Toxigenic effects of sponges and benthic diatoms on marine invertebrates: possible biotechnological applications

doi:10.21203/rs.3.rs-3916716/v1

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Toxigenic effects of sponges and benthic diatoms on marine invertebrates: possible biotechnological applications

https://doi.org/10.21203/rs.3.rs-3916716/v1

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published 25 Oct, 2024

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Secondary metabolites play important physiological roles being bioactive as defences against other organisms, or attractive signals used for various purposes, including reproduction. Their production and the emission in the environment may be viewed as an adaptive feature subjected to evolutionary selection. They were demonstrated to be useful for applications in various biotechnological fields, such as pharmaceutical, nutraceutical and cosmeceutical. Sponges and microalgae, including diatoms, are the most promising sources of bioactive compounds from the sea. We aimed at detecting the ecotoxicological effects of crude extracts and fractions obtained from three marine sponges, Geodia cydonium, Haliclona (Halichoclona) vansoesti and Agelas oroides and two benthic diatoms, Nanofrustulum shiloi and Cylindrotheca closterium on model marine organisms. We tested their effects on the Mediterranean purple sea urchin, Paracentrotus lividus, and on two diatoms, Phaeodactylum tricornutum and Cylindrotheca closterium, chosen because they are considered standard indicators for assessment of ecological impacts. Our results showed that extracts and fractions from both sponges and diatoms may be harmful for model invertebrates. However, eggs appeared “protected” from sponge allelochemicals when still unfertilized. The majority of sponge fractions exhibited noticeable impacts during the post-fertilization treatments. In contrast, fractions from diatoms notably increased the rate of malformations compared to the control, both in pre- and post-fertilization treatments.

Biological sciences/Biotechnology

Biological sciences/Ecology

Researches in the field of drug discovery are leading to the characterization of compounds from several marine organisms^[1;2]. In fact, their secondary metabolites exhibited various physiologic roles, and demonstrated an allelopathic activity when involved in defence and predation. Some of them have been applied to biotechnologies as antifouling and antimicrobial substances^[3]. They may be, as well, involved in spawning and in symbiotic relationship and, in this case, they may be applied to medical and nutraceutical biotechnologies. The life competition, as well as various environmental pressures, pushed towards a wide chemical biodiversity during the evolution, that characterizes all marine environments, and it has no counterpart in the terrestrial environments. Among marine organisms, microalgae (mainly diatoms) and sponges represent the most challenging sources of bioactive compounds for biotechnological applications in pharmacological, nutraceutical and cosmeceutical fields^[4;5]. In contrast, insufficient data are available on the actual effects of secondary metabolites derived from diatoms and sponges on marine model organisms. The pioneer investigation on the toxicity of chemical extract from marine sponges on marine invertebrates reported by Cariello et al.^[6] showed that compounds isolated from an ethanolic extract of the sponge Dysidea avara were toxic for the egg development of the sea urchin Sphaerechinus granularis, causing delayed development and block of the cell division. Three main compounds were indicated to be responsible for this activity, viz the Avarol (sesquiterpenoid hydroquinone) and other two chemically correlated to avarol compounds obtained with butanol extraction, named DA and DB^[6]. The extracts from the sponges Rossella fibulata, Rossella sp. and Isodictya verrucosa displayed toxic effect on the embryos of Sterechinus neumayeri at low concentrations (1 mg/mL and 0.05 mg/mL, respectively)^[7]. The highest concentration triggered block of embryo development prior to reach the blastula stage. In the same study, extracts from Iophon sp. and Mycale acerate demonstrated toxigenic activity on the sea urchin sperm at low concentrations (1, 0.5 and 0.05 mg/mL, respectively), causing inhibition of the sperm mobility. Another study^[8] demonstrated that two compounds, Mycalosides A and G, extracted from the marine sponge Mycale laxissima, inhibited the fertilization of the sea urchin Strongylocentrotus nudus eggs, acting as spermostatics.

