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

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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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction 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 mobilit y. 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., 7 E ,12 E ,20 Z -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. Results 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. Discussion 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. Methods 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 × 10 6 x − 3466.5 Where, y is the optical density and x is the corresponding cell density. The R 2 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. Declarations 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. References Proksch, P. Defensive roles for secondary metabolites from marine sponges and sponge-feeding nudibranchs. Toxicon . 32(6) , 639-655 (1994). Malve, H. Exploring the ocean for new drug developments: Marine pharmacology. J. Pharm. Bioallied. Sci. 8(2) , 83 (2016). Protopapa, et al . of antifouling potential and ecotoxicity of secondary metabolites derived from red algae of the genus Laurencia . Mar. drugs . 17(11) , 646 (2019). Esposito, R. et al . 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Satheesh, S., Soniamby, A. R., Sunjaiy Shankar, C. V., Mary Josephine Punitha, S. Antifouling activities of marine bacteria associated with sponge ( Sigmadocia sp.). Ocean Univ. China . 11 , 354-360 (2012). Xin, X., et al . Potential antifouling compounds with antidiatom adhesion activities from the sponge-associated bacteria, Bacillus pumilus . J. Adhes. Sci. Technol. 31(9) , 1028-1043 (2017). Buttino, I., Miralto, A., Ianora, A., Romano, G., Poulet, S. A. Water-soluble extracts of the diatom Thalassiosira rotula induce aberrations in embryonic tubulin organisation of the sea urchin Paracentrotus lividus . Mar. Biol. 134 , 147-154 (1999). Romano, G., Miralto, A., Ianora, A. Teratogenic effects of diatom metabolites on sea urchin Paracentrotus lividus embryos. Mar. Drugs. 8(4) , 950-967 (2010). Varrella, S., et al. Molecular response to toxic diatom-derived aldehydes in the sea urchin Paracentrotus lividus . Mar. Drugs . 12(4), 2089-2113 (2014). Varrella, S., et al . First morphological and molecular evidence of the negative impact of diatom-derived hydroxyacids on the sea urchin Paracentrotus lividus . Toxicol. Sci. 151(2) , 419-433 (2016). Gudimova, E., Eilertsen, H. C., Jørgensen, T. Ø., Hansen, E. In vivo exposure to northern diatoms arrests sea urchin embryonic development. Toxicon. 109 , 63-69 (2016). Cutignano, A., et al . Development and application of a novel SPE-method for bioassay-guided fractionation of marine extracts. Mar. Drugs . 13(9) , 5736-5749 (2015). Nuzzo, G., Manzo, E., Gallo, C., d’Ippolito, G., Fontana, A. Fractionation Protocol of Marine Metabolites. Mar. Genomics . 307-313 (2022). Marrone, V., et al. Defensome against toxic diatom aldehydes in the sea urchin Paracentrotus lividus . PLoS One . 7(2) , e31750 (2012). Ruocco, N., et al. Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7. Aquat. Toxicol . 176 , 128-140 (2016). Ruocco N.; Costantini M.; Santella L. New insights into negative effects of lithium on sea urchin Paracentrotus lividus embryos. Sci. Rep. 6 , 32157 (2016d) Lyon, E. P., & Shackell, L. F. On the increased permeability of sea urchin eggs following fertilization. Science . 32(816) , 249-251 (1910). Adams, S. L., Kleinhans, F. W., Mladenov, P. V., Hessian, P. A. Membrane permeability characteristics and osmotic tolerance limits of sea urchin ( Evechinus chloroticus ) eggs. Cryobiology. 47(1) , 1-13 (2003). Nemer, M. Characteristics of the utilization of nucleosides by embryos of Paracentrotus lividus. J. Biol. Chem. 237(1), 143-149 (1962). Killian, C. E., Nishioka D. Ribonucleoside uptake and phosphorylation during fertilization and early development of the sea‐urchin, Strongylocentrotus purpuratus . European J. Mol. Biol. Biochem . 1 65(1), 91-98 (1987). Galmarini, C. M., Mackey, J. R., Dumontet, C. Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol. 3(7), 415-424 (2002). Galmarini, C. M., Popowycz, F., Joseph, B. Cytotoxic nucleoside analogues: different strategies to improve their clinical efficacy. Curr. Med. Chem. 15(11), 1072-1082 (2008). El Kertaoui, N., et al . Key nutritional factors and interactions during larval development of pikeperch ( Sander lucioperca ). Sci. Rep. 9(1), 7074 (2019). Rodríguez, J. F., Dynarowicz-Latka, P., Conde, J. M. How unsaturated fatty acids and plant stanols affect sterols plasma level and cellular membranes? Review on model studies involving the Langmuir monolayer technique. Chem. Phys. Lipids. 232 , 104968 (2020). Stowe, S. D., et al . Anti-biofilm compounds derived from marine sponges. Mar Drugs . 