Zinc speciation promotes distinct effects on dinoflagellate growth and coral trypsin-like enzyme activity

doi:10.21203/rs.3.rs-4836952/v1

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Zinc speciation promotes distinct effects on dinoflagellate growth and coral trypsin-like enzyme activity

https://doi.org/10.21203/rs.3.rs-4836952/v1

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published 15 Jan, 2025

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Zinc is an essential metal to living organisms, including corals and their symbiotic microalgae (Symbiodiniaceae). Both Zn(II) deprivation and overload are capable of leading to dysfunctional metabolism, coral bleaching, and even organism death. The present work investigated the effects of chemically defined Zn species (free Zn, ZnO nanoparticles, and the complexes Zn-histidinate and Zn-EDTA) over the growth of the dinoflagellates Symbiodinium microadriaticum, Breviolum minutum, and Effrenium voratum, and on the trypsin-like proteolytic activity of the hydrocoral Millepora alcicornis. B. minutum was the most sensitive strain to any form of added Zn. For the other strains, the complex [Zn(His)₂] better translated metal load into growth. This complex was the only tested compound that did not interfere with the trypsin-like activity of Millepora alcicornis extracts. Also, histidine was able to recover the activity of the enzyme inhibited by zinc. [Zn(His)₂] is a potential biocarrier of zinc for microalgae or coral cultivation. These findings suggest that the control of chemical speciation of an essential metal could lead to useful compounds that assist autotrophy, while not affecting heterotrophy, in the coral holobiont.

zinc

Symbiodiniaceae

histidine

sunscreen

Millepora

In the biological space, zinc is a very important trace nutrient, with both structural (e.g., zinc fingers, superoxide dismutase 1) and regulatory (e.g., carbonic anhydrase) roles in crucial processes such as DNA transcription, antioxidant defense, photosynthesis, and carbon dioxide fixation (Fraústo da Silva & Williams, 2001). In the marine environment with oligotrophic waters, metals such as Zn may be absorbed preferentially by dinoflagellate symbionts of corals in order to sustain growth and photosynthetic ability (Reich et al., 2020; Rodriguez et al., 2016). Being present in seawater at the 1.0–2.0 µg/L range (Neff, 2002), it is conceivable that Zn supplements could be beneficial to promote growth of symbiotic microalgae (Symbiodiniaceae) in aquaria or in nurseries for coral recovery, with positive outcomes for industries such as aquarism, fisheries and tourism (Kropat et al., 2011; Nguyen-Deroche et al., 2012).

Coral reefs are currently a threatened environment, most notably by the anthropogenic emission of carbon dioxide (de Barros Marangoni et al., 2017; Luz et al., 2018). Chemical contamination is another potential threat to coral health, which includes the runoff of sunscreens worn by bathers on beaches, as around 14,000 tons of sunscreen are estimated to be released in coral reef areas annually (Downs et al., 2016). The UV filters used in sunscreen formulations may be organic or inorganic. Zinc oxide (ZnO) has been applied in cosmetic formulations since 1990, as it has a broad range of protection against both UVA and UVB (Dransfield, 2000; Ma & Yoo, 2021; Mancebo et al., 2014; Mancuso et al., 2017). Currently, ZnO nanoparticles (ZnO-NPs) are the most used form of this ingredient in sunscreens (Adler & DeLeo, 2020; Bocca et al., 2018; Lu et al., 2015). The FDA and the European Commission both allow up to 25% of ZnO in sunscreens (Ma & Yoo, 2021), and according to the FDA, only zinc oxide and titanium dioxide are generally recognized as safe and effective among all the allowed UV filters (Adler & DeLeo, 2020; Narla & Lim, 2020), regardless of the classification of ZnO as hazardous to the aquatic life (ECHA, 2022; Moeller et al., 2021). Coral reefs are exposed to ZnO-NPs (Osmond & McCall, 2010) and to all their transformation products in marine environments, such as labile “free” zinc and organic complexes (Baysal et al., 2019, 2020; Esteban-Tejeda et al., 2017; Gelabert et al., 2014; Lai et al., 2020; L. Li et al., 2020; M. Li et al., 2011; Miao et al., 2010; Noventa et al., 2018; Reed et al., 2012). Some organic filters, such as oxybenzone, have been banned from certain countries due to their toxicity towards corals; nonetheless, no bans regarding inorganic filters exist (Miller et al., 2021).