Remarkably, sponges host a number of microorganisms responsible for the synthesis of bioactive compounds. Regueiras et al.^[9] tested aqueous and organic extracts from twelve cyanobacteria associated to several sponges from Portugal on P. lividus embryos. The most active organic extract derived from a cyanobacterial strain ascribed to Chroococcales (6MA13ti), associated to the sponge Tedania ignis. Sea urchin embryos exposed to this organic extract exhibited complete arrest of development, and were unable to reach the pluteus stage. Similarly, aqueous extracts from Synechoccales cyanobacteria (LEGE11384) and Phormidium spp. (25J1tp) isolated from Polymastia sp. and Tedania pilarriosae, respectively, induced a reduced number of embryos reaching the pluteus stage^[9].

The effect of sponge extracts on algae has been less explored. In 2002 Tsoukatou et al.^[10] demonstrated that extracts from three sponges belonging to the genus Ircinia inhibited the growth of several diatoms (Amphora coffeaformis, P. tricornutum and Cylindrotheca closterium). More recently, the extract of Ircinia oros was demonstrated to inhibit the growth of the diatom P. tricornutuum^[11]. In addition, sponge-derived polybrominated diphenyl ether (3,5-dibromo-2-(2’,4’-dibromophenoxy)-phenol A) exhibited antifouling activity on the diatom A coffeaeformis ^[12]. Other three compounds extracted from the marine sponge Semitaspongia bactriana (i.e., 7E,12E,20Z-variabilin, cavernosolide, lintenolide A) showed efficient antifouling properties towards the diatom Nitzschia closterium^[13]. The anti-fouling activity vs. a Chlorella sp. species can be due to compounds produced by micro-organisms associated to sponges, as in the case of the strain SS05 of Bacillus cereus, associated to the sponge Sigmadocia sp. ^[14]. Another extract from sponge-associated bacteria (Bacillus pumilus) inhibited the growth of N. closterium ^[15] .

In parallel, diatom-derived extracts were demonstrated to influence the physiology of sea urchin embryos. The incubation of embryos of the sea urchin P. lividus with crude extract of the diatom Thalassiosira rotula led to a disorganization of tubulin and impairment of the mitotic spindle^[16]. The end-products of the lipoxygenase/hydroperoxide lyase metabolic pathway of planktonic diatoms (primed by wounding of cells, as done by grazers) caused malformations and cell cycle arrest on embryos of the sea urchin P. lividus. These compounds, mainly represented by Polyunsaturated Fatty Acids (PUFAs)^[17], Polyunsaturated Aldehydes (PUAs)^[18] and hydroxyacids^[19] are grazing deterrents. Gudimova et al.^[20] demonstrated that even the simple exposure of embryos of Strongylocentrotus droebranchiensis and Echinus acutus to intact cells of various diatoms arrested embryonic development. Skeletonema marinoi resulted to be the most effective, priming acute mortality in S. droebachiensis embryos after four hours, as well as Thalassiosira gravida, which caused acute mortality after 24 hours of exposure.

Taking into account these data, we aimed at detecting the ecotoxicological effects of total extracts and fractions (according to Cutignano et al.^[21] and Nuzzo et al.^[22]) of three marine sponges, G. cydonium, H. (H.) vansoesti and A. oroides and two benthic diatoms N. shiloi and C. closterium, on marine model organisms. In particular, they were tested on the Mediterranean sea urchin P. lividus, extensively used for ecotoxicological studies in response to natural and anthropogenic toxins, because of its easy manipulation in laboratory ^[23;24]. Two diatom species were also adopted as targets for sponge and diatom metabolites: i.e. P. tricornutum, a well-established and standardized bioindicator, widely recognized for its sensitivity to environmental stressors and commonly employed in ecotoxicological assessments; ii. C. closterium, a cosmopolitan diatom quite common in the Mediterranean Sea, in order to study local strains in their native environments.

