ORIGINAL PAPER

Screening of the Antarctic marine sponges (Porifera) as a sourceof bioactive compounds

Sabina Berne1 • Martina Kalauz1,2 • Marko Lapat1,2 • Lora Savin1,2 •

Dorte Janussen3 • Daniel Kersken3 • Jerneja Ambrozic Avgustin1 •

Spela Zemljic Jokhadar4 • Domen Jaklic5 • Nina Gunde-Cimerman1,6 •

Mojca Lunder7 • Irena Roskar7 • Tina Elersek8 • Tom Turk1 • Kristina Sepcic1

Received: 18 March 2015 / Revised: 5 November 2015 / Accepted: 10 November 2015 / Published online: 20 November 2015

� Springer-Verlag Berlin Heidelberg 2015

Abstract Sponges (Porifera) currently represent one of

the richest sources of natural products and account for

almost half of the pharmacologically active compounds of

marine origin. However, to date very little is known about

the pharmacological potential of the sponges from polar

regions. In this work we report on screening of ethanolic

extracts from 24 Antarctic marine sponges for different

biological activities. The extracts were tested for cytotoxic

effects against normal and transformed cell lines, red blood

cells, and algae, for modulation of the activities of selected

physiologically important enzymes (acetylcholinesterase,

butyrylcholinesterase, and a-amylase), and for inhibition of

growth of pathogenic and ecologically relevant bacteria

and fungi. An extract from Tedania (Tedaniopsis) oxeata

was selectively cytotoxic against the cancer cell lines and

showed growth inhibition of all of the tested ecologically

relevant and potentially pathogenic fungal isolates. The

sponge extracts from Isodictya erinacea and Kirkpatrickia

variolosa inhibited the activities of the cholinesterase

enzymes, while the sponge extracts from Isodictya lan-

kesteri and Inflatella belli reduced the activity of a-amy-

lase. Several sponge extracts inhibited the growth of

multiresistant pathogenic bacterial isolates of different

origins, including extended-spectrum beta-lactamase and

carbapenem-resistant strains, while sponge extracts from K.

variolosa and Myxilla (Myxilla) mollis were active against

a human methicillin-resistant Staphylococcus aureus strain.

We conclude that Antarctic marine sponges represent a

valuable source of biologically active compounds with

pharmacological potential.

Keywords Antarctic marine sponges � Antibacterialactivity � Antifungal activity � Antialgal activity � Enzyme

inhibition � Cytotoxicity

Introduction

Due to the rapid progress in the development of separation,

analytical and high-throughput screening techniques over

the last two decades, natural products are again attracting

attention as sources of therapeutic agents for the treatment

of human and animal diseases (Molinski et al. 2009).

Although marine organisms were underestimated for a long

time as sources of biologically active compounds, they are

already providing more pharmacologically interesting

natural products than terrestrial organisms (Munro et al.

1999; Leal et al. 2012). It is estimated that more than

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00300-015-1835-4) contains supplementarymaterial, which is available to authorized users.

& Kristina Sepcic

kristina.sepcic@bf.uni-lj.si

1 Department of Biology, Biotechnical Faculty, University of

Ljubljana, Ljubljana, Slovenia

2 Department of Biotechnology, University of Rijeka, Rijeka,

Croatia

3 Senckenberg Research Institute and Nature Museum,

Frankfurt am Main, Germany

4 Faculty of Medicine, Institute of Biophysics, University of

Ljubljana, Ljubljana, Slovenia

5 Medex d.o.o., Ljubljana, Slovenia

6 Centre of Excellence for Integrated Approaches in Chemistry

and Biology of Proteins (CIPKeBiP), Jamova 39,

1000 Ljubljana, Slovenia

7 Chair of Pharmaceutical Biology, Faculty of Pharmacy,

University of Ljubljana, Ljubljana, Slovenia

8 National Institute of Biology, Ljubljana, Slovenia

123

Polar Biol (2016) 39:947–959

DOI 10.1007/s00300-015-1835-4

20,000 natural compounds have been discovered in marine

organisms since the 1960s (Hu et al. 2011), and about 500

natural products from marine organisms are now being

discovered each year (Hu et al. 2011; Leal et al. 2012).

Almost half of these bioactive natural compounds, which

are mainly terpenoids and alkaloids, have been purified

from sponges (Porifera), which are considered as the most

bioprospective marine taxon (Hu et al. 2011; Leal et al.

2012).

Sampling strategies that are used to discover new

bioactive natural products target mainly new and still

unexplored groups of organisms, or under-sampled eco-

logical niches (Hu et al. 2011; Gerwick and Moore 2012).

Polar regions have been unexplored for a long time due to

their inaccessibility, harsh climatic conditions, and the

complexity of logistics associated with sampling; as such,

they still represent a considerable challenge (Abbas et al.

2011). Although the bioactivity levels of marine natural

products from polar regions are comparable with those

recorded in more temperate marine environments (Avila

et al. 2008), to date, only ca. 3 % of the discovered marine

natural products originate from the polar regions (Lebar

et al. 2007; Abbas et al. 2011; Leal et al. 2012). The

majority of these have been purified from Antarctic

organisms (Abbas et al. 2011), as mainly sponges and

echinoderms (Leal et al. 2012).

The Southern Ocean comprises ca. 10 % of the total

world ocean area (McClintock et al. 2005). Sponges rep-

resent some of the most abundant macroinvertebrates of

Antarctic benthic communities, and they serve both as

substrates for colonizing epibionts and endobionts, and as

food sources for predators (McClintock et al. 2005; Ker-

sken et al. 2014). Thus, it can be expected that these sessile

filter feeders will have developed efficient chemical

strategies to protect themselves, e.g., from fouling by

marine microorganisms and from predators that feed on

sponges.

To obtain new insights into the pharmacological

potential of marine sponges from polar regions, we tested

ethanolic extracts obtained from 24 sponge species for their

biological potential in a series of high-throughput assays.

These sponges were sampled on the deep Antarctic shelf in

three large-scale areas around the tip of the Antarctic

Peninsula.

Materials and methods

Sponge collection

Antarctic Porifera (Demospongiae and Hexactinellida) that

included 24 species (Table 1) were collected in three large-

scale areas around the Antarctic Peninsula: Bransfield

Strait, Drake Passage, and Weddell Sea (61�–64� S; 54�–61� W; Fig. 1). Bottom trawling with an Agassiz trawl was

conducted at depths between 101 and 779 m during the

ANT XXIX/3 Antarctic expeditions of the German

Research Vessel ‘‘Polarstern’’ (January 22 to March 18,

2013). Most specimens were identified to species level

(with exception of one Haliclona sp., one Mycale sp., and

Rossella spp.). On board, subsamples of these specimens

were immediately frozen and kept at -20 �C. These

sponge samples were lyophilized prior to the extractions.