The coral holobiont may nurture itself either autotrophically (photosynthesis) or heterotrophically by the capture and digestion of prey (Apprill, 2020; Titlyanov & Titlyanova, 2020). Corals under stress are susceptible to bleaching, a process in which the host expels the microalgae symbiont, leading to a reduction in photosynthesis and loss of pigments (Rodriguez et al., 2016). Even though the heterotrophic pathway might provide nutrients for the bleached coral, it may not suffice for long term survival (Ferrier-Pagès et al., 2018). Furthermore, the dysregulation of the microbiota of the coral can weaken immunity and increase the possibility of infections that may facilitate the bleaching process (Garren & Azam, 2012; Krediet et al., 2013; Nguyen-Kim et al., 2015; Rosenberg & Kushmaro, 2011).

Both zinc deprivation and zinc overload are deleterious to corals. Therefore, considerable attention has been given to the effects of Zn species on microalgae (growth promotion or inhibition, effects on photosynthetic proteins, metal accumulation in intracellular media, and EC/IC50 (Aruoja et al., 2009; Franklin et al., 2007; Goh & Chou, 1997; Hardefeldt & Reichelt-Brushett, 2015; Kuzminov et al., 2013; R. J. Miller et al., 2010; Spisni et al., 2016)) and cnidarians (fertilization success, zinc uptake, EC/LC50, photochemical response, symbiont density, bleaching, prokaryotic/viral abundance, and the activity of some antioxidant enzymes (Brock & Bielmyer, 2013; Corinaldesi et al., 2018; Deschaseaux et al., 2018; Duckworth et al., 2017; Fel et al., 2019; Howe et al., 2014b, 2014a; Reichelt-Brushett & Harrison, 1999, 2005; Tang et al., 2017)). Since emissions of zinc and ZnO-NPs possibly induce bleaching (Freitas Neto & Espósito, 2023), continued research is necessary, especially considering environmentally relevant scenarios and the chemical changes these nanoparticles may undergo in seawater (Freitas Neto & Espósito, 2023; Miller et al., 2021; Moeller et al., 2021).

In a context of potential new contaminants in seawater, such as zinc compounds in cosmetic formulations, it is important to have accessible and reliable parameters to screen environmental toxicity. Thus, in this study, we investigated the varying effects of chemically defined zinc species (free Zn(II), ZnO-NPs, zinc edetate and zinc histidinate) on the growth of three species of Symbiodiniaceae (Symbiodinium microadriaticum, Breviolum minutum, and Effrenium voratum) and on the trypsin-like proteolytic activity of Millepora alcicornis. These approaches were proposed as a means of gaining insight on the effects of zinc over both autotrophic and heterotrophic coral nutrition (Hernández-Elizárraga et al., 2022; Sebé-Pedrós et al., 2018). Millepora are the preeminent reef builders in Brazil, widely distributed (Amaral et al., 2008). Most common symbionts of Brazilian M. alcicornis are the genera Breviolum¸ followed by Symbiodinium (Garrido et al., 2022). This bioinorganic investigation aims to contribute basic knowledge on how the chemical speciation of zinc can modulate its toxicity to these important environmental targets.

Organisms

i. Microalgae

The species under study were Symbiodinium microadriaticum (BMAK215), Breviolum minutum (BMAK213) and Effrenium voratum (BMAK212) from the Aidar and Kutner Bank of Microrganisms (BMAK) of the Oceanographic Institute of University of São Paulo. Their ITS2 was checked by gDNA extraction, PCR amplification, and sequencing and correspondence to the A1, B1 and E1 ITS2 (Mies et al., 2018). The clades were from the Buffalo Undersea Reef Research Collection (Buffalo University) and have been kept in BMAK since 2013. The strains codes at Buffalo Collection are, respectively, a194-CassKb8, B184-Ap04, and E202Rt-383.