9(10), 2010-2035 (2011). Dobretsov, S., Dahms, H. U., & Qian, P. Y. Antibacterial and anti-diatom activity of Hong Kong sponges. Aquat. Microb. Ecol. 38(2), 191-201 (2005). Ruocco, N., et al . Toxigenic effects of two benthic diatoms upon grazing activity of the sea urchin: Morphological, metabolomic and de novo transcriptomic analysis. Sci. Rep . 8(1) , 5622 (2018). Sansone, C., et al . Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells. PLoS One. 9(7), e101220 (2014). Bhattacharya, T., et al . Nutraceuticals and bio-inspired materials from microalgae and their future perspectives. Curr Top Med Chem. 21(12), 1037-1051 (2021). Butler, T., Kapoore, R. V., Vaidyanathan, S. Phaeodactylum tricornutum : a diatom cell factory. Trends Biotechnol . 38(6) , 606-622 (2020). Bertolino, M., et al. First certain record of Demospongiae class (Porifera) alien species from the Mediterranean Sea. Mar. Genomics 63, 100951 (2022). Gharbi, M., et al. Scale-Up of an Aquaculture Plant for Reproduction and Conservation of the Sea Urchin Paracentrotus lividus: Development of Post-Larval Feeds. J. Mar. Sci. Eng. 11(1), 154 (2023). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 25 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Aug, 2024 Reviews received at journal 10 Aug, 2024 Reviewers agreed at journal 06 Aug, 2024 Reviews received at journal 25 Jul, 2024 Reviewers agreed at journal 16 Jul, 2024 Reviewers invited by journal 22 Feb, 2024 Editor assigned by journal 22 Feb, 2024 Editor invited by journal 21 Feb, 2024 Submission checks completed at journal 21 Feb, 2024 First submitted to journal 01 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Zupo","email":"","orcid":"","institution":"Stazione Zoologica Anton Dohrn, Ischia Marine Centre","correspondingAuthor":false,"prefix":"","firstName":"Valerio","middleName":"","lastName":"Zupo","suffix":""}],"badges":[],"createdAt":"2024-02-01 08:49:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3916716/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3916716/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-74100-5","type":"published","date":"2024-10-25T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51560340,"identity":"4e98efad-2007-4746-8f71-e08521ba4cc4","added_by":"auto","created_at":"2024-02-23 17:43:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":293474,"visible":true,"origin":"","legend":"\u003cp\u003ePost-fertilization exposure of sea urchin \u003cem\u003eP. lividus \u003c/em\u003eembryos to the total extracts (AORO EXT) and fractions (AORO 2B, AORO 2C, AORO 2D, AORO 2E) from \u003cem\u003eA. oroides\u003c/em\u003e at the three concentrations tested: C1 = 0.125 mg/L; C2 = 0.250 mg/L; C3 = 0.500 mg/L.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/fed7269870df601b9d4115b4.png"},{"id":51560339,"identity":"1538c160-d3e2-48b4-b50b-61edffc6b863","added_by":"auto","created_at":"2024-02-23 17:43:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":228308,"visible":true,"origin":"","legend":"\u003cp\u003ePre-fertilization experiments of sea urchin \u003cem\u003eP. lividus \u003c/em\u003eembryos to the total extract (AORO EXT) and fractions (AORO 2B, AORO 2C, AORO 2D, AORO 2E) from \u003cem\u003eA.oroides \u003c/em\u003eat the three concentrations: C1 = 0.125 mg/L; C2 = 0.250 mg/L; C3 = 0.500 mg/L.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/236e3885908a4faab996a588.png"},{"id":51560591,"identity":"2410630b-e5eb-4530-beaf-59243f5158dd","added_by":"auto","created_at":"2024-02-23 17:51:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259772,"visible":true,"origin":"","legend":"\u003cp\u003ePre-fertilization experiments of sea urchin \u003cem\u003eP. lividus\u003c/em\u003e embryos to the total extract (NSHII EXT) and fractions (NSHII 2B, NSHII 2C, NSHII 2D, NSHII 2E) from \u003cem\u003eN. shiloi\u003c/em\u003e at the three concentrations: C1 = 0.125 mg/L; C2 = 0.250 mg/L; C3 = 0.500 mg/L.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/6330c3afd4d89dcce9b5146c.png"},{"id":51560345,"identity":"a10b58c4-b368-4d8d-9d2a-acd2dc917a1c","added_by":"auto","created_at":"2024-02-23 17:43:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":255150,"visible":true,"origin":"","legend":"\u003cp\u003ePre-fertilization experiments of sea urchin \u003cem\u003eP. lividus \u003c/em\u003eembryos to the total extract (CCLO EXT) and fractions (CCLO 2B, CCLO 2C, CCLO 2D, CCLO 2E) from \u003cem\u003eC. closterium\u003c/em\u003e at the three concentrations: C1 = 0.125 mg/L; C2 = 0.250 mg/L; C3 = 0.500 mg/L.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/c1458104a8b974dee486281d.png"},{"id":51560344,"identity":"ec81f3fa-b194-4bef-806c-e958bf728819","added_by":"auto","created_at":"2024-02-23 17:43:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86352,"visible":true,"origin":"","legend":"\u003cp\u003eShort-term preliminary tests at the highest concentration (C3 = 0.500 mg/L) of sponge extracts AORO, HVAN, and GCYD on \u003cem\u003eP. tricornutum\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e. Negative values indicate a growth stimulation.