The sponges here investigated are deposited in the Porifera

collection of the Senckenberg Nature Museum, preserved

in 96 % ethanol. All specimens were inventoried with SMF

numbers, and the data are available online within the

SESAM database which is part of the Senckenberg website

(http://sesam.senckenberg.de/).

Preparation of extracts

Freeze-dried sponge samples (0.25 g) were homogenized

(MixerMill MM 400; Retsch, Germany) in 96 % ethanol

Table 1 Antarctic sponges which are included in this study

Species SMF Station

Haliclona sp. SMF11136 162-7

Rossella sp. SMF11409 162-7

Myxodoryx hanitschi SMF11411 162-7

Tedania (Tedaniopsis) charcoti SMF11139 162-7

Myxilla (Myxilla) mollis SMF11626 162-7

Anoxycalyx (Scolymastra) joubini SMF11143 164-4

Rossella sp. SMF11144 164-4

Isodictya lankesteri SMF11421 164-4

Guitarra sigmatifera SMF11154 185-3

Clathria (Clathria) pauper SMF11466 193-9

Tentorium papillatum SMF11192 196-8

Tedania (Tedaniopsis) massa SMF11475 197-4

Isodictya erinacea SMF11476 197-4

Iophon unicorne SMF11478 197-4

Kirkpatrickia variolosa SMF11202 197-5

Tedania (Tedaniopsis) oxeata SMF11208 197-5

Inflatella belli SMF11209 198-5

Iophon gaussi SMF11211 198-5

Mycale (Mycale) tridens SMF11218 199-4

Mycale (Oxymycale) acerata SMF11498 199-4

Rossella cf. vanhoeffeni SMF11228 217-6

Tetilla cf. leptoderma SMF11510 220-2

Rossella cf. racovitzae SMF11519 240-3

Cinachyra antarctica SMF11520 240-3

All specimens are part of AGT catches from PS81 ANT XXIX/3

(LASSO)

948 Polar Biol (2016) 39:947–959

123

(Merck, Germany) and extracted overnight by orbital

shaking (600 rpm) at 37 �C. After the removal of the debris

by centrifugation (15,000g, 30 min, 22 �C), the extracts

were vacuum-dried, resuspended in fresh 96 % ethanol at

2 mg dried extract/mL, and stored at -20 �C.

Cytotoxic activity

The cytotoxic activities were determined using sponge

extracts SMF11136, SMF11139, SMF11626, SMF11421,

SMF11154, SMF11466, SMF11192, SMF11476, SMF11202,

SMF11208, SMF11498, SMF11228, SMF11510, SMF11519,

and SMF11520. The cell lines used were: V-79-379 A (V-79)

cells (diploid lung fibroblasts from Chinese hamster); CaCo-2

cells (human colon adenocarcinoma); and HeLa cells (human

adenocarcinoma). The V-79 and HeLa cells were cultured

in advanced Eagle’s minimal essential medium (Gibco,

Invitrogen, UK), and the CaCo-2 cells were cultured in

advanced RPMI 1640 (Gibco), both at 37 �C in a CO2

incubator (5 % CO2, 95 % air, 95 % relative humidity).

Both of these culture media were supplemented with

2 mM L-glutamine, 100 lg/mL penicillin, 100 lg/mL

streptomycin, and 5 % (v/v) fetal bovine serum (all from

Gibco). For the in vitro cytotoxicity assays, the cells were

plated in 96-well microtiter plates (100 lL; TPP,

Switzerland) at 5,000 cell/well (V-79, HeLa cells) or

10,000 cell/well (CaCo-2 cells). After a 3-h incubation, the

ethanol-dissolved extracts prepared in the respective media

without serum were added to a final concentration of

100 lg dried extract/mL, and the incubations were carried

out for 1 h (under their respective cell culture conditions).

Ethanol added in the respective media was used as the

control. The cells were then washed once with their

respective medium, and their fresh medium with fetal

bovine serum was added for a further 48 h (as before). The

cytotoxicity was determined using the MTS test (i.e.,

3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-

2-[4-sulfophenyl]-2H-tetrazolium; CellTiter 96 AQueous

Reagent, Promega, USA). Here, 20 lL MTS was added to

the cell cultures in each well. After 1 h, the absorbance at

490 nm was measured using a microplate reader (Bio-Tek

Instruments Inc., USA). The absorption corresponded to

the amount of soluble formazan produced, which is directly

proportional to the number of viable cells. The cell via-

bility was expressed as the ratios of the absorbance at

490 nm of the treated and control cells, expressed as per-

centages. These data are presented as mean ± SD of three

independent experiments. The differences were analyzed

using Student’s t tests on two populations, with p\ 0.05

considered significant.

Hemolytic activity

Aliquots of 100 lL of a suspension of fresh bovine ery-

throcytes in erythrocyte buffer (140 mM NaCl, 20 mM

Tris–HCl, pH 7.4) with an initial apparent absorption of 0.5

AU at 650 nm were pipetted into each microplate well. The

ethanolic sponge extracts were then added to each well at

different final concentrations of the dry extracts, and their

hemolytic activities were determined using a microplate

VIS absorption reader (Dynex, USA), as described previ-

ously (Turk et al. 2013). The ethanol concentration in each

well did not exceed 20 % (an experimentally verified non-

lytic concentration). The time course of hemolysis was

Fig. 1 AGT deployments where sponge specimens were collected; maps created with ocean data view 4.7.2 (Schlitzer 2015)

Polar Biol (2016) 39:947–959 949

123

monitored over 30 min at 25 �C, and the hemolytic activity

was expressed as the half-time of the hemolysis (t50), i.e.,

the time in which the apparent absorbance at 650 nm

dropped from 0.5 to 0.25 AU. All of these measurements

were carried out in triplicate.