2.1. Sea urchin bioassay

Our experimental approaches on sea urchins, with exposition of eggs to extracts before or after the fertilization, produced contrasting results. In fact, when eggs were exposed after fertilization to extracts and fractions obtained from the sponges G. cydonium and H. (H.) vansoesti, the first mitotic division of the fertilized eggs was blocked at all the tested concentrations (see Supplementary Tables S1 and S2), and several delayed embryos were detected still at the gastrula stage, with evident apoptotic signals, very similar to those reported by Ruocco et al.^[25](Supplementary figures S1-S2). Moreover, several embryos reaching the pluteus stage showed morphological malformations, mainly consisting in alterations of arms, spicules and apices, as reported in Varrella et al.^[18]. The impact was proportional to the tested concentrations, becoming more significant at the highest concentration.

Different results were obtained when embryos were exposed to extracts and fractions from the sponge A. oroides. The most active one was AORO 2D that both at the concentrations C3 (0.500 mg/L) and C2 (0.250 mg/L) triggered embryo malformations (75% and 70%, respectively), whereas at the lowest concentration C1 (0.125 mg/L), it showed antimitotic activity, leading to a high percentage (73%) of fertilized eggs not entering the first mitotic division (Fig. 1). In the fractions AORO 2B and 2C tested at concentrations C3 and C2 (0.5 and 0.250 mg/L, respectively), the malformed plutei in the fraction 2B were 42% and 43% (respectively at C3 and C2), whereas the percentage of the malformed plutei in the fraction AORO 2C were and 51% and 44% of the total number of embryos for the highest (C3) and intermediate concentration (C2), respectively. Also in this case, the effect of the extract and its fractions were dose-dependent and more evident at the highest concentration (Supplementary Table S3). When eggs were exposed to the extract and the fractions before fertilization (Supplementary figures S3-S4 and Supplementary tables S4-S5-S6) no effects were detected, with the only exception of the fraction AORO 2C, which induced embryo malformations similar to the ones observed by Varrella et al.^[18], mainly affecting arms, spicules and apices. The percentages of malformed plutei prompted by this fraction were 64%, 40% and 23% at the highest (C3), medium (C2) and lower (C1) concentrations, respectively (Fig. 2).

The total extract and the fraction NSHII 2D obtained from N. shiloi, induced a significant percentage of malformed plutei in the pre-fertilization treatment accounting for 51% at the higher concentration (C3) and 43% both at the medium (C2) and the low concentration (C1). (Fig. 3 and Supplementary Table S7). This fraction caused malformation in the plutei, also when embryos were exposed after fertilization, accounting for 44% at C3, 40% at C2 and 41% at C1, respectively (Supplementary Figure S5 and Supplementary Table S8). In contrast, no effects were prompted by C. closterium extract and fractions, with the only exception of the fraction 2D (Fig. 4 and Supplementary figure S6), showing low percentage of malformed plutei in the pre-fertilization treatment, equal for all three concentrations (23%), similar to the ones obtained in the post-fertilization treatment (26%, 24% and 24% respectively at C3, C2 and C1). All percentages of malformations are reported in the Supplementary Tables S9-S10.

2.2 Algal growth bioassay

The tests on the diatoms P. tricornutum and C. closterium exposed to sponge extracts, specifically the fractions from H. (H.) vansoesti (HVAN), A. oroides (AORO) and G. cydonium (GCYD), yielded complex patterns of results. Short- term preliminary tests provided with a stimulatory effect as compared to the control group at the same three tested concentrations. Figure 5 showed the results obtained at the highest concentration, where short-term preliminary tests yielded negative values for three sponge extracts (AORO, HVAN, GCYD), suggesting a bio-stimulatory effect on both diatoms. Specifically, AORO and HVAN consistently exhibited a notable bio-stimulatory effect, with negative values indicating moderate or strong stimulation on both P. tricornutum and C. closterium. GCYD displayed a varied response at this concentration, with some concentrations showing a weaker biostimulatory effect. However, a block of the growth was prompted by both diatoms, upon a longer-term exposure.