Innocula (1 mL) were transferred to Erlenmeyer flasks containing 100 mL of Guillard f/2 culture medium (Guillard, 1975) without silicate (prepared with autoclaved seawater with salinity adjusted to 35 and pH adjusted to 8.0). Cultures were incubated at 20 ºC under a 12:12h photoperiod with daylight type lamps (100 µE m^− 2 s^− 1). Growth was monitored by sampling the cultures and determining cell density in Nageotte chambers under a Leika microscope. Upon reaching exponential growth phase, cultures were transferred to a new culture medium without silicate nor zinc (f/2 – Zn), in 1 L flasks, to prepare the cultures to be used in the tests. Growth was again monitored until reaching exponential growth phase. The exclusion of zinc at this stage allowed us to control to which zinc species the microalgae would be subjected, for the duration of the experiment.

ii. Coral

The source of trypsin-like enzymes were fragments from five colonies of Millepora alcicornis (Cnidaria, Hydrozoa; ~25 cm in diameter) collected in Porto de Galinhas (Ipojuca, Pernambuco, Brazil; -8.507, -35.000) under license 53583 (SISBIO/ICMBio). The fragments were kept under ice until arrival at the Laboratory of Enzimology (LABENZ, UFPE, Recife, Pernambuco, Brazil), where they were kept at -20^oC. The coral extract was obtained by the simultaneous mechanical grind and ultrasonication of 30 g of the thawed fragments in 60 mL of filtered, autoclaved seawater under an ice bath for 30 min. The extract was then centrifuged (10,000 × g/20 min/4 ºC) and the protein content of the supernatant was determined by the Bradford test.

b. Zinc compounds

Zinc compounds were synthesized by published methods: zinc histidinate ([Zn(His)₂]) (Ghasemi et al., 2013) and zinc edetate ([ZnEDTA]) (Crespi et al., 1993; Kula, 1965; G. H. Reed & Kula, 1971; Reichle et al., 1975; Top & Çetinkaya, 2015). The structures were confirmed by FTIR spectroscopy and elemental analysis (CHN). Zinc oxide nanoparticles (ZnO-NPs) were from Sigma-Aldrich (nanopowder, < 100 nm; catalogue # 544906).

c. Microalgae exposure to zinc

Aliquots of 6 mL from the mother culture (~ 30,000 cells) were diluted to 20 mL with f/2 – Zn in stoppered borosilicate flasks. Tests were run in triplicates. The control treatment had 7.7·10^− 2 µM Zn(II). Samples from each clade were submitted to ZnO-NPs, ZnSO₄.7H₂O, [ZnEDTA] or [Zn(His)₂] at either 0.09 mg Zn L^− 1 (1.4 µM Zn) or 50 mg Zn L^− 1 (760 µM Zn), which correspond to the upper limit allowed for zinc in seawater (CONAMA, 2005) or a situation of extreme zinc contamination (El-Sorogy et al., 2012), respectively. Cultures were incubated at 20 ºC under a 12:12h photoperiod with daylight type lamps (320 µE m^− 2 s^− 1). 500 µL aliquots from each test replicate were sampled at 0, 24, 48, 120, 216, 360 and 480 h after exposition, fixed with 10 µL of glutaraldehyde 25%, vortexed and cooled. Cell density was determined in an Attune® NxT Acoustic Focusing Cytometer (ThermoFischer Scientific). Results were expressed as normalized cell density in relation to the initial density (ratio between measured cellular density and the cellular density of initial day – 1500 cells·mL^− 1). One-way ANOVA was performed to compare the values obtained within each sampling time.

For determining intracellular zinc contents at 480 h, the contents of the same replicates were pooled and centrifuged at 3,000 rpm for 5 min. The pellets were washed 3 times with 5 mL of a buffer (HEPES 20 mM, EDTA 1 mM, salinity 35 and pH 7.42) designed to remove non-adsorbed metal ions. The pellets were dried in an oven (60 ºC/12h) and their dry biomass was determined. After that, 300 µL of bidistilled HNO₃ was added to each vial at 80 ºC for 30 minutes, followed by the addition of 150 µL of H₂O₂ 32% (Romero et al., 2022). The final volume of each digestion solution was brought to 5 mL with milli-Q water and zinc was quantified by ICP-OES.

Millepora alcicornis trypsin-like enzyme inhibition and recovery of activity

Trypsin-like activity was assessed in 96-well microplates by incubation of quadruplicate samples of coral extract (20 µL), 80 µL of NaCl 0.9%, 50 µL 0.1 M Tris-HCl pH 8.0, and 20 µL of the inhibitor (benzamidine or zinc compound). After 30 min at room temperature, 30 µL of the trypsin specific substrate benzoyl-_DL-arginine p-nitroanilide (BApNA) (8 mM in DMSO) was added. The change in absorbance at 405 nm (Ɛ = 9100 M^− 1cm^− 1) was recorded after 24 h in a microtitre reader (Bio-Rad xMark™). Blanks replacing BApNA with DMSO or seawater were run in parallel. Results are expressed in percentage relative to control (Bezerra et al., 2005). The enzyme activity of the extract (blank) was determined as 0.11 mU/mg. Results were compared with one-way ANOVA, considering control activity as 100%.