Antialgal activity

The screening for the effects of sponge extracts on algal

growth was carried out according to the guideline of the

Organisation for Economic Cooperation and Development

(OECD) TG 201 Growth Inhibition Test (2011). We used

the green alga Pseudokirchneriella subcapitata, which was

obtained from the algae collection (SAG 61.81) of the

University of Gottingen, Germany. The alga was cultivated

under defined conditions in terms of the OECD liquid

medium composition, temperature (24 ± 2 �C), constantshaking (100 trembling movement/min), and constant

illumination with light intensity 80–120 lmol photons

m-2 s-1. The dried ethanolic sponge extracts were dis-

solved in 25 % dimethyl sulfoxide in water and added to

the algal cultures at an initial density of 5 9 104 cell/mL,

which were then grown in glass flasks for 72 h. The final

concentration of the extracts was 4 lg dried extract/mL. In

spite of the relatively short testing time, effects over sev-

eral algal generations can be assessed in this way. The final

dimethyl sulfoxide concentration was 0.2 %, which

exceeds the OECD recommended value (0.01 %), but as

demonstrated previously (Brezovsek et al. 2014), 0.2 %

dimethyl sulfoxide does not affect the growth of the alga

used here. The final concentration of 0.2 % dimethyl sul-

foxide was added also to the control cultures. The cell

densities in each flask were determined on days 2 and 3 by

cell counting (Burker-Turk hemocytometer). The observed

endpoint was growth inhibition, which is expressed as the

difference in the logarithmic increase in the cell number

(mean specific growth rate) in comparison with the control

over the exposure period of 3 days. The statistical signifi-

cance (p\ 0.05) of the extract effects in comparison with

the control was determined by nonparametric ANOVA

(Kruskal–Wallis tests) with Bonferroni post hoc tests at a

95 % confidence interval.

Cholinesterase activities

The cholinesterase inhibition assays were performed

according to the method of Ellman et al. (1961). Acetyl-

cholinesterase (AChE; EC 3.1.1.7) from electric eel and

butyrylcholinesterase (BChE; EC 3.1.1.8) from equine

serum (both Sigma, USA) were each dissolved in 100 mM

phosphate buffer (pH 7.4) to a concentration of 500 EU/

mL. Prior to the assays, the enzymes were 100-fold diluted

in the same buffer. Ellman reagent (0.5 mM 5,5-dithiobis-

2-nitrobenzoic acid; 100 lL) in 50 mM phosphate buffer

(pH 7.4) containing the substrate acetylcholine (for

1 mM final assay concentration) was added to each

microplate well. The sponge extracts (5 lL) and then

45 lL of AChE or BChE were added to start the reac-

tions. Ethanol (5 lL/well) was used as the control. The

time courses of the enzymatic reactions were monitored

over 5 min at 25 �C, at 405 nm using a VIS microplate

reader (Dynex, USA). All of the measurements were

performed in triplicate.

a-Amylase activity

A previously described method (Ali et al. 2006) was

modified and adapted to perform the a-amylase activity

assay in 96-well microplates. Porcine pancreatic a-amylase

(EC 3.2.1.1, type IV, Sigma-Aldrich) was dissolved in ice-

cold distilled water, and the dried ethanolic sponge extracts

were dissolved in 25 % (v/v) dimethyl sulfoxide in water.

The reactions were started by the addition of 0.5 % potato

starch solution (Sigma-Aldrich) to the pre-incubated mix-

ture of 1.75 EU/mL enzyme solution and the sponge

extracts at 25 lg dried extract/mL, in a final volume of

100 ll. The plates were incubated at room temperature for

3 min, followed by sample transfer into separate sealable

plates containing the 3,5-dinitro salicylic acid color reagent

(Bernfeld 1955). The plates were placed in a thermo-block

at 95 �C for 15 min, and then the samples were diluted

with distilled water and transferred to new 96-well plates.

The a-amylase activities were determined by measuring

the absorbance of the samples at 540 nm. The inhibition

assays were performed in triplicate, and the mean absor-

bance was calculated. For the blank incubations, the 3,5-

dinitro salicylic acid reagent was first added to the mixture

of each sponge extract in the 25 % dimethyl sulfoxide and

substrate, to allow for the absorbance produced by the

sponge extracts. The enzyme solution was added afterward,

and the mixtures were incubated at 95 �C to also measure

the absorbance due to the lactose present in the enzyme

reagent. The absorbance due to the maltose generated was

calculated as:

A540nm 100% control or sponge extractð Þ¼ A540nmTest �A540nm Blank ð1Þ

The remaining a-amylase activity was calculated as A540nm

(extract)/A540nm (100 % control).

Antibacterial activity

The antibacterial activities of the sponge extracts were

determined using the disk diffusion method adapted from

Gopi et al. (2012a, b) using a variety of different Gram-

negative and Gram-positive bacterial strains, which

950 Polar Biol (2016) 39:947–959

123

included: (1) ecologically relevant Gram-negative marine

bacteria from Arctic sea water and Arctic ice: Pseu-

domonas spp. ARK13, Pseudomonas spp. ARK14, Pseu-

domonas spp. ARK285; (2) laboratory strains of

Escherichia coli DH5 and Salmonella enterica serovar

typhimurium TL747; (3) environmental isolates of Sta-

phylococcus saprophyticus L572, Bacillus subtilis L519,

Bacillus cereus L593, and Paenibacillus L564; (4) patho-

genic Streptococcus canis, Listeria monocytogenes, and

Enterococcus spp. isolates; and (5) multiresistant patho-

genic isolates of different origins: carbapenem-resistant

Klebsiella pneumoniae ATCC� BAA-1705TM-(CRKP),

Acinetobacter baumannii 12588, Pseudomonas aeruginosa

12599, extended-spectrum beta-lactamase (ESBL) pro-

ducing E. coli KM128, sequence type ST131, E. coli Z39,

E. coli 3273, methicillin-resistant Staphylococcus pseu-

dointermedius (MRSP) 1342, methicillin-resistant Staphy-

lococcus aureus (MRSA) 3797, S. aureus 2315, and

vancomycin-resistant Enterococcus faecalis.

The strains were obtained from ATCC (USA), from the

Genetic Laboratory Microbes culture collections of the

Chair of Molecular Genetics and Biology of Microorgan-

isms of the Biotechnical Faculty (preserved in the Ex

Culture Collection of the Infrastructural Centre Mycosmo

[MRICUL] of the Department of Biology, Biotechnical

Faculty), University of Ljubljana, from the Institute of

Microbiology and Parasitology, Veterinary Faculty,

University of Ljubljana, and from the Institute of Micro-

biology and Immunology, Faculty of Medicine, University

of Ljubljana.