The growth responses of the two diatoms exposed to diatom extracts (NSHI and CCLO) is shown in Figs. 6 and 7. P. tricornutum exhibited an exponential growth pattern when exposed to NSHI extracts, characterized by an initial lag phase and followed by an exponential increase in the cell density, particularly at the concentration C3. In contrast, C. closterium displayed a linear increase of the growth when exposed to diatom extracts, tough at a lower rate than P. tricornutum. When subjected to CCLO extracts, both species exhibited concentration-dependent growth increases, yet the growth patterns remained distinct.

3.1. Effects of sponge bioactive compounds

Contrasting results were obtained when P. lividus eggs were treated with extracts and fractions of sponges before and after fertilization. Only in the case of exposure after fertilization all sponge extracts affected the first mitotic division and caused death of gastrulae, whereas in both cases malformed plutei were present. It must be considered that sea urchin eggs have a different permeability to molecules before and after the fertilization, as it has been demonstrated in previous studies where a decrease in the electrical resistance of sea-urchin eggs following fertilization leaded to permeability increase to water and solutes^[26;27]. In this view, eggs could result “protected” from sponge allochemicals when still unfertilized.

The majority of sponge fractions exhibited noticeable impacts during the post-fertilization treatment. For example, all sponge subfraction 2B, which according the extraction protocol^[21] contained nucleosides, induced plutei malformation and inhibited the initial mitotic division. As shown in previous investigations, the intake of exogenous nucleosides in the sea urchins P. lividus and S. purpuratus increases after fertilization^[28;29]. Specifically, the fertilized eggs of P. lividus are capable of intake about 20 times more nucleosides just one hour post fertilization, as compared to their unfertilized counterparts, and these exogenous components are actively used in the embryonic metabolism^[28]. It was demonstrated that some nucleoside analogues have cytotoxic effect and are used as anticancer drugs, due to their effect as competitors of nucleotides and eventually, interaction with intracellular targets to induce cytotoxicity^[30;31].

Exposing embryos to fractions 2D resulted in similar effects when derived from G. cydonium and H. (H.) vansoesti, leading to an increase in malformed plutei and fertilized but undivided eggs. However, the most potent impact was observed when embryos were treated with the subfraction AORO 2D obtained from A. oroides. In fact, this fraction caused malformations (at C3 and C2) or block of the first mitotic division (at C1) of the fertilized eggs. These results demonstrated that the classes of compounds present in this fraction (mainly sterols and free fatty acids according Cutignano et al.^[21]) could penetrate in the embryos and interfere with the larval embryogenesis. As also demonstrated for diatoms, this class of compounds was toxic both for adults and their larval stage. Hence, it is likely that that sterols and free fatty acids deriving from sponges are as toxic as the ones present in diatoms. Embryos exposed to fractions 2E, which mainly includes triglycerides^[21], exhibited similar impacts, resulting in malformed plutei, apoptotic gastrulae and eggs that did not undergo the first mitotic division across all three tested concentrations in the post-fertilization treatment. Embryos treated with fraction 2C (containing mainly glycolipids and phospholipids, according to Cutignano et al.^[21]) showed similar effects in the post-fertilization assays regardless of the sponge from which this fraction was obtained. However, in the pre-fertilization treatment, the fraction 2C was more potent when derived from A. oroides, causing a higher percentage of malformed plutei than the control. It is worth-noting that fatty acids and sterols have been also shown to be important nutrients during larval development of several organisms, from nematodes to fish^[32], but their effects as allochemicals are scarcely investigated^[33]. The activity of these fractions of A. oroides is in agreement with the results obtained on cancer cell lines, showing a strong cytotoxic activity (unpublished data).