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/c78fdd28f74015f7337b2ff9.png"},{"id":51560595,"identity":"8506f865-6849-4b8a-8e47-4ac3be11fe79","added_by":"auto","created_at":"2024-02-23 17:51:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119470,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of long-term exposure to the fractions from \u003cem\u003eN. shiloi\u003c/em\u003e at the three concentrations (C1 = 0.125 mg/L; C2 = 0.250 mg/L; C3 = 0.500 mg/L) on diatoms: A), B), C), D), and E) \u003cem\u003eP. tricornutum\u003c/em\u003e, and F), G), H), I), and J) \u003cem\u003eC. closterium.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/fd09b94ee35a8c4be538b741.png"},{"id":51560346,"identity":"3fb61b29-3ebb-4d5e-a7d6-1b0d58faa74c","added_by":"auto","created_at":"2024-02-23 17:43:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":118935,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of long-term exposure to the fractions from \u003cem\u003eC. closterium\u003c/em\u003e at the three concentrations (C1 = 0.125 mg/L; C2 = 0.250 mg/L; C3 = 0.500 mg/L) on diatoms: A), B), C), D), and E) \u003cem\u003eP. tricornutum\u003c/em\u003e, and F), G), H), I), and J) \u003cem\u003eC. closterium.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/f750b4df248c9497511bf031.png"},{"id":51560348,"identity":"c5550ff2-e334-4d1d-9932-ca0e6526e8b2","added_by":"auto","created_at":"2024-02-23 17:43:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":318972,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design showing the main steps of the experimental assay, consisting in collection and culture of samples, chemical extraction and fractionation, and test of the extracts / fractions on sea urchin eggs/embryos and on algal cultures.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/78dd5b292b68855331f79946.png"},{"id":67681622,"identity":"d59fb8a2-5fd1-4453-b3ab-c4f5923921dc","added_by":"auto","created_at":"2024-10-28 16:07:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2234093,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/40b6fda4-65e5-4a20-bd95-79e992cc7a36.pdf"},{"id":51560341,"identity":"0300f5cf-f9c0-405a-b35f-00bd9dd95b47","added_by":"auto","created_at":"2024-02-23 17:43:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1555025,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3916716/v1/1c4383bd68436494974bbff7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Toxigenic effects of sponges and benthic diatoms on marine invertebrates: possible biotechnological applications","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eResearches in the field of drug discovery are leading to the characterization of compounds from several marine organisms\u003csup\u003e[1;2]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[4;5]\u003c/sup\u003e. 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.\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e showed that compounds isolated from an ethanolic extract of the sponge \u003cem\u003eDysidea avara\u003c/em\u003e were toxic for the egg development of the sea urchin \u003cem\u003eSphaerechinus granularis\u003c/em\u003e, 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\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. The extracts from the sponges \u003cem\u003eRossella fibulata\u003c/em\u003e, \u003cem\u003eRossella\u003c/em\u003e sp. and \u003cem\u003eIsodictya verrucosa\u003c/em\u003e displayed toxic effect on the embryos of \u003cem\u003eSterechinus neumayeri\u003c/em\u003e at low concentrations (1 mg/mL and 0.05 mg/mL, respectively)\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The highest concentration triggered block of embryo development prior to reach the blastula stage. In the same study, extracts from \u003cem\u003eIophon\u003c/em\u003e sp. and \u003cem\u003eMycale acerate\u003c/em\u003e 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 mobilit\u003cb\u003ey.\u003c/b\u003e Another study\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e demonstrated that two compounds, Mycalosides A and G, extracted from the marine sponge \u003cem\u003eMycale laxissima\u003c/em\u003e, inhibited the fertilization of the sea urchin \u003cem\u003eStrongylocentrotus nudus\u003c/em\u003e eggs, acting as spermostatics.\u003c/p\u003e \u003cp\u003eRemarkably, sponges host a number of microorganisms responsible for the synthesis of bioactive compounds. Regueiras et al.