetidus sponges provided extracts with moderate anticancer activity, whether
they derived from wild or farmed populations. More specically, wild explants exhibited
IC50 values in the range of 70.8–71.5 μg/mL, while their cultivated counterparts showed a
more profound variation, with values starting from 80.5 μg/mL and reaching 41.2 μg/mL.
However, the average cytotoxicity against HTC-116 cells, which was derived from the IC50
values of the three tested sponge extracts, was evaluated as statistically similar between
the two populations (t-test, p = 0.7). This was found to be as high as 70.9 ± 0.5 μg/mL for
wild sponges and 65.3 ± 21.1 μg/mL for the farmed ones.
Based on these observations, it can be assumed that S. foetidus extracts contain me-
tabolites that are more active against HCT-116 cells than those detected in A. oroides. How-
ever, previous studies have indicated the cytotoxicity of Agelas species against various
carcinoma cells, but the associated results concern the activity of isolated metabolites, like
oroidin [137] and sceptrin derivatives [138], and not the whole sponge extract. Although
these metabolites were present in our tested Agelas extracts, it is likely that the levels at
which they were produced, remained relatively low to exhibit bioactivity, or other com-
ponents might counteract their inhibitory properties. Nevertheless, the highest percentage
-1 0 1 2 3
0
50
100
150
1BB
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(a)
-1 0 1 2 3
0
50
100
150
2BB
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(b)
-1 0 1 2 3
0
50
100
150
3BB
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(c)
-1 0 1 2 3
0
50
100
150
4BB
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(d)
-1 0 1 2 3
0
50
100
150
5BB
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(e)
-1 0 1 2 3
0
50
100
150
6BB
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(f)
Figure 7.
HCT-116 cells viability determined by the MTS assay after a 72-h treatment with extracts
derived from wild (
a
–
c
) and farmed (
d
–
f
)A. oroides specimens at different concentrations (presented
in a logarithmic scale). All data points are expressed as mean
±
standard error of the mean from
at least three independent experiments. DMSO was used as vehicle control, and 10
µ
M 5-Fu as
positive control.
Mar. Drugs 2023, 21, x 17 of 28
of HCT-116 cell inhibition, observed for one of our farmed A. oroides specimens, was
within the range reported by Ang et al. (2023) for various crude extracts of Agelas species
against the HCT-116 carcinoma cell line [139]. However, it should be mentioned that the
laer study revealed the lowest percent of cell viability (i.e., 75.8%) at 30 μg/mL, which
was four times lower than our “active” concentration (i.e., 125 μg/mL) for crude Agelas
extracts.
Figure 8. HCT-116 cells viability determined by the MTS assay after a 72-h treatment with extracts
derived from wild (a–c) and farmed (d–f) S. foetidus specimens at dierent concentrations (presented
in a logarithmic scale). All data points are expressed as mean ± standard error of the mean from at
least three independent experiments. DMSO was used as vehicle control, and 10 μM 5-Fu as positive
control.
Regarding S. foetidus, it can be observed that all study extracts, either derived from
wild or farmed specimens, showed 50% inhibition against HCT-116 cells. However, ac-
cording to the American National Cancer Institute guidelines (NCI), crude extracts achiev-
ing 50% anti-proliferative activity are regarded as cytotoxic at less than 30 μg/mL after a
72-h exposure [140]. Based on this, we can consider that only one extract, which belongs
to a farmed S. foetidus specimen (i.e., #2), is marginally cytotoxic, given its mean IC50 value
of 41.2 μg/mL. This bioactivity was two times lower than the one demonstrated by the rest
of farmed and wild S. foetidus counterparts, revealing a higher presence of cytotoxic com-
pounds in its composition. By scrutinizing the % abundance and weight-normalized in-
tensity values of metabolites among extracts, it is observed that indole-3-methylethanoate
is present at signicantly higher levels in the cytotoxic extract. Specically, its abundance
reaches 0.3% in the farmed extract #2, while it is detected at levels less than 0.1% in the
rest of the tested samples. In terms of production levels, the weight-normalized intensity
values of indole-3-methylethanoate dier by an order of magnitude between farmed ex-
tract #2 and the rest of analyzed S. foetidus samples. Although there is no report indicating
the anticancer potential of this specic metabolite, indole compounds derived from ma-
rine sources, including sponges, have long been viewed to possess cytotoxic properties
against tumor cell lines [141,142].