The long-term exposure of the model marine diatoms (P. tricornutum and C. closterium) to sponge extracts showed complete block of the growth. According to various studies^[4;34], sponges produce unique compounds retarding the formation of biofilms on their surfaces. Additionally, sponge extracts directly tested on diatoms were effective and limited the fouling adhesion^[10;35]. Nevertheless, these findings do not exclude the possibility that the same sponge extracts and fractions could be effective if tested on other diatoms.

3.2. Effects of diatoms as producers of bioactive compounds

P. lividus embryos and eggs treated with extracts from N. shiloi (NSHI) and C. closterium (CCLO) yielded similar results. Fraction 2D obtained from both diatoms and used both for pre- and post-fertilization treatments notably prompted an increase of malformations than the control. Nevertheless, this effect is still less potent than the one obtained with sponge extracts. The efficacy of fraction 2D was in line with the results of Ruocco et al.^[36], showing the effect on adult P. lividus fed on N. shiloi and C. closterium for one month. The study demonstrated a toxigenic impact on embryos obtained from eggs produced by sea urchin females fed on these benthic diatoms. Within the same study, a chemical examination indicated an exclusive production of polyunsaturated aldehydes by N. shiloi, while both diatoms exhibited notable production of various oxylipins, known for their cytotoxic effects on grazers and cancer cell lines^[36;37]. Moreover, sterols and fatty acids are contained in the fraction D according to the extraction protocol by Cutignano et al.^[21]. Our data are with previous findings showing the toxicity of diatom-derived secondary metabolites.

Apparently, extracts and fractions from diatoms seem to be natural occurring supplements because they triggered increased growth. A slight concentration-dependent stimulation was found within the replicates treated with the same extract. There was a difference in the pattern among the P. tricornutum and C. closterium growth enhancement pattern thus indicating specie-specific variations in growth dynamics response to the same natural extracts. Scarce information is available about the chance of diatoms extract used as growth supplements, although it could appear scarcely useful to culture diatoms to prime the growth of other microalgae. Anyway, diatoms were mainly studied as sustainable sources of nutritious compounds for humans^[38] and the effect of diatoms herein demonstrated might be simply ascribed to the addition of organic materials, which are composed by bacteria and produce nutrients for other microalgae. Diatom extracts and their purified compounds, however, find large exploitation in the bio-pharmacological and nutraceutical field^[5].

Moreover, P. tricornutum genome is well-characterized, and there is a rich toolbox of engineering tools available for straightforward gene manipulation of the algae^[39]. These findings may introduce research towards genetic modifications aimed at enhancing the production of specific compounds with various industrial applications. The integration of diatom extracts with genetic engineering methodologies holds the potential for sustainable and versatile solutions within the fields of biotechnology and bioengineering, further underscoring the remarkable promise of natural diatom-derived supplements.

4.1 Experimental design and sample collection

Our experimental design comprised: i. collection and culture of organisms; ii. extraction and fractionation of cultured biomasses of sponges and diatoms; iii. replicated tests of extracts and fractions produced on the survival rates and malformations of sea urchin eggs and embryos, as well as on the survival and growth of two target diatoms (see Fig. 8), compared to controls.

Sponge samples were collected as follows: G. cydonium in Secca delle Fumose, Parco Sommerso di Baia (40°49ʹN, 14°5ʹE) and A. oroides in Punta San Pancrazio (Ischia Island, 40°42ʹN, 13°57ʹE) in the Gulf of Napes; H. (H.) vansoesti in The Faro Lake (Sicily; 38^◦ 16′ 07′′ N, 15^◦38′ 13′′E) (see Bertolino et al.^[40]). The two benthic diatoms N. shiloi and C. closterium were previously isolated from the leaves of Posidonia oceanica and identified using both morphological and molecular approaches^[37].