\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e tested aqueous and organic extracts from twelve cyanobacteria associated to several sponges from Portugal on \u003cem\u003eP. lividus\u003c/em\u003e embryos. The most active organic extract derived from a cyanobacterial strain ascribed to \u003cem\u003eChroococcales\u003c/em\u003e (6MA13ti), associated to the sponge \u003cem\u003eTedania ignis\u003c/em\u003e. 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 \u003cem\u003eSynechoccales\u003c/em\u003e cyanobacteria (LEGE11384) and \u003cem\u003ePhormidium spp.\u003c/em\u003e (25J1tp) isolated from \u003cem\u003ePolymastia\u003c/em\u003e sp. and \u003cem\u003eTedania pilarriosae\u003c/em\u003e, respectively, induced a reduced number of embryos reaching the pluteus stage\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effect of sponge extracts on algae has been less explored. In 2002 Tsoukatou et al.\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e demonstrated that extracts from three sponges belonging to the genus \u003cem\u003eIrcinia\u003c/em\u003e inhibited the growth of several diatoms (\u003cem\u003eAmphora coffeaformis\u003c/em\u003e, \u003cem\u003eP. tricornutum\u003c/em\u003e and \u003cem\u003eCylindrotheca closterium)\u003c/em\u003e. More recently, the extract of \u003cem\u003eIrcinia oros\u003c/em\u003e was demonstrated to inhibit the growth of the diatom \u003cem\u003eP. tricornutuum\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In addition, sponge-derived polybrominated diphenyl ether (3,5-dibromo-2-(2\u0026rsquo;,4\u0026rsquo;-dibromophenoxy)-phenol A) exhibited antifouling activity on the diatom \u003cem\u003eA coffeaeformis\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Other three compounds extracted from the marine sponge \u003cem\u003eSemitaspongia bactriana\u003c/em\u003e (i.e., 7\u003cem\u003eE\u003c/em\u003e,12\u003cem\u003eE\u003c/em\u003e,20\u003cem\u003eZ\u003c/em\u003e-variabilin, cavernosolide, lintenolide A) showed efficient antifouling properties towards the diatom \u003cem\u003eNitzschia closterium\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The anti-fouling activity vs. a \u003cem\u003eChlorella\u003c/em\u003e sp. species can be due to compounds produced by micro-organisms associated to sponges, as in the case of the strain SS05 of \u003cem\u003eBacillus cereus\u003c/em\u003e, associated to the sponge \u003cem\u003eSigmadocia\u003c/em\u003e sp. \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Another extract from sponge-associated bacteria (\u003cem\u003eBacillus pumilus\u003c/em\u003e) inhibited the growth of \u003cem\u003eN. closterium\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eIn parallel, diatom-derived extracts were demonstrated to influence the physiology of sea urchin embryos. The incubation of embryos of the sea urchin \u003cem\u003eP. lividus\u003c/em\u003e with crude extract of the diatom \u003cem\u003eThalassiosira rotula\u003c/em\u003e led to a disorganization of tubulin and impairment of the mitotic spindle\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. 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 \u003cem\u003eP. lividus\u003c/em\u003e. These compounds, mainly represented by Polyunsaturated Fatty Acids (PUFAs)\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, Polyunsaturated Aldehydes (PUAs)\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e and hydroxyacids\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e are grazing deterrents. Gudimova et al.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e demonstrated that even the simple exposure of embryos of \u003cem\u003eStrongylocentrotus droebranchiensis\u003c/em\u003e and \u003cem\u003eEchinus acutus\u003c/em\u003e to intact cells of various diatoms arrested embryonic development. \u003cem\u003eSkeletonema marinoi\u003c/em\u003e resulted to be the most effective, priming acute mortality in \u003cem\u003eS. droebachiensis\u003c/em\u003e embryos after four hours, as well as \u003cem\u003eThalassiosira gravida\u003c/em\u003e, which caused acute mortality after 24 hours of exposure.\u003c/p\u003e \u003cp\u003eTaking into account these data, we aimed at detecting the ecotoxicological effects of total extracts and fractions (according to Cutignano et al.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e and Nuzzo et al.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e of three marine sponges, \u003cem\u003eG. cydonium, H.\u003c/em\u003e (\u003cem\u003eH.