These bacterial strains were grown in Luria–Bertani

broth (Sigma, USA) for 20 h at 37 �C, except for the

Arctic isolates, which were incubated at 18 �C. Subse-quently, a suspension was prepared in sterile saline

solution (0.9 %). The turbidity of the suspension was

adjusted to match that of a 0.5 McFarland standard. Then,

100 ll of each suspension was spread onto Mueller–

Hinton agar. Sterile filter-paper disks of 6-mm diameter

were placed on the culture-spread Mueller–Hinton agar

plates at suitable spacing. The disks were subsequently

impregnated with 10 ll of a sponge extract. Ethanol was

used for one disk on each plate as the solvent controls.

The positive control commercial disks contained the

antibiotics rifampicin (25 lg), ciprofloxacin (5 lg), gen-tamicin (10 lg), chloramphenicol (30 lg), tetracycline

(5 lg), and ampicillin (10 lg) (additional data are given

in Online Resource files ESM_1.doc and ESM_2.doc).

The plates were incubated for 24 h at 37 �C, except forthe plates seeded with the Arctic isolates that were incu-

bated at 18 �C. The antibacterial activities were deter-

mined by measurement of the diameters of the inhibition

zones around the disks.

Antifungal activity

The antifungal activities were determined using microdi-

lution tests according to two modified reference methods:

the broth dilution antifungal susceptibility testing of yeast

(M27-A3CLSI Approved Standard) and the test using fil-

amentous fungi (M38-A2; CLSI Approved Standard). All

of the fungal strains were subcultured on malt extract agar

and incubated at 25 �C for 3–5 days.

The fungal strains were obtained from the Ex Culture

Collection of Extremophilic Fungi, which is part of the

MRICUL in the Department of Biology, Biotechnical

Faculty, University of Ljubljana, Slovenia. The ecologi-

cally relevant fungal strains tested included: (1) cultures

isolated from Arctic subglacial ice: Rhodosporidium lusi-

tanie (EX-3935), Cryptococcus carnescens (EX-1551),

Cryptococcus victoriae (EX-1623), and Aureobasidum

subglaciale (EX-2481); (2) cultures isolated from Arctic

sea water: Debaryomyces hansenii (EX-4023), and Rho-

dotorula mucilaginosa (EX-4015); (3) opportunistic

pathogens from dishwashers: Candida parapsilosis (EX-

9370), Exophiala dermatitidis (EX-5721), and Fusarium

dimerum (EX-9424); (4) an opportunistic pathogen from

kitchen surfaces (dish strainer): Candida albicans (EX-

9382); and (5) opportunistic pathogens from potable water

and rubber of a kitchen drain: Aureobasidium melano-

genum (EX-9454) and (EX-9467), respectively.

The fungal inoculi were prepared in 5 mL RPMI med-

ium using Neubauer mesh, to obtain concentrations of 105

cell/conidia/mL. Then, 95 lL of the different fungal ino-

culi was pipetted into the wells of 96-well microtiter plates,

and 5 lL of the ethanolic sponge extracts was added, to a

final concentration of 100 lg dried extract/mL. After a

3-day incubation at 25 �C, the microtiter test plates were

analyzed at 630 nm using a microtiter plate reader (model

MRX; Dynatech Laboratories, USA). The turbidity was

used as a measure to determine the fungal susceptibilities

to the sponge extracts. The antifungal activities are

expressed as percentage inhibition compared to the nega-

tive control.

Results and discussion

Consistent with our previous study on ethanolic extracts

from 28 species of deep-sea marine sponges collected in

the Antarctic waters (Turk et al. 2013), ethanolic extracts

from the Antarctic sponge species tested in the present

study showed a range of bioactivities. These were, how-

ever, not as large compared to ethanolic extracts of pre-

viously tested tropical marine sponges using a similar

experimental set-up (Sepcic et al. 2010).

Polar Biol (2016) 39:947–959 951

123

One of the most notable bioactivities observed in the

present study was selective cytotoxicity, which is a char-

acteristic of certain compounds that can lead to the

development of new chemotherapeutics (Laport et al.

2009). Indeed, in 10 of the 15 sponge extracts tested, the

cytotoxic activity was significantly greater against the

transformed cells (i.e., human colon adenocarcinoma cells;

Table 2). At least some of these cytotoxic effects can be

ascribed to already known compounds. For example,

Kirkpatrickia variolosa has been reported to produce a

potent cytotoxic and antiviral guanidine alkaloid, variolin

B (Perry et al. 1994; McClintock et al. 2005). Similarly,

sponges from the genus Mycale have been reported to

synthesize several metabolites with cytotoxic activities,

including pateamine, peloruside, and mycalamide (Hood

et al. 2001; Singh et al. 2010), and a highly cytotoxic

macrolide cinachyrolide A was isolated from Cinachyra

antarctica (Fusetani et al. 1993). Here, the extracts from

both species of Tedania that were included (SMF11139,

SMF11208) showed selective cytotoxicity against the

CaCo-2 cells. Although cytotoxic activities have not been

mentioned in the literature for these sponge species, it is

known that a Caribbean ‘‘fire sponge’’ of the same genus

[i.e., Tedania (Tedania) ignis] produces highly cytotoxic

macrolides, tedanolides (Schmitz et al. 1984). The

ethanolic extract of Tetilla leptoderma (SMF11510)

showed selective cytotoxicity here against this tumor cell

line. To the best of our knowledge, bioactivities for extracts

from this particular sponge species have not been reported

previously in the literature. However, it is interesting to

note that in our previous study (Turk et al. 2013), an

ethanolic extract of T. leptoderma did not show any cyto-

toxic activity when tested using similar experimental set-

up. A possible explanation here might derive from the fact

that the production of bioactive secondary metabolites in

marine sponges can be affected by several environmental

factors (hydrodynamics, depth, water temperature, habitat)

and also by the sponge physiology, including its size,

reproductive stage, and response to stress (Thompson et al.

1987; Thakur and Anil 2000; Duckworth and Battershill

2001; Page et al. 2005; Abdo et al. 2007; Ferretti et al.

2009; Sacristan-Soriano et al. 2012). The present study and

some previous studies (Taboada et al. 2010; Turk et al.

2013) show that some Rosella spp. can have selective

cytotoxicities that are associated with an as yet undefined

metabolite. Finally, the cytotoxicity of the ethanolic extract

of the sponge Myxilla (Myxilla) mollis (SMF11626) seen

here has not been described previously in the literature,

although cytotoxic metabolites were found in the fungus

Beauveria bassiana isolated from the sponge Myxilla

(Myxilla) incrustans collected in the North Sea (Neumann

2008).