4.2 Chemical extraction and solid-phase fractionation of methanol extracts

After lyophilization, 35 g of dried sponges and 2 g of dry diatoms were sonicated and extracted with MeOH (3x100 mL). The organic phase was decanted and dried under vacuum. The obtained extracts were weighed and 60 mg of sponges and 30 mg of diatoms were further fractioned using a vacuum manifold CHROMABOND® HR-X cartridges (6 mL/500 mg), according to the method reported in Cutignano et al.^[21] and Nuzzo et al.^[22] (Supplementary figure S7), to obtain 5 enriched fractions (A-E). Briefly, after a washing step with water to remove salts (fraction A), the organic fraction were eluted with CH3OH/H2O 50:50 (fraction B) to CH3CN/H2O 70:30 (fraction C), 100% CH3CN (fraction D) and, finally, CH2Cl2/CH3OH 90:10 (fraction E) (see Table 1 for extracts and fractions obtained for sponges and diatoms). All fractions were analysed by Thin Layer Chromatography (TLC) on KieselGel 60 F254 plates (Merck, Darmstadt, Germany) using developing solvent eluent and revealed by spraying with a Ce(SO4)2 acidic solution, followed by plate heating.

Table 1

Extracts and fractions obtained from the sponges and diatoms (and abbreviations) used for the ecotoxicological tests.
Sponge	Extract/Fraction	Abbreviation
Agelas oroides	Total extract	AORO EXT
	Fraction B	AORO 2B
	Fraction C	AORO 2C
	Fraction D	AORO 2D
	Fraction E	AORO 2E
Geodia cydonium	Total extract	GCYD EXT
	Fraction B	GCYD 2B
	Fraction C	GCYD 2C
	Fraction D	GCYD 2D
	Fraction E	GCYD 2E
Haliclona (Halichoclona) vansoesti	Total extract	HVAN EXT
	Fraction B	HVAN 2B
	Fraction C	HVAN 2C
	Fraction D	HVAN 2D
	Fraction E	HVAN 2E
Diatom
Nanofrustulum shiloi	Total extract	NSHII EXT
	Fraction B	NSHII 2B
	Fraction C	NSHII 2C
	Fraction D	NSHII 2D
	Fraction E	NSHII 2E
Cylindrotheca closterium	Total extract	CCLO EXT
	Fraction B	CCLO 2B
	Fraction C	CCLO 2C
	Fraction D	CCLO 2D
	Fraction E	CCLO 2E

4.2 Sea urchin exposure

Adult sea urchins were hand collected by scuba divers in the Gulf of Naples at a depth of about 10 meters. Collected individuals had a size between 4 and 6 cm (diameter of tests) and they were immediately stored in a cool-box and transported to the laboratory to be reared in aerated recirculating tanks for ten days. A chemical stimulation was performed to collect their gametes, by injecting 1 mL of 0.5 M KCl through their peribuccal membrane^[41]. The gamete donors were stored in aerated recirculating tanks immediately after the collection of sperms and eggs. Sperm was collected dry from males with a plastic pipette and kept undiluted at 5°C until the fertilization. Eggs were collected in glass dishes filled with filtered sea water (FSW) and then washed-up several times to remove faecal pellets and contaminants. Pools of 120 eggs were treated according two experimental procedures: i. eggs were incubated for 10 minutes with sponge or diatom extracts and fractions (obtained as described in paragraph 2.2; see also Table 1), at three different concentrations (C1 = 0.125 mg/L; C2 = 0.250 mg/L; C3 = 0.500 mg/L) and then fertilized; ii. eggs were fertilized and then incubated with the extracts and fractions (same concentrations as above). The embryos produced were incubated in a thermostatic chamber at 18°C with a 12/12 h light/dark cycle, and their development was monitored, from the fertilization to the first mitotic division, until forty-eight hours post-fertilization (hPF), normally corresponding to the pluteus stage. The embryos were fixed with 0.5% glutaraldehyde and morphological observations were performed to evaluate and record the percentage of normal plutei (N.P.), malformed plutei (M.P.), apoptotic gastrulae (A.G.) and fertilized eggs exhibiting no first mitotic division (N.D.).