\u003c/em\u003e) \u003cem\u003evansoesti\u003c/em\u003e and \u003cem\u003eA. oroides\u003c/em\u003e and two benthic diatoms \u003cem\u003eN. shiloi\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e, on marine model organisms. In particular, they were tested on the Mediterranean sea urchin \u003cem\u003eP. lividus\u003c/em\u003e, extensively used for ecotoxicological studies in response to natural and anthropogenic toxins, because of its easy manipulation in laboratory \u003csup\u003e[23;24]\u003c/sup\u003e. Two diatom species were also adopted as targets for sponge and diatom metabolites: i.e. \u003cem\u003eP. tricornutum\u003c/em\u003e, a well-established and standardized bioindicator, widely recognized for its sensitivity to environmental stressors and commonly employed in ecotoxicological assessments; ii. \u003cem\u003eC. closterium\u003c/em\u003e, a cosmopolitan diatom quite common in the Mediterranean Sea, in order to study local strains in their native environments.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1. Sea urchin bioassay\u003c/h2\u003e\n\u003cp\u003eOur 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 \u003cem\u003eG. cydonium\u003c/em\u003e and \u003cem\u003eH.\u003c/em\u003e (\u003cem\u003eH.\u003c/em\u003e) \u003cem\u003evansoesti\u003c/em\u003e, the first mitotic division of the fertilized eggs was blocked at all the tested concentrations (see \u003cstrong\u003eSupplementary Tables S1 and S2\u003c/strong\u003e), and several delayed embryos were detected still at the gastrula stage, with evident apoptotic signals, very similar to those reported by Ruocco et al.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e(\u003cstrong\u003eSupplementary figures \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e-S2\u003c/strong\u003e). 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.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The impact was proportional to the tested concentrations, becoming more significant at the highest concentration.\u003c/p\u003e\n\u003cp\u003eDifferent results were obtained when embryos were exposed to extracts and fractions from the sponge \u003cem\u003eA. oroides.\u003c/em\u003e 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). 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 (\u003cstrong\u003eSupplementary Table S3\u003c/strong\u003e). When eggs were exposed to the extract and the fractions before fertilization (\u003cstrong\u003eSupplementary figures S3-S4\u003c/strong\u003e and \u003cstrong\u003eSupplementary tables S4-S5-S6)\u003c/strong\u003e 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.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe total extract and the fraction NSHII 2D obtained from \u003cem\u003eN. shiloi\u003c/em\u003e, 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cstrong\u003eSupplementary Table S7\u003c/strong\u003e). 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 (\u003cstrong\u003eSupplementary Figure S5\u003c/strong\u003e and \u003cstrong\u003eSupplementary Table S8\u003c/strong\u003e). In contrast, no effects were prompted by \u003cem\u003eC. closterium\u003c/em\u003e extract and fractions, with the only exception of the fraction 2D (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cstrong\u003eSupplementary figure S6\u003c/strong\u003e), 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 \u003cstrong\u003eSupplementary Tables S9-S10\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2 Algal growth bioassay\u003c/h2\u003e\n\u003cp\u003eThe tests on the diatoms \u003cem\u003eP. tricornutum\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e exposed to sponge extracts, specifically the fractions from \u003cem\u003eH.\u003c/em\u003e (\u003cem\u003eH.\u003c/em\u003e) \u003cem\u003evansoesti\u003c/em\u003e (HVAN), \u003cem\u003eA. oroides\u003c/em\u003e (AORO) and \u003cem\u003eG. cydonium\u003c/em\u003e (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\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e 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 \u003cem\u003eP. tricornutum\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e. 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.\u003c/p\u003e\n\u003cp\u003eThe growth responses of the two diatoms exposed to diatom extracts (NSHI and CCLO) is shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e. \u003cem\u003eP. tricornutum\u003c/em\u003e 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, \u003cem\u003eC. closterium\u003c/em\u003e displayed a linear increase of the growth when exposed to diatom extracts, tough at a lower rate than \u003cem\u003eP. tricornutum\u003c/em\u003e. When subjected to CCLO extracts, both species exhibited concentration-dependent growth increases, yet the growth patterns remained distinct.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effects of sponge bioactive compounds\u003c/h2\u003e \u003cp\u003eContrasting results were obtained when \u003cem\u003eP. lividus\u003c/em\u003e 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\u003csup\u003e[26;27]\u003c/sup\u003e. In this view, eggs could result \u0026ldquo;protected\u0026rdquo; from sponge allochemicals when still unfertilized.