0123
-50
0
50
100
150
7B
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(a)
0123
-50
0
50
100
150
8B
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(b)
0123
-50
0
50
100
150
9B
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(c)
0123
-50
0
50
100
150
10B
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(d)
0123
-50
0
50
100
150
11B
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(e)
0123
-50
0
50
100
150
12B
log (conc), g/mL
MTS metabolism
(fold to DMSO, %)
(f)
Figure 8.
HCT-116 cells viability determined by the MTS assay after a 72-h treatment with extracts
derived from wild (
a
–
c
) and farmed (
d
–
f
)S. foetidus specimens at different concentrations (presented
in a logarithmic scale). All data points are expressed as mean
±
standard error of the mean from
at least three independent experiments. DMSO was used as vehicle control, and 10
µ
M 5-Fu as
positive control.
Mar. Drugs 2023,21, 612 17 of 27
Table 4.
Marine sponge crude extracts with anticancer activity against human colorectal carcinoma
cell line HCT-116. Results are expressed as mean IC
50
values, determined by MTS assay. DMSO was
used as vehicle control, and 10 µM 5-Fu as positive control.
Species Type of Population Replicate IC50 (µg/mL) 95% CI
Sarcotragus
foetidus
Wild
#1 70.8 70.3–80.4
#2 70.4 65.7–74.7
#3 71.45 68.4–73.8
Farmed
#1 80.5 74.1–93.3
#2 41.2 30.1–50.0
#3 74.2 70.8–79.2
Regarding S. foetidus, it can be observed that all study extracts, either derived from wild
or farmed specimens, showed 50% inhibition against HCT-116 cells. However, according
to the American National Cancer Institute guidelines (NCI), crude extracts achieving 50%
anti-proliferative activity are regarded as cytotoxic at less than 30
µ
g/mL after a 72-h
exposure [
140
]. Based on this, we can consider that only one extract, which belongs to a
farmed S. foetidus specimen (i.e., #2), is marginally cytotoxic, given its mean IC
50
value
of 41.2
µ
g/mL. This bioactivity was two times lower than the one demonstrated by the
rest of farmed and wild S. foetidus counterparts, revealing a higher presence of cytotoxic
compounds in its composition. By scrutinizing the % abundance and weight-normalized
intensity values of metabolites among extracts, it is observed that indole-3-methylethanoate
is present at significantly higher levels in the cytotoxic extract. Specifically, its abundance
reaches 0.3% in the farmed extract #2, while it is detected at levels less than 0.1% in the rest
of the tested samples. In terms of production levels, the weight-normalized intensity values
of indole-3-methylethanoate differ by an order of magnitude between farmed extract #2
and the rest of analyzed S. foetidus samples. Although there is no report indicating the
anticancer potential of this specific metabolite, indole compounds derived from marine
sources, including sponges, have long been viewed to possess cytotoxic properties against
tumor cell lines [141,142].
3. Materials and Methods
3.1. Sponge Material
Farmed individuals of A. oroides and S. foetidus were introduced in integrated aquacul-
ture in June 2020, attached on cultivation structures (individual cages made from plastic
mesh) in proximity (5–10 m distance) to the fish cages of an operating fish farm in Souda
Bay, NW Crete, Aegean Sea (35.4801/24.1117), at 7–10 m depth. Farmed sponges originated
from explants (i.e., biomass cuttings) collected from adjacent natural populations (Souda
Bay, 35.4783/24.1091 for A. oroides and Stavros Bay, 35.5879/24.0785 for S. foetidus). For a
continuous timespan of 19 months, the explants remained in open-sea cultivation, exhibit-
ing negligible mortality and showing regeneration and positive growth (Vernadou et al.,
in preparation). Tissue sampling from three replicate individuals of each sponge species
from wild and farmed sponge specimens (12 samples in total) were obtained in February
2022, according to Varamogianni-Mamatsi et al. (2022) [
16
]. Sampling locations for the
wild individuals corresponded to the locations of collection for the initial seeding of the
experimental sponge cultivation. Sampling was performed selectively by diving, and
care was taken to partially collect excess biomass, thus, allowing the donor individuals
to regenerate. In both cases (farmed and wild sponges), tissue samples were extracted
underwater from a parent sponge, using a razor blade, and held in individual labeled
sterile bags, prior to preservation in cooler boxes with ice packs and transportation to the
facilities of Hellenic Centre for Marine Research within 3 h, where they were kept in
−
20
◦
C
until further analysis.
The studied demosponge species are commonly distributed in high abundances along
eastern Mediterranean habitats [
143
] with emerging bioremediation [
16
,
24
] and bioproduc-
Mar. Drugs 2023,21, 612 18 of 27
tion potential [
144
] to be included in integrated aquaculture systems. The first sponge of
interest, Agelas oroides (Schmidt, 1864), is a massive, variably lobate-digitate, vivid orange-
colored demosponge that can reach 25 cm in height. It typically occurs in 2–40 m water
depth, preferably in habitats with low light intensity [
145
,
146
]. Sponges of the genus
Agelas, including the species A. oroides, are well-known alkaloid producers (e.g., pyrrole
and terpenoid alkaloids) [
49
]. The second case study demosponge is the species Sarcotragus
foetidus (Schmidt, 1862). This concerns a variably dark-colored, rather common Mediter-
ranean keratose sponge, which approximates an irregularly globular to massive growth
form, generally reaching 1 m in diameter and 50 cm in height. It is commonly found in
shallow habitats exposed to light, but also in darker zones up to 400 m in depth [
147
]. This
demosponge is known to host numerous symbiotic bacteria (e.g., heterotrophic bacteria
and cyanobacteria [
148
]) and fungi [
149
]. Sarcotragus sponges are widely recognized to be
prolific sources of a variety of bioactive compounds, such as terpenoids, indoles, as well as
lipids [51].
3.2. Extraction
Freeze-dried specimens of wild and farmed sponges were ground in a conventional
mixer to obtain a dry powder (1 to 4 g per specimen), which was subsequently extracted
three times in an ice-cold sonication bath (15 min each round) using a solvent mixture of
methanol/dichloromethane (20 mL of MeOH:DCM 1:1, v/vper gram of sponge). After
each extraction, sponge suspensions were centrifuged (8000
×
g, 7 min, 20
◦
C), and col-
lected supernatants were passed through a filter paper and evaporated to dryness using a
centrifugal vacuum evaporator (EZ-2 Plus; Genevac, United Kingdom). The dry extracts
were redissolved in 4 mL MeOH:DCM 1:1, transferred into 50 mL Falcon tubes, and mixed
with 16 mL acetonitrile. After overnight protein precipitation at
−
20
◦
C, the samples
were centrifuged (10,000
×
g, 10 min, 4
◦
C) and collected supernatants were evaporated to
dryness. For the removal of neutral lipids, each sample was applied onto a glass column
(8 mm i.d.) packed with 1.5 g of silica gel (silica gel 60, particle size: 0.060–0.200 mm, Merck;
activated at 300
◦
C for 3 h) and elution was performed using 2% v/vethyl acetate in hexane
(20 mL), ethyl acetate (15 mL), and MeOH (15 mL). The latter two fractions were combined,
evaporated to dryness, and stored at +4 ◦C until further analysis.
3.3. LC−MS/MS Analysis
3.3.1. Chemicals and Reagents
Analytical solvents, MeOH, and acetonitrile solvents at Ultra-High-Performance Liq-
uid Chromatography-Mass Spectrometry (UHPLC–MS) grade and formic acid at LC–MS
grade were supplied from Carlo Erba
®
Reagents S.A.S (Le Vaudreuil, France). Ultrapure
water was supplied from a Milli-Q
®
ultrapure water system equipped with a Milli-Q
®
Reference and a Q-POD®element.
3.3.2. Instrumental LC–MS/MS Analysis
The LC–MS/MS analysis was performed using a Dionex
®
Ultimate 3000 System (UH-
PLC, Thermo Scientific, Germany) coupled to a TSQ QuantisTM triple-stage quadrupole
mass spectrometer (Thermo Scientific, Waltham, MA, USA). The UHPLC was equipped
with four modules, a SR
−
3000 Solvent Rack, an LPG-3400RS pump, an WPS-3000TRS
auto sampler with temperature control, and a TCC-3000RS column compartment. The
triple-stage quadrupole mass spectrometer was equipped with an electrospray ionization
(ESI) source. The LC
−
MS/MS operation and acquisition data system was controlled by the
XCaliburTM 4.1 Thermo Scientific SP1 (0388-00CD-7B33, USA) software.
3.3.3. Sample Preparation for LC–MS/MS Analysis
All plastic materials and glassware were cleaned carefully to avoid contamination.
Organic solvents (LC
−
MS grade) and distilled water were analyzed before use, to minimize
background interferences.
Mar. Drugs 2023,21, 612 19 of 27
Before injection into the LC–MS/MS system, all extracts were dissolved in 1 mL MeOH,
filtered with a 13-mm, 0.22-
µ
m nylon syringe filter (Filter-Lab
®
, Sant Pere de Riudebitlles,
Spain) using a 500
µ
L syringe (Gastight 1750 Hamilton
®
, Vernon Hills, IL, USA), and
diluted 10 times with MeOH. The liquid extract was transferred using a syringe filter (Filter-
Lab
®
, Sant Pere de Riudebitlles, Spain) into a conical insert into a sterile 2-mL vial (9-425
C0000752) with a screw cap and red PTFE/white silicone septa (Alwsci
®
Technologies,
Shaoxing, China).
3.3.4. Chromatographic and Mass Spectrometry Conditions
The separation of compounds was achieved using an Accurore
TM
RP-MS Column
(2.6
µ
m, 150
×
2.1 mm, Thermo Fisher Scientific), by setting the sample injection volume
to 10
µ
L. The gradient mobile phase consisted of water with 0.1% formic acid (A) and
acetonitrile (B). Samples were injected and eluted at a flow of 0.200 mL/min with the
following linear gradients: a 1 min re-equilibration phase at 5% B, 0.0–15.0 min at 5–50% of
B, 15.0–20.0 min at 50–99% of B, 20.0–29.0 min at 99% of B, and 29.0–30.0 min at 99–5% of B.
Mass spectrometry (MS) analysis was carried out using the triple-stage quadrupole
mass spectrometer. The electrospray ionization (ESI) parameters were set as follows:
spray voltage, +3500/
−
3000 V; sheath gas flow, 50 L/min; auxiliary gas flow, 10 L min/L;
sweep gas flow, 0 L min/L; ion transfer tube temperature, 320
◦
C; vaporizer temperature,
30
◦
C. The cycle time was set to 0.5 s, using a calibrated radio frequency (RF) lens and
a collision induced dissociation (CID) gas of 1.5 mTorr. The collision energy was tested
to 10, 20, and 40 V. Samples were injected in select reaction monitoring (SRM) mode,
using multiple reaction monitoring (MRM). Selective MRM transitions were monitored
for each targeted analyte, according to Tables S1 and S4 settings, and are provided, along
with the instrumental parameter fragmentation settings used for the mass spectrometry
conditions, as supplementary material. An intensive literature review was performed for
each sponge’s species under study, on previously reported compounds that have been
identified along with their mass ions, adducts and their fragments. Briefly, the identification
and classification of detected metabolites was performed by reference to the literature and to
public databases (e.g., DrugBank, FoodB, GNPS, HMDB, MoNA, Metabolomics Workbench,
and PubChem).
3.4. Antibacterial Assays
The antibacterial activity of the crude sponge extracts was evaluated by performing
growth inhibition assays for two strains belonging to the Gram-positive and -negative
human opportunistic pathogenic bacteria. The tested strains were the methicillin-resistant
Staphylococcus aureus strain COL (MRSA) and the Escherichia coli strain ATCC 25922, re-
spectively. The protocol followed was based on the one proposed by Pinto-Almeida et al.
(2022) [
150
]. In detail, S. aureus strains were cultured in tryptic soy broth (TSB; Becton
Dickinson, Germany), and E. coli cells in Lysogeny broth (NZYtech), at 37
◦
C. The assays
were performed in 96-well polystyrene flat bottom microplates. Bacterial overnight cultures
were diluted to an optical density (OD
600nm
) of 0.005 and were incubated statically in the
presence of different concentrations of each crude extract, solubilized in DMSO (1% w/v).
All cultures were two-fold serially diluted, resulting in final concentrations of the extracts
ranging from 250 to 0.4935
µ
g/mL. After 24 h of incubation at 37
◦
C, the minimal inhibitory
concentration (MIC) value was determined by visual inspection. The latter is defined as
the lowest concentration of an antimicrobial agent that inhibits the visible growth of a
microorganism after overnight incubation [
151
]. The resulting values were compared with
a positive control (vancomycin for MRSA, and tetracycline for E. coli), a DMSO solvent
control, and a negative control (inoculated medium without any extract addition) on the
same plate.
Mar. Drugs 2023,21, 612 20 of 27
3.5. Anticancer Assays
The sponge crude extracts of wild and farmed populations of A. oroides and S. foetidus
were tested
in vitro
against the human colorectal carcinoma cell line HCT-116 (ECACC
91091005, Porton Down, UK), according to Florindo et al. (2016) [
152
] and Prieto-Davó
et al. (2016) [
153
]. Cells were cultured in McCoy’s 5A medium, supplemented with 10%
FBS and 1% antibiotic/antimycotic solution (Gibco, Thermo Fisher Scientific, Paisley, UK)
and maintained at 37
◦
C under a humidified atmosphere of 5% CO
2
. For cell viability
assays, HCT-116 cells were seeded in 96-well plates (0.5
×
10
4
cells/well). After 24 h, treat-
ment with the marine sponges’ extracts (concentrations ranging from 0.30 to 125
µ
g/mL),
DMSO (vehicle control), or 10
µ
M 5-fluorouracil (5-Fu, positive control) was followed for
72 h. Cell viability was assessed through MTS metabolism, using CellTiter 96
®
Aqueous
Non-Radioactive Cell Proliferation Assay (Promega, Fitchburg, WI, USA), following the
manufacturer’s instructions. Absorbance signal (490 nm) was recorded using a Glomax
®
-
MultiDetection System (Promega). All data were expressed as the mean
±
standard error of
the mean (SEM) from at least three independent experiments. Data analysis was performed
using GraphPad Prism 8.4.2 software (La Jolla, CA, USA). Dose–response curves were es-
tablished and IC
50
best-fit values determined using the log-(inhibitor) vs. response–variable
slope (four parameters).
3.6. Statistical Analysis
Principal component analysis (PCA) was performed using the XLSTAT software
(version 2016; Addinsoft Inc., New York, NY, USA) to identify similarities/differences
between the extracts obtained from wild and farmed sponge populations, with respect to
their chemical composition. The content of each detected metabolite in the extracts was
determined by using the peak intensity data generated from the LC–MS/MS analysis. Peak
intensity percentages of constituents were used as active variables in PCA. Metabolites that
gave zero-peak intensity values were excluded from the analysis. t-tests were performed
using MetaboAnalyst 5.0. online platform (http://www.metaboanalyst.ca; accessed on
13 March 2023) to investigate significant differences in the content and production levels
(expressed as weight-normalized intensity values) of the various metabolites present in both
wild and farmed sponge populations. The same was applied to the IC
50
values obtained
from the different sponge populations, in order to compare their respective anticancer
activities. The level of significance was set to p = 0.05.
4. Conclusions
Open-sea sponge farming is regarded as a promising source of high added-value
natural products, in addition to effective cleanup technology. In this study, cultivations
of the sponges A. oroides and S. foetidus were assessed for their metabolic profiling, as
well as their bioactivities, and the results were compared with those obtained from their
wild counterparts.
LC–MS/MS analysis revealed an array of natural products present in the studied
extracts, with bioactive compounds belonging to alkaloids, benzenoids, indoles, lipids,
polyketides, and other chemical classes. However, the biosynthesis of metabolites seemed to
be species-specific, with alkaloids being the predominant constituents of A. oroides extracts,
and lipids representing the major components for S. foetidus. In both cases, farming did
not impose inhibition or alterations to the compositions and production levels of the
sponge-related metabolites, while in some cases it even promoted the production of the
bioactive compounds.
In terms of bioactivity, farmed sponge extracts had similar or slightly lower antimi-
crobial potency against Gram-positive strains than that demonstrated by their wild coun-
terparts. A reverse effect was observed only in the case of A. oroides sponges, regarding
Gram-negative bacteria. Extracts belonging to S. foetidus populations showed consistent,
but marginal, anticancer activity against the human colon carcinoma cell line HCT-116, a
bioactivity that was not detected in A. oroides.