For the recovery of enzyme activity assays, the above procedure was repeated in a zinc-preloaded extract (obtained by incubating coral extract and ZnCl₂ for 30 min; final Zn concentration: 100 mM), and substituting the inhibitor for the same volume of solutions of 21 amino acids (final concentration: 0.2 mM) or hydrolyzed collagen (Puravida’s Collagen Protein Longevity Drink). Results were compared with one-way ANOVA, considering control activity as 100%.

a. Effect of zinc species over the growth and metal load of Symbiodiniaceae

Growth curves obtained in each treatment and clade are displayed in Fig. 1. Full analysis of variance (ANOVA) for each microalga, zinc species, concentration, and sampling time are displayed in Table S1; here, the most important findings are highlighted. Under control conditions, S. microadriaticum displayed the smallest population after 480 h, while B. minutum reached more than twice this level.

In S. microadriaticum under the lowest Zn load (0.09 mg L^− 1; Fig. 1a), no differences were found in the first 24 h among Zn treatments. Until 216 h, all treatments (Fig. 1a) resulted in decreased cellular density compared to the control. By the end of the experiment (480 h), ZnO-NPs and [ZnEDTA] lead to decreased cell growth (p < 0.05). However, under the highest Zn load (50 mg L^− 1; Fig. 1b), this trend was inverted: growth promoted by ZnO-NPs and [ZnEDTA] were statistically identical to the control, while ZnSO₄ and [Zn(His)₂] caused decreased cellular density.

A similar pattern was identified in E. voratum. Under the lowest Zn load (Fig. 1c), all Zn compounds led to significantly decreased growth for most of the time. However, under the highest load (Fig. 1d), ZnSO₄ and [Zn(His)₂] were much more toxic to the microalgae.

B. minutum proved to be the most sensitive clade to the Zn supplementation. Even at the lowest Zn load (Fig. 1e), still considered environmentally acceptable, B. minutum growth was already affected after 24 h by ZnO-NPs and [Zn(His)₂]. By the end of the experiment, all Zn compounds led to decreased growth in relation to the control. At the highest metal load (Fig. 1f), all Zn compounds led to severe growth impairment compared to the control, with some of them starting to be cytostatic as soon as 24 h after exposure (ZnO-NPs).

The amount of Zn per unit of dry Symbiodiniaceae biomass after each Zn treatment at the end of the experiment is listed in Table 1.

Table 1

Zn levels in the dinoflagellates after 480 h of exposure.
Species	Excess Zn load (mg L^− 1)	Zn source	Zn concentration in the dry biomass (µg mg ^− 1)
	Control	-	0.04
Symbiodinium microadriaticum	0.09	ZnSO₄	0.12
		ZnO-NPs	0.31
		[ZnEDTA]	0.01
		[Zn(His)₂]	0.11
	50	ZnSO₄	63.25
		ZnO-NPs	4.01
		[ZnEDTA]	1.21
		[Zn(His)₂]	0.32
	Control	-	0.05
Effrenium voratum	0.09	ZnSO₄	0.11
		ZnO-NPs	0.14
		[ZnEDTA]	0.12
		[Zn(His)₂]	0.07
	50	ZnSO₄	28.23
		ZnO-NPs	1.63
		[ZnEDTA]	1.77
		[Zn(His)₂]	0.22
	Control	-	0.0048
Breviolum minutum	0.09	ZnSO₄	0.0066
		ZnO-NPs	0.0033
		[ZnEDTA]	0.012
		[Zn(His)₂]	0.0061
	50	ZnSO₄	47.88
		ZnO-NPs	3.09
		[ZnEDTA]	0.13
		[Zn(His)₂]	0.44

A correlation diagram of cell density after 480 h and Zn concentration in the biomass (Figure S1) helped us verify some trends. Free zinc (ZnSO₄) consistently caused high metal loads that did not translate into growth (compared to the control), irrespective of the initial amount of Zn (but the effect was more striking at 50 mg L^− 1). The only exception was for S. microadriaticum treated with 0.09 mg L^− 1 ZnSO₄. At the lowest concentration, ZnO-NPs (for S. microadriaticum and E. voratum) or [ZnEDTA] (for E. voratum and B. minutum) also led to higher Zn loads associated with lower cell density. This suggests that these forms of the metal may be linked to toxicity by metal overload. Data on ZnO-NPs at 50 mg L^− 1 should be interpreted with caution, as most of the insoluble material deposited in the bottom of the flasks and might not be available for the dinoflagellates.