\u003c/p\u003e \u003cp\u003eThe majority of sponge fractions exhibited noticeable impacts during the post-fertilization treatment. For example, all sponge subfraction 2B, which according the extraction protocol\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e 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 \u003cem\u003eP. lividus\u003c/em\u003e and \u003cem\u003eS. purpuratus\u003c/em\u003e increases after fertilization\u003csup\u003e[28;29]\u003c/sup\u003e. Specifically, the fertilized eggs of \u003cem\u003eP. lividus\u003c/em\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[30;31]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExposing embryos to fractions 2D resulted in similar effects when derived from \u003cem\u003eG. cydonium\u003c/em\u003e and \u003cem\u003eH.\u003c/em\u003e (\u003cem\u003eH.\u003c/em\u003e) \u003cem\u003evansoesti\u003c/em\u003e, 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 \u003cem\u003eA. oroides\u003c/em\u003e. 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.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e) 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\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, 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.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e) 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 \u003cem\u003eA. oroides\u003c/em\u003e, 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\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, but their effects as allochemicals are scarcely investigated\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. The activity of these fractions of \u003cem\u003eA. oroides\u003c/em\u003e is in agreement with the results obtained on cancer cell lines, showing a strong cytotoxic activity (\u003cem\u003eunpublished data\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe long-term exposure of the model marine diatoms (\u003cem\u003eP. tricornutum\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e) to sponge extracts showed complete block of the growth. According to various studies\u003csup\u003e[4;34]\u003c/sup\u003e, 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\u003csup\u003e[10;35]\u003c/sup\u003e. Nevertheless, these findings do not exclude the possibility that the same sponge extracts and fractions could be effective if tested on other diatoms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Effects of diatoms as producers of bioactive compounds\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. lividus\u003c/em\u003e embryos and eggs treated with extracts from \u003cem\u003eN. shiloi\u003c/em\u003e (NSHI) and \u003cem\u003eC. closterium\u003c/em\u003e (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.\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, showing the effect on adult \u003cem\u003eP. lividus\u003c/em\u003e fed on \u003cem\u003eN. shiloi\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e 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 \u003cem\u003eN. shiloi\u003c/em\u003e, while both diatoms exhibited notable production of various oxylipins, known for their cytotoxic effects on grazers and cancer cell lines\u003csup\u003e[36;37]\u003c/sup\u003e. Moreover, sterols and fatty acids are contained in the fraction D according to the extraction protocol by Cutignano et al.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Our data are with previous findings showing the toxicity of diatom-derived secondary metabolites.\u003c/p\u003e \u003cp\u003eApparently, 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 \u003cem\u003eP. tricornutum\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoreover, \u003cem\u003eP. tricornutum\u003c/em\u003e genome is well-characterized, and there is a rich toolbox of engineering tools available for straightforward gene manipulation of the algae\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e4.1 Experimental design and sample collection\u003c/h2\u003e\n\u003cp\u003eOur 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e), compared to controls.\u003c/p\u003e\n\u003cp\u003eSponge samples were collected as follows: \u003cem\u003eG. cydonium\u003c/em\u003e in Secca delle Fumose, Parco Sommerso di Baia (40\u0026deg;49ʹN, 14\u0026deg;5ʹE) and \u003cem\u003eA. oroides\u003c/em\u003e in Punta San Pancrazio (Ischia Island, 40\u0026deg;42ʹN, 13\u0026deg;57ʹE) in the Gulf of Napes; \u003cem\u003eH.\u003c/em\u003e (\u003cem\u003eH\u003c/em\u003e.) \u003cem\u003evansoesti\u003c/em\u003e in The Faro Lake (Sicily; 38\u003csup\u003e◦\u003c/sup\u003e 16\u0026prime; 07\u0026prime;\u0026prime; N, 15\u003csup\u003e◦\u003c/sup\u003e38\u0026prime; 13\u0026prime;\u0026prime;E) (see Bertolino et al.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e). The two benthic diatoms \u003cem\u003eN. shiloi\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e were previously isolated from the leaves of \u003cem\u003ePosidonia oceanica\u003c/em\u003e and identified using both morphological and molecular approaches\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e4.2 Chemical extraction and solid-phase fractionation of methanol extracts\u003c/h2\u003e\n\u003cp\u003eAfter 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\u0026reg; HR-X cartridges (6 mL/500 mg), according to the method reported in Cutignano et al.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e and Nuzzo et al.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e (\u003cstrong\u003eSupplementary figure S7\u003c/strong\u003e), 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\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e 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.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eExtracts and fractions obtained from the sponges and diatoms (and abbreviations) used for the ecotoxicological tests.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSponge\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eExtract/Fraction\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAbbreviation\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eAgelas oroides\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal extract\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAORO EXT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAORO 2B\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAORO 2C\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAORO 2D\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAORO 2E\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eGeodia cydonium \u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal extract\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGCYD EXT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGCYD 2B\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGCYD 2C\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGCYD 2D\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGCYD 2E\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eHaliclona\u003c/em\u003e (\u003cem\u003eHalichoclona\u003c/em\u003e) \u003cem\u003evansoesti\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal extract\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHVAN EXT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHVAN 2B\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHVAN 2C\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHVAN 2D\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHVAN 2E\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDiatom\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eNanofrustulum shiloi\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal extract\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNSHII EXT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNSHII 2B\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNSHII 2C\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNSHII 2D\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNSHII 2E\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eCylindrotheca closterium\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal extract\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCLO EXT\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCLO 2B\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCLO 2C\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCLO 2D\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFraction E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCCLO 2E\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e4.2 Sea urchin exposure\u003c/h2\u003e\n\u003cp\u003eAdult 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\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. 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\u0026deg;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\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), at three different concentrations (C1\u0026thinsp;=\u0026thinsp;0.125 mg/L; C2\u0026thinsp;=\u0026thinsp;0.250 mg/L; C3\u0026thinsp;=\u0026thinsp;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\u0026deg;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.).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e4.3 Algal growth assays\u003c/h2\u003e\n\u003cp\u003eAxenic cultures of marine diatoms, \u003cem\u003eP. tricornutum\u003c/em\u003e and \u003cem\u003eC. closterium\u003c/em\u003e, 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 \u003cem\u003eSkeletonema\u003c/em\u003e sp. and \u003cem\u003eP.tricornutum\u003c/em\u003e by substances and mixtures contained in sea water or by environmental water samples). Cultures were kept at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;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 \u0026micro;L of each solution (using spiked sponge extract solutions in synthetic culture medium), 125 \u0026micro;L of a culture medium, and 125 \u0026micro;L of inoculum consisting of microalgae collected during the exponential growth phase (20 *10 \u003csup\u003e4\u003c/sup\u003e cell/mL). The prepared wells were then moved on a horizontal shaker, at a speed of 50 rpm, for 72 hours at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under a continuous light intensity of 6,700 lux. The solutions spiked with sponge extracts correspond to the three concentrations C1\u0026thinsp;=\u0026thinsp;0.125 mg/L, C2\u0026thinsp;=\u0026thinsp;0.250 mg/L and C3\u0026thinsp;=\u0026thinsp;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.\u0026nbsp;(1):\u003c/p\u003e\n\u003cp\u003e(1)\u003cem\u003e Y\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003ex\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;3466.5\u003c/p\u003e\n\u003cp\u003eWhere, \u003cem\u003ey\u003c/em\u003e is the optical density and \u003cem\u003ex\u003c/em\u003e is the corresponding cell density.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e 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 \u003cem\u003eP. tricornutum\u003c/em\u003e, and \u003cem\u003eC. closterium\u003c/em\u003e, 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.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe axenic cultures of \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e and \u003cem\u003eCylindrotheca closterium\u003c/em\u003e 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eProksch, P. Defensive roles for secondary metabolites from marine sponges and sponge-feeding nudibranchs. \u003cem\u003eToxicon\u003c/em\u003e. \u003cstrong\u003e32(6)\u003c/strong\u003e, 639-655 (1994).\u003c/li\u003e\n\u003cli\u003eMalve, H. Exploring the ocean for new drug developments: Marine pharmacology.\u003cem\u003e J. Pharm. Bioallied. 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Eng.\u003c/em\u003e \u003cstrong\u003e11(1),\u003c/strong\u003e 154 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3916716/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3916716/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSecondary 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, \u003cem\u003eGeodia cydonium\u003c/em\u003e, \u003cem\u003eHaliclona\u003c/em\u003e (\u003cem\u003eHalichoclona\u003c/em\u003e) \u003cem\u003evansoesti\u003c/em\u003e and \u003cem\u003eAgelas oroides\u003c/em\u003e and two benthic diatoms, \u003cem\u003eNanofrustulum shiloi\u003c/em\u003e and \u003cem\u003eCylindrotheca closterium\u003c/em\u003e on model marine organisms. We tested their effects on the Mediterranean purple sea urchin, \u003cem\u003eParacentrotus lividus\u003c/em\u003e, and on two diatoms, \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e and \u003cem\u003eCylindrotheca closterium\u003c/em\u003e, 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 \u0026ldquo;protected\u0026rdquo; 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.\u003c/p\u003e","manuscriptTitle":"Toxigenic effects of sponges and benthic diatoms on marine invertebrates: possible biotechnological applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-23 17:43:21","doi":"10.21203/rs.3.rs-3916716/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-12T09:31:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-10T09:06:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226447265412907196433863351514578592873","date":"2024-08-06T15:35:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-25T17:47:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83356530368298189202902061646811555644","date":"2024-07-16T18:05:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-22T10:00:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-22T09:56:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-21T16:00:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-21T12:45:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-01T08:46:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b4e37c6-6cb8-4cdc-ab42-757e9aa578e6","owner":[],"postedDate":"February 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28924088,"name":"Biological sciences/Biotechnology"},{"id":28924089,"name":"Biological sciences/Ecology"}],"tags":[],"updatedAt":"2024-10-28T15:58:52+00:00","versionOfRecord":{"articleIdentity":"rs-3916716","link":"https://doi.org/10.1038/s41598-024-74100-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-25 15:56:58","publishedOnDateReadable":"October 25th, 2024"},"versionCreatedAt":"2024-02-23 17:43:21","video":"","vorDoi":"10.1038/s41598-024-74100-5","vorDoiUrl":"https://doi.org/10.1038/s41598-024-74100-5","workflowStages":[]},"version":"v1","identity":"rs-3916716","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3916716","identity":"rs-3916716","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}