The hemolytic activity represents the disruption of the

membranes of red blood cells, and this was associated with

14 of the 24 sponge extracts tested (Table 3). Indeed, this

activity was particularly strong in the extracts of Tedania

Table 2 Cytotoxic activities of

the most active sponge extracts,

expressed as viability of the

V-79, CaCo-2, and HeLa cell

lines treated with 100 lg dried

extract/mL

Sponge species extract SMF no. Cell viability (% control)

V79 cells CaCo-2 cells HeLa cells

Haliclona sp. SFM11136 88.6 ± 6.0* 86.8 ± 2.4** n.d.

Tedania (Tedaniopsis) charcoti SFM11139 105.1 ± 2.5 97.0 ± 2.5** n.d.

Myxilla (Myxilla) mollis SFM11626 100.6 ± 3.2 88.9 ± 3.3** n.d.

Isodictya lankesteri SFM11421 97.2 ± 5.5 77.7 ± 6.3 n.d.

Guitarra sigmatifera SFM11154 99.5 ± 4.3 69.3 ± 4.9 n.d.

Clathria (Clathria) pauper SFM11466 95.9 ± 3.4 82.7 ± 3.5 n.d.

Tentorium papillatum SFM11192 99.6 ± 3.4 72.2 ± 4.4 n.d.

Isodictya erinacea SFM11476 86.3 ± 3.1** 79.8 ± 3.7 n.d.

Kirkpatrickia variolosa SFM11202 37.5 ± 3.0** 23.1 ± 2.5** n.d.

Tedania (Tedaniopsis) oxeata SFM11208 94.6 ± 4.6 54.4 ± 2.2** 81.7 ± 0.03**

Mycale (Oxymycale) acerata SFM11498 90.0 ± 4.5 60.7 ± 3.7** 73.2 ± 0.04*

Rossella cf. vanhoeffeni SFM11228 91.7 ± 3.3* 67.7 ± 3.8* n.d.

Tetilla cf. leptoderma SFM11510 100.4 ± 3.9 61.7 ± 4.7** n.d.

Rossella cf. racovitzae SFM11519 98.9 ± 3.7 71.3 0 ± 2.6* n.d.

Cinachyra antarctica SFM11520 106.4 ± 3.4 64.6 ± 3.0** 87.9 ± 0.02**

Controls were treated with the same volume of ethanol only

n.d. not determined

* p\ 0.05; ** p\ 0.01, significant differences in cytotoxic activity between control and treated V-79,

CaCo-2, and HeLa cell lines

952 Polar Biol (2016) 39:947–959

123

(Tedaniopsis) oxeata (SMF11208) and Mycale (Oxymy-

cale) acerata (SMF11498), which suggests that the above-

mentioned cytotoxicity of these extracts derives from their

interactions with cell membranes.

It is also interesting to note that 21 of these sponge

extracts inhibited the growth of the green freshwater alga

P. subcapitata, with this inhibition ranging from 9 to 70 %

on day 3, in comparison with the control (Table 3). Only

three of these extracts did not have any impact on the algal

growth, according to the comparisons with the control, and

as tested by ANOVA and Bonferroni post hoc tests:

Rosella cf. vanhoeffeni (SMF11228), Rosella cf. racovitzae

(SMF11519), and Haliclona sp. (SMF11136). The use of

algal inhibitors, once also tested on other algae, would be

very useful in places where algal growth is unwanted, such

as fountains, pools, monuments, outside works of art,

boats, rafts, and tourist caves.

Acetylcholinesterase is a key enzyme in the nervous

system that is involved in the transmission of signals across

cholinergic synapses, through its degradation of the neu-

rotransmitter acetylcholine (Pohanka 2011). BChE is found

in the blood plasma of vertebrates (Pezzementi and Cha-

tonnet 2010), and it is less specific for different substrates

than AChE. As such, it is assumed that it can serve as a

‘‘back-up’’ for AChE, especially when AChE activity is

compromised or absent, thus further supporting and regu-

lating cholinergic transmission (Li et al. 2000). Use of

AChE inhibitors to prevent the hydrolysis of acetylcholine

has been suggested as one of the strategies for the treatment

of patients with Alzheimer’s disease, as well as for the

treatment of glaucoma and the autoimmune disorder

myasthenia gravis, and for the recovery from neuromus-

cular block during surgery (Kaur and Zhang 2000; Munoz-

Torrero 2008). Marine sponges have already been shown to

be an important source of new cholinesterase inhibitors

(Orhan 2013). In particular, in our previous bioactivity

screening of Antarctic marine sponges (Turk et al. 2013),

ethanolic extracts of sponges of a Latrunculia sp. induced

Table 3 Hemolytic activities

of the sponge extracts and

antialgal activities of the sponge

extracts against green alga

Pseudokirchneriella subcapitata

Sponge species SMF no. Hemolytic activitya Antialgal activityb

Haliclona sp. SMF11136 0.05 2

Rossella sp. SMF11409 55*

Myxodoryx hanitschi SMF11411 28*

Tedania (Tedaniopsis) charcoti SMF11139 0.18 51*

Myxilla (Myxilla) mollis SMF11626 0.09 54*

Anoxycalyx (Scolymastra) joubini SMF11143 59*

Rossella sp. SMF11144 0.08 24*

Isodictya lankesteri SMF11421 0.13 63*

Guitarra sigmatifera SMF11154 70*

Clathria (Clathria) pauper SMF11466 0.06 14*

Tentorium papillatum SMF11192 0.07 27*

Tedania (Tedaniopsis) massa SMF11475 0.08 70*

Isodictya erinacea SMF11476 0.06 33*

Iophon unicorne SMF11478 40*

Kirkpatrickia variolosa SMF11202 70*

Tedania (Tedaniopsis) oxeata SMF11208 0.82 9*

Inflatella belli SMF11209 37*

Iophon gaussi SMF11211 0.07 9*

Mycale (Mycale) tridens SMF11218 70*

Mycale (Oxymycale) acerata SMF11498 0.80 54*

Rossella cf. vanhoeffeni SMF11228

Tetilla cf. leptoderma SMF11510 0.14 65*

Rossella cf. racovitzae SMF11519

Cinachyra antarctica SMF11520 0.23 70*

Empty spaces in columns denote that the tested sponge extract exhibited no activity

* Significant difference in antialgal activity between control and treated algae (* p\ 0.05)a Expressed as 1/t50 (min-1) at 400 lg dried extract/mL in the assayb Expressed as % of growth inhibition (in comparison with the control) 3 days after the addition of

extracts in final concentration of 4 lg dried extract/mL

Polar Biol (2016) 39:947–959 953

123

50 % inhibition of AChE activity at a few ng dried extract/

mL. The purification, structural characterization, and bio-

logical activity of this new bioactive compound that can act

as a reversible competitive AChE inhibitor will be pub-

lished elsewhere. In the present study, two additional

sponge extracts showed moderate anti-cholinesterase

potential that have not yet been mentioned in the literature:

those of Isodictya erinacea (SMF11476) and K. variolosa

(SMF11202) (Table 4). It is, however, interesting to note

that variolins, cytotoxic macrolides from K. variolosa

acting via an inhibition of cyclin-dependent kinases (Si-

mone et al. 2005), have been proposed as hypothetical

agents that could block the neurodegeneration in Alzhei-

mer’s disease (Sagar et al. 2013).

a-Amylase is one of the main secretory products of the

pancreas and salivary glands. As such, it has important

roles in starch and glycogen digestion. The delay of glu-

cose absorption through inhibition of carbohydrate-hy-

drolyzing enzymes represents an important strategy to

blunt postprandial glucose levels and thereby to prevent

diabetes-related complications. Moreover, inhibition of

starch hydrolysis to glucose in the oral cavity can prevent

unwanted plaque formation and subsequent dental caries

and periodontal diseases, as this glucose can be used as a

food source for oral bacteria, and further metabolized into

lactic acid (Scannapieco et al. 1993). Many natural

resources, plants in particular, have been investigated with

respect to a-amylase inhibition (Sales et al. 2012). Inhibi-

tory effects of extracts of Antarctic marine sponges against

carbohydrate metabolizing enzymes have not been inves-

tigated to date. Furthermore, only one study has investi-

gated the effects of marine sponge extracts (i.e., from four

varieties of Red Sea sponges, of the genera Smenospongia,

Callyspongia, Niphates, Stylissa) on a-amylase and other

carbohydrate-hydrolyzing enzymes (Shaaban et al. 2012).

This previous study demonstrated important inhibitory

effects of a Callyspongia extract that were attributed to

phenolic compounds that interacted with and/or inhibited

these enzymes. In the present study, four of the 24

ethanolic sponge extracts showed inhibitory effects against

a-amylase, whereby two of them induced 50 % reductions

in the a-amylase activity at 25 lg dried extract/mL: Isod-

ictya lankesteri (SMF11421) and Inflatella belli

(SMF11209) (Table 4). These results show that with fur-

ther compound characterization, such marine sponge

extracts might provide important drug leads for the man-

agement of type-2 diabetes, obesity, and oral diseases.

The production of antibacterial compounds is one of the

most prominent characteristics of sessile marine organisms

and of sponges in particular. These compounds protect

sponges against fouling by different microorganisms and

can also represent an important source of new antibiotics,

especially in view of the appearance of multiresistant

bacterial strains (Laport et al. 2009). Antibacterial and

antifungal activities are particularly noted in sponges living

in moderate and tropical waters, where organic extracts of

almost all of the sponge species tested have shown inhi-

bition of bacterial growth (Sepcic et al. 2010). In com-

parison with sponges from temperate or tropical waters, the

sponges in polar environments appear to produce fewer

numbers of antibacterial compounds that show generally

weaker activities (McClintock and Gauthier 1992; Lippert

et al. 2003; Abbas et al. 2011; Turk et al. 2013). Never-

theless, polar marine sponges should not be neglected as

potential sources of new antibiotics. Indeed, recent studies

have shown that Antarctic sponges have a remarkable

potential against several plant bacterial pathogens (Xin

et al. 2011), while bacteria associated with Antarctic

sponges have been shown to effectively inhibit the growth

of human opportunistic multiresistant pathogenic bacteria

(Papaleo et al. 2013). In the present study, the ethanolic

sponge extracts were tested on a variety of pathogenic and

multiresistant Gram-negative and Gram-positive bacterial

strains that are environmentally or clinically relevant, or

under laboratory use. The ethanolic extracts from the

Antarctic marine sponges tested in our previous study

showed greater antibacterial potential against ecologically

relevant bacteria obtained from a polar environment (i.e.,

from Arctic ice), which probably reflects their ecological

role (Turk et al. 2013). The analysis of the data obtained in

Table 4 Inhibitory activities of

sponge extracts against

acetylcholinesterase (AChE),

butyrylcholinesterase (BChE),

and a-amylase. Only the

bioactive sponge extracts are

shown

Sponge species extract SMF no. Inhibitory activity (lg/mL, 50 % inhibition)

AChE BChE a-Amylase

Isodictya lankesteri SMF11421 25

Tentorium papillatum SMF11192 [25

Isodictya erinacea SMF11476 140

Kirkpatrickia variolosa SMF11202 9.5 25

Inflatella belli SMF11209 25

Mycale (Oxymycale) acerata SMF11498 [25

Empty spaces in columns denote that the tested sponge extract showed no activity

954 Polar Biol (2016) 39:947–959

123

the present study (Tables 5,6) confirms these findings,

whereby 20 out of the 24 sponge extracts inhibited the

growth of at least one ecologically relevant strain of

Pseudomonas spp. isolated from Arctic ice. Furthermore,

all of these three Pseudomonas spp. strains showed con-

siderably higher susceptibility to the sponge extracts tested

when compared to the multiresistant pathogenic isolate of

P. aeruginosa.

All of these tested sponge extracts showed weak-to-

moderate inhibitory activities against at least one strain of

the bacteria tested, regardless of the Gram staining. In this

regard, extracts from Haliclona sp. (SMF11136), Tedania

(Tedaniopsis) charcoti (SMF11139), M. (Myxilla) mollis

(SMF11626), Iophon gaussi (SMF11211), and R. cf. ra-

covitzae (SMF11519) inhibited the growth of the largest

number of the bacterial strains tested (45–55 %). Polar

sponges of the genus Haliclona have been reported to

synthesize antibacterial 3-alkylpyridinium alkaloids (Timm

et al. 2010). However, antimicrobial activities of extracts

from polar species of the genus Tedania have not been

reported in the literature, although it has been shown that

organic extracts from tropical sponges that belong to this

Table 5 Antibacterial activities of the sponge extracts against environmental, laboratory, commensal, and clinically relevant (multiresistant)

Gram-negative bacterial strains

Sponge species extract SMF no. Antibacterial activity (inhibitory zone diameter, mm)a

ARK

13

ARK

14

ARK

285

12599 DH5 3273 Z

39

KM

128

TL

747

KPC

1705

12588

Haliclona sp. SMF11136 9 9 10 9 8 9 9 10

Rossella sp. SMF11409 8 8 8

Myxodoryx hanitschi SMF11411 8 9 9

Tedania (Tedaniopsis)

charcoti

SMF11139 9 7 9 8 9 9

Myxilla (Myxilla) mollis SMF11626 9 9 8 8 9

Anoxycalyx (Scolymastra)

joubini

SMF11143 9 13 8 9 8 12 10

Rossella sp. SMF11144 9 8

Isodictya lankesteri SMF11421 8 8 9

Guitarra sigmatifera SMF11154 9 8 9 10

Clathria (Clathria) pauper SMF11466 8 9

Tentorium papillatum SMF11192 8 8 9

Tedania (Tedaniopsis) massa SMF11475 10 9 9 8 10

Isodictya erinacea SMF11476 9 8

Iophon unicorne SMF11478 9 8

Kirkpatrickia variolosa SMF11202 9 8

Tedania (Tedaniopsis) oxeata SMF11208 9 8 9

Inflatella belli SMF11209 9 9

Iophon gaussi SMF11211 9 8 11 8 8 8 9

Mycale (Mycale) tridens SMF11218 10 8 8 8 7 9

Mycale (Oxymycale) acerata SMF11498 8 10 10

Rossella cf. vanhoeffeni SMF11228 10 9 8 8

Tetilla cf. leptoderma SMF11510 8 10

Rossella cf. racovitzae SMF11519 9 11 9 12 9

Cinachyra antarctica SMF11520 9 8 9

ARK 13, ARK 14, ARK 285, Arctic strains of Pseudomonas spp.; 12599, multiresistant P. aeruginosa (clinical isolate); DH5, Escherichia coli

(laboratory strain); 3273, extended-spectrum beta-lactamase-producing (ESBL) E. coli (urinary tract infection isolate); Z 39, ESBL E. coli (food

contaminating isolate); KM 128, ESBL E. coli (sequence type ST131; human respiratory tract infection); TL 747, Salmonella enterica ser.

Typhimurium (laboratory strain); KPC 1705, carbapenemase-producing (KPC) Klebsiella pneumonia (human urine isolate); 12588, carbapen-

emase-producing (NDM) Acinetobacter baumannii (human clinical isolate)

Empty spaces in columns denote that the tested sponge extract showed no antibacterial activitya With 2 mg dried extract/mL in assay (see ‘‘Materials and methods’’ section)

Polar Biol (2016) 39:947–959 955

123

genus can have strong antifungal and antimicrobial activ-

ities (Muricy et al. 1993). In the Chinese species Tedania

(Tedania) anhelans, the antimicrobial activities appear to

be related to different cultivable bacterial endosymbionts

(Zhen et al. 2013). The antibacterial potential of Antarctic

sponges belonging to the genera Myxilla and Rossella was

reported in our previous study (Turk et al. 2013), and this

appears to be related to as yet unknown metabolite(s). It is

interesting to note that when tested here against multire-

sistant Gram-negative pathogenic isolates of different ori-

gins, several of these sponge extracts showed good

antibacterial potential compared with the commercial

antibiotic disks. These pathogen examples include the

extended-spectrum beta-lactamase producing E. coli 3273,

which was inhibited by nine of these sponge extracts

compared to only one of the commercial antibiotics tested,

and the carbapenem-resistant isolates of K. pneumoniae

and A. baumannii, which were inhibited by nine and 10 of

these sponge extracts, respectively. These results are very

promising considering that resistance to the third-genera-

tion cephalosporins and carbapenems is a major concern

for public health. Antibacterial potential of selected sponge

extracts was also observed against the human and animal

methicillin-resistant S. aureus (MRSA) isolates. The

growth inhibition of human MRSA by the extract of K.

variolosa (SMF11202) (which corresponded to 20 lgextract dry weight) was the highest among all of the

antibacterial activities reported in the present study. In this

Table 6 Antibacterial activities of the sponge extracts against environmental, laboratory, clinically relevant, and pathogenic multiresistant

Gram-positive bacterial strains

Sponge species extract SMF no. Antibacterial activity (inhibitory zone diameter, mm)a

L 572 2315 3797 1342 S. canis L 545 L 606 L 519 L 593 L 564 12809

Haliclona sp. SMF11136 11 8 8 9

Rossella sp. SMF11409 8 10

Myxodoryx hanitschi SMF11411 9 8 8 8 8

Tedania (Tedaniopsis) charcoti SMF11139 8 8 8 10 8 8

Myxilla (Myxilla) mollis SMF11626 8 11 9 8 8 8 9

Anoxycalyx (Scolymastra) joubini SMF11143 8

Rossella sp. SMF11144 8 9 8 9

Isodictya lankesteri SMF11421 11 9

Guitarra sigmatifera SMF11154 8

Clathria (Clathria) pauper SMF11466 8 8 8

Tentorium papillatum SMF11192 8 8 9

Tedania (Tedaniopsis) massa SMF11475 9 9

Isodictya erinacea SMF11476

Iophon unicorne SMF11478 8 9 9

Kirkpatrickia variolosa SMF11202 13 7

Tedania (Tedaniopsis) oxeata SMF11208 8

Inflatella belli SMF11209 8 8

Iophon gaussi SMF11211 9 9

Mycale (Mycale) tridens SMF11218 8 10

Mycale (Oxymycale) acerata SMF11498 9 9

Rossella cf. vanhoeffeni SMF11228 8 8 10

Tetilla cf. leptoderma SMF11510 8 8

Rossella cf. racovitzae SMF11519 9 8 8 10 10

Cinachyra antarctica SMF11520 8 9 9

L 572, Staphylococcus saprophyticus (environmental); 2315, methicillin-resistant Staphylococcus aureus (MRSA-human isolate); 3797,

methicillin-resistant Staphylococcus aureus (MRSA-animal isolate); 1342, methicillin-resistant Staphylococcus pseudintermedius (MRSP-animal

isolate); S. canis, Streptococcus canis (animal isolate); L 545, Enterococcus spp. (animal isolate); L 606, Listeria monocytogenes (animal

isolate); L 519, Bacillus subtilis (environmental isolate); L 593, Bacillus cereus (environmental isolate); L 564, Paenibacillus sp. (environmental

isolate); 12809, vancomycin-resistant Enterococcus spp. (VRE-human clinical isolate)

Empty spaces in columns denote that the tested sponge extract showed no antibacterial activitya With 2 mg dried extract/mL in assay (see ‘‘Materials and methods’’ section)

956 Polar Biol (2016) 39:947–959

123

regard, K. variolosa antimicrobial metabolites, the exis-

tence of which has already been reported (McClintock and

Gauthier 1992), deserve further investigation. The same

applies to the extract obtained from M. (M.) mollis

(SMF11626), which also showed inhibition of the MRSA

isolates, and to the extract from Rosella sp, which was

active against the tested vancomycin-resistant Enterococ-

cus sp.