Mar. Drugs 2023,21, 612 21 of 27
Our findings demonstrate the significance of sponge mariculture in reproducing indi-
viduals of similar chemical fingerprints and bioactivities when compared with their wild
donors. Although open-sea sponge farming can potentially serve as an additional source of
profit for aquaculture-related enterprises, the “sponge-driven bioproduction/bioremediation”
concept is still new and unexplored. Altogether, our study emphasizes the significance of
sponge mariculture as a promising prospect for diversifying fish farm productivity through
the growth of biotechnologically important marine invertebrates, with the possibility for
future bioremediation applications and bioactive metabolites supply.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/md21120612/s1, Table S1: selected compounds for A. oroides extracts.
Experimental retention time (RT), mode polarity (mode), collision energy, precursor ion, fragment
ions, and bibliographic references for SRM ions. Table S2: the relative percentages of the metabolite
components identified among the A. oroides extracts. Table S3: weight-normalized peak intensity
values of the metabolite components identified among the A. oroides extracts, related to their pro-
duction levels within sponges. Table S4: selected compounds for S. foetidus extracts. Experimental
retention time (RT), mode polarity (mode), collision energy, precursor ion, fragment ions, and bibli-
ographic references for SRM ions. Table S5: the relative percentages of the metabolite components
identified among the S. foetidus extracts. Table S6: weight-normalized peak intensity values of the
metabolite components identified among the S. foetidus extracts, related to their production levels
within sponges [70,71,81,101,105,154–157].
Author Contributions:
Conceptualization, M.M.; methodology, S.P.G., M.J.N., R.G.S., C.M.P.R., M.M.,
E.V. and T.D.; software, M.J.N. and D.V.-M.; validation, S.P.G., M.M., M.J.N., R.G.S. and C.M.P.R.;
formal analysis, M.J.N., D.V.-M., V.M. and R.G.S.; investigation, D.V.-M., M.J.N., V.M., T.I.A. and E.K.;
bioresources, E.V. and T.D.; data curation, M.J.N., D.V.-M., C.M.P.R., R.G.S. and S.P.G.; writing—original
draft preparation, D.V.-M., M.J.N. and V.M.; writing—review and editing, M.M., S.P.G., T.D., R.G.S., V.M.,
C.M.P.R., M.J.N. and N.K.; visualization, all authors; supervision, S.P.G., M.M., M.J.N., R.G.S., T.D.
and N.K.; project administration, S.P.G. and M.M.; funding acquisition, M.M., S.P.G., L.C.B., R.G.S.,
C.M.P.R. and T.D. All authors have read and agreed to the published version of the manuscript.
Funding:
This study was implemented in the framework of the research project SPINAQUA (Grant
No 239) funded by the Hellenic Foundation for Research and Innovation (HFRI) and the General
Secretariat for Research and Technology (GSRT) under the “1st call for H.F.R.I. Research Projects
for the support of Post-doctoral Researchers”. This work was also financed by national funds from
FCT—Fundação para a Ciência e a Tecnologia, IP, in the scope of the project UIDP/04378/2020
of the Research Unit on Applied Molecular Biosciences–UCIBIO and the project LA/P/0140/2020
of the Associate Laboratory Institute for Health and Bioeconomy. Associate Laboratory for Green
Chemistry-LAQV (UIDB/50006/2020 and UIDP/50006/2020) for the financial support of this work.
This publication is based upon work from COST Action CA18238 (Ocean4Biotech), supported by
COST (European Cooperation in Science and Technology) program, which provided Short Term
Scientific Mission (STSM) grant support to D.V.-M. to perform the experimental work at NOVA-FCT.
C.M.P.R. is financially supported by grants from FCT—Fundação para a Ciência e Tecnologia (Grant
No PTDC/MED-FAR/3492/2021) and La Caixa Foundation (Grant No LCF/PR/HR21/52410028).
Institutional Review Board Statement: Not applicable.
Data Availability Statement:
The original data presented in the study are included in the arti-
cle/Supplementary Material; further inquiries can be directed to the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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