4.3 Algal growth assays

Axenic cultures of marine diatoms, P. tricornutum and C. closterium, were cultivated in artificial seawater medium supplemented with nutrients (ISO 10253:2016, a method for the determination of the inhibition of growth of the unicellular marine algae Skeletonema sp. and P.tricornutum by substances and mixtures contained in sea water or by environmental water samples). Cultures were kept at a temperature of 22 ± 1°C under a light-dark cycle of 16:8 hours with a light intensity of 6,700 lux. To assess algal growth inhibition, a 72-hour test was conducted according to the guidelines of ISO 10253:2016 and ISO 8692:2012 (a method for the determination of the growth inhibition of unicellular green algae by substances and mixtures contained in water or by waste water). 24-well plates were used for the test. For each concentration, three wells were filled with 2,250 µL of each solution (using spiked sponge extract solutions in synthetic culture medium), 125 µL of a culture medium, and 125 µL of inoculum consisting of microalgae collected during the exponential growth phase (20 *10 ⁴ cell/mL). The prepared wells were then moved on a horizontal shaker, at a speed of 50 rpm, for 72 hours at 22 ± 1°C under a continuous light intensity of 6,700 lux. The solutions spiked with sponge extracts correspond to the three concentrations C1 = 0.125 mg/L, C2 = 0.250 mg/L and C3 = 0.500 mg/L. After 72-hour of exposure, spectrophotometric measurements at 670 nm of the samples were performed using a DR5000-SC UV-Vis Laboratory Spectrophotometer (ach Srl). These measurements yielded the cell density according to the Eq. (1):

(1) Y = 9 × 10⁶ x − 3466.5

Where, y is the optical density and x is the corresponding cell density.

The R² coefficient for this equation was 0.9802, indicating a strong correlation between optical density and cell density. The growth rates were compared to the control, following the guidelines of ISO 2016 and 2012. Additional algal growth inhibition tests were conducted with P. tricornutum, and C. closterium, extending the test duration to 7 days and modifying the standard protocol, in order to be conducted under static conditions. All of the aforementioned tests were conducted in triplicate, to ensure the accuracy of the results. The use of ISO guidelines ensured that the tests were standardized, to assure reliable and comparable results.

Acknowledgments

The axenic cultures of Phaeodactylum tricornutum and Cylindrotheca closterium at the Hygiene Laboratory of the Department of Biology of the University of Naples Federico II. SF received support through a Ph.D. fellowship co-funded by the Stazione Zoologica Anton Dohrn (Naples, Italy) and the University of Genoa. This work was partially funded by the National Biodiversity Future Centre (NBFC) Program, Italian Ministry of University and Research, PNRR, Missione 4 Componente 2 Investimento 1.4 (Project: CN00000033).

Author contributions

S.F. performed the ecotoxicological tests on the sea urchin, as well as on two diatoms, with A.S., M.S. M.G. R.E., N.R., G.N. A.C. performed chemical extractions. M.B. identified the sponges. M.G., M.P., M.C. and V.Z. were involved in the supervision of S.F. S.F., A.S. M.C. and V.Z. contributed in writing original draft of the manuscript. All the authors contributed in review and editing of the manuscript.

Data availability statement

All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interest statement

The authors declare no competing interests.

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No competing interests reported.

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Toxigenic effects of sponges and benthic diatoms on marine invertebrates: possible biotechnological applications

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Abstract

Figures

1. Introduction

2. Results

2.1. Sea urchin bioassay

2.2 Algal growth bioassay

3. Discussion

3.1. Effects of sponge bioactive compounds

3.2. Effects of diatoms as producers of bioactive compounds

4. Methods

4.1 Experimental design and sample collection

4.2 Chemical extraction and solid-phase fractionation of methanol extracts

4.2 Sea urchin exposure

4.3 Algal growth assays

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References

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Supplementary Files

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