In line with these antibacterial activities, these ethanolic

extracts from Antarctic marine sponges also showed anti-

fungal potential, although due to the lack of material, this

was tested with only 10 of the sponge extracts (Table 7).

The selected fungal species belong to ecologically relevant

polar isolates and to thermotolerant, oxidative-stress-re-

sistant, and generally stress-tolerant fungi that are recog-

nized as opportunistic human pathogens (Gostincar et al.

2009). These were recently isolated from the extreme

environments of household appliances, including dish-

washers (Zalar et al. 2011), washing machines (Novak

Babic et al. 2015), and various kitchen surfaces. The

majority of Exophiala spp. are opportunistic pathogens that

can cause cutaneous and subcutaneous infections, and lung

and neurotropic infections (de Hoog et al. 2009). Both R.

mucilaginosa and C. parapsilosis have been reported to be

new emerging pathogens, as they are primarily responsible

for catheter-related infections and opportunistic

nosocomial fungemias in immunocompromised patients

(Neofytos et al. 2007; Pfaller et al. 2007; van Asbeck

et al. 2009; Miceli et al. 2011). Various Fusarium spp. are

causative agents of approximately 80 % of human fungal

infections. They produce mycotoxins and can cause

localized subcutaneous infections, sinusitis, and ony-

chomycosis (O’Donnell et al. 2010; Sutton and Brandt

2011; Garnica and Nucci 2013). In contrast to the bacteria

tested here and previously (Turk et al. 2013), the fungal

strains isolated from subglacial Arctic ice were not more

susceptible to these sponge extracts. Among the fungi

tested, C. parapsilosis was the most susceptible, with its

growth inhibited by all of the sponge extracts tested. The

sponge extract that showed the broadest range of anti-

fungal activities was also the most efficient in comparison

with the others tested, and this was from Tedania

(Tedaniopsis) massa (SMF11475). At 100 lg dried

extract/mL, this extract strongly inhibited the growth of

all of the fungi tested. The antifungal metabolites of T.

(T.) massa (SMF11475), the presence of which has also

been described in the tropical species T. (T.) ignis

(Muricy et al. 1993), are worth further investigation.

Moderate, but still broad, antifungal activities were also

seen for the extracts of Anoxycalyx (Scolymastra) joubini

(SMF11143), I. belli (SMF11209), and Mycale (Mycale)

tridens (SMF11218).

Table 7 Antifungal activities of the sponge extracts against ecologically relevant and selected opportunistic pathogenic fungi

Sponge species SMF no. EX

3935

EX

4023

EX

4015

EX

1551

EX

2481

EX

9382

EX

9370

EX

5721

EX

9424

EX

9454

EX

9467

Rossella sp. SMF11409 ±

Myxilla (Myxilla) mollis SMF11626 ± ± ± ±

Anoxycalyx (Scolymastra)

joubini

SMF11143 ± ± ± ? ± ± ± ±

Rossella sp. SMF11144 ±

Guitarra sigmatifera SMF11154 ± ± ±

Tedania (Tedaniopsis)

massa

SMF11475 ?? ? ?? ? ?? ?? ?? ? ± ? ??

Kirkpatrickia variolosa SMF11202 ? ±

Inflatella belli SMF11209 ± ? ± ± ±

Mycale (Mycale) tridens SMF11218 ± ± ± ?? ? ± ?

Rossella cf. vanhoeffeni SMF11228 ?

The following fungal strains from subglacial Arctic ice or from Arctic sea water were tested: Rhodosporidium lusitanie (EX—3935),

Debaryomyces hansenii (EX—4023), Rhodotorula mucilaginosa (EX—4015), Cryptococcus carnescens (EX—1551), and Aureobasidum sub-

glaciale (EX—2481). The opportunistic pathogenic strains were: Candida albicans (EX—9382) from kitchen dish strainer, Candida parapsilosis

(EX—9370) from dishwasher, Exophiala dermatitidis (EX 5721) from dishwasher, Fusarium dimerum (EX—9424) from dishwasher, Aure-

obasidum melanogenum (EX—9454) from potable water, A. melanogenum (EX—9467) from rubber on kitchen drain. The antifungal activity is

expressed as follows: ±, 0–25 % growth inhibition as compared to the control, ?, 26–50 % growth inhibition as compared to the control, ??,

51–75 % growth inhibition as compared to the control, ???, 76–100 % growth inhibition as compared to the control. Empty spaces in columns

denote that the tested sponge extract did not exhibit any antifungal activity. Final concentrations of ethanolic sponge extracts in the test were

100 lg dried extract/mL

Polar Biol (2016) 39:947–959 957

123

Conclusions

The data from the present study broaden our knowledge of

the biological activities of natural products associated with

marine sponges from polar regions. Considering the high

diversity and abundance of marine sponges in the Southern

Ocean (Janussen and Downey 2014), the potential of

Antarctic sponges for the production of bioactive com-

pounds can be considered as particularly high. Our data

show that Antarctic sponges can provide valuable resources

for new pharmaceutical lead compounds.

Acknowledgments The authors gratefully acknowledge the Slove-

nian Research Agency (Research Programmes P1-0207, P4-0127, P1-

0055, and P1-0198), the ERASMUS Student Mobility Programme for

financial support to MK, LS and ML, and Deutsche Forschungsge-

meinschaft for financial support for the Antarctic sponge research

project by DJ (JA-1063/17-1). We acknowledge the financial support

received from the Ministry of Education, Science and Sport and the

University of Ljubljana via the ‘‘Innovative scheme for co-financing

of doctoral studies,’’ the Slovenian Research Agency through the

Infrastructural Centre Mycosmo, MRIC UL, and the Centre of

Excellence for Integrated Approaches in Chemistry and Biology of

Proteins (CIPKeBiP). Dr. Chris Berrie is greatly acknowledged for

editing and appraisal of the manuscript.

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