On the other hand, in other instances, reduced growth was associated with low intracellular levels of Zn. This was observed for [ZnEDTA] (0.09 mg L^− 1 in S. microadriaticum), ZnO-NPs (0.09 mg L^− 1 in B. minutum), and [Zn(His)₂] (50 mg L^− 1 in E. voratum and B. minutum). This might be due to either decreased ability of the dinoflagellate to acquire the Zn nutrient, or to enhanced reactivity leading to toxicity in those models.

In summary, [Zn(His)₂] at environmentally safe levels (0.09 mg Zn L^− 1) led to high cell density with comparably low metal levels, being the best compound that translated intracellular added Zn into growth. The only exception was in B. minutum, which was little tolerant to any form of added Zn.

b. Effect of zinc species over trypsin-like activity from M. alcicornis

The effect of all Zn species, as well as of the ligands used to synthesize the complexes (His and EDTA), on the trypsin-like activity of the M. alcicornis coral is shown in Fig. 2. Two concentrations of Zn were used in the test (0.1 and 1 mM). Free Zn was a very efficient inhibitor of the enzymatic activity, even at the lower concentration. Its EC50 (15.6 ± 1.1 µM; Figure S2) is at the low micromolar level, and it is almost three times a better inhibitor than for example ZnO-NPs (EC50 = 56.2 ± 1.1 µM; Figure S3), a common inorganic filter of sunscreens. Strong inhibiton was also observed for [ZnEDTA] as well as for other simple Zn complexes (citrate and glycinate, Figure S3). None of the ligands used to synthesize the complexes were inhibitors except for the strong chelator EDTA.

The remarkable exception for the inhibition of trypsin-like enzymes in M. alcicornis was [Zn(His)₂] (Fig. 2). Zinc histidinate did not affect the enzyme activity even at the highest concentration.

The finding that zinc histidinate did not cause enzyme inhibition led us to verify the possibility of using other amino acids (Fig. 3) or collagen hydrolysates (Fig. 4) to recover the activity of Zn-loaded trypsin. Histidine was indeed the best amino acid to recover enzyme activity, and this behavior was mirrored by the peptide as well.

a. Effect of zinc species over the growth and metal load of symbiodiniaceae

At environmentally safe levels of Zn (0.09 mg Zn L^− 1), E. voratum was the most resilient clade, irrespective of the nature of the Zn compound. On the other hand, B. minutum was the most sensitive to any Zn supplementation. This observation, allied to the fact that B. minutum is also thermosensitive (Reich et al., 2021; Romero et al., 2022), suggests that it is an interesting candidate for a model organism in standard ecotoxicological assays with dinoflagellates (I. B. Miller et al., 2022; Moeller et al., 2021), especially in the Brazilian coast and in the Caribbean (LaJeunesse et al., 2018; Picciani et al., 2016; Romero et al., 2022).

The toxic effect of both free Zn and nanoparticulate zinc oxide (ZnO-NPs) is well known for several microalgae (Aruoja et al., 2009; Baek et al., 2020; Franklin et al., 2007; Hardefeldt & Reichelt-Brushett, 2015; Kuzminov et al., 2013; R. J. Miller et al., 2010; Spisni et al., 2016; Zhang et al., 2016). To the best of our knowledge, only the work of Goh and Chou (1997) dealt with the effect of free Zn(II) on the growth of S. microadriaticum. Our results (at 0.09 mg L^− 1) and theirs (at 0.5 mg L^− 1) agree in the sense that no toxic effects were observed for relatively low levels of Zn in the water (Goh & Chou, 1997).

Excess Zn is known to cause metabolic changes and tamper with the structure of several biomolecules, leading to impaired function (Liang et al., 2020). Besides, it can prompt oxidative stress by lipid peroxidation and inhibit the activity of several important antioxidant enzymes (M. Li et al., 2006). In the marine dinoflagellate Ostreococcus tauri, it was observed that Zn interferes with iron homeostasis (Lelandais et al., 2016), possibly leading to dysfunctional metabolism. This may also impact the production of photosynthesizing pigments (Kumar et al., 2014).

On the other hand, the mechanism of ZnO-NPs toxicity is less established. This particulate material can slowly release toxic levels of soluble Zn(II) in the marine environment (Hazeem, 2022; Liang et al., 2020; Vargas-Estrada et al., 2020), or it can be adsorbed by humic substances (Hazeem, 2022), in both cases increasing Zn bioavailability. ZnO-NPs may agglutinate around the cells, preventing access to nutrients and light (Liang et al., 2020; Song et al., 2010; Vargas-Estrada et al., 2020). This was observed in the microalgae Micromonas commode and Chlorella vulgaris, where damage to the integrity of the plasma membrane was possibly determinant for toxicity (Genevière et al., 2020; Suman et al., 2015).

For high Zn exposure at 50 mg L^− 1, beyond environmentally safe (or normal) levels, it is possible to observe differences between both Zn complexes, [ZnEDTA] and [Zn(His)₂] (Fig. 1b, d, f). While at 480 h [ZnEDTA] did not affect the growth of E. voratum or S. microadriaticum, [Zn(His)₂] had a striking negative effect. Besides, in B. minutum [ZnEDTA] performed better than [Zn(His)₂]. Different chelators (EDTA and His) may give the metal access to different cell compartments, however the higher thermodynamic stability of [ZnEDTA] when compared to [Zn(His)₂] (log K_f = 16.44 and 12.06, respectively) (Smith & Martell, 1976) suggests that [ZnEDTA] is a less efficient source of zinc overload to the microalgae.

At environmentally safe Zn levels (0.09 mg Zn L^− 1), basically no differences in growth were observed for any of the treatments (Fig. 1a, c, e), suggesting that any of the compounds could be a vehicle for the supplementation of this micronutrient. EDTA is routinely used to prevent metal hydrolysis and precipitation; however, it is a very strong synthetic chelator displaying several environmental problems, being considered an emergent contaminant (Houtman, 2010; Stefanakis & Becker, 2020). In addition, as observed above, [Zn(His)₂] caused a more efficient translation of intracellular Zn into growth in S. microadriaticum and E. voratum. Therefore, while both [ZnEDTA] and [Zn(His)₂] are potential carriers for Zn supplementation in cultured microalgae, the latter should be preferred as it is based on a natural, atoxic chelator.

Therefore, the effects in both cellular density and zinc uptake vary with the speciation of zinc. It still remains necessary to assess the response of the dinoflagellates in symbiosis and in the coral holobiont, a more complete target system.

Effect of zinc species over trypsin-like activity from M. alcicornis

The activity of trypsin-like enzymes is an interesting indicator of the heterotrophic metabolism in cnidarians. This family of enzymes is ubiquitous in the animal kingdom; serine proteases were detected in the aqueous extracts of stony corals (García-Arredondo et al., 2016) and trypsins have been identified in the nematocysts of Millepora complanata (Hernández-Elizárraga et al., 2022). Furthermore, they are found in zymogen cells and digestive filaments of cnidarians (Sebé-Pedrós et al., 2018; Steinmetz, 2019). The proteolytic activity of trypsin-like enzymes is inhibited by a number of divalent metal ions, such as zinc(II) (Bezerra et al., 2005). The chemical speciation of zinc, however, interferes on its inhibition activity (Fig. 2 and Figure S3). [Zn(His)₂] was the only compound that did not inhibit trypsin-like enzymes. As mentioned above, histidine forms a relatively stable complex with Zn(II), therefore its dissociation would not increase metal levels to an inhibitory level. While EDTA forms an even more stable complex with Zn, it should be noticed that EDTA itself is a potent inhibitor of proteases (Ayache et al., 2006; Burchacka et al., 2022; Kohara et al., 1991), therefore even small amounts of dissociated [ZnEDTA] would have toxic effects to the enzyme. Again, this observation suggests that the strategy of using EDTA as a solubilizing agent for metals in mineral supplements or growth buffers is problematic.

ZnO-NPs used as inorganic UV filters in sunscreens is an ecological concern for coral reefs in some parts of the world (Freitas Neto & Espósito, 2023), therefore we decided to investigate its inhibitory activity on the trypsin-like enzymes of M. alcicornis (Fig. 3). Even though it is less active than the free metal, it is still inhibitory at the same order of magnitude, which is worrisome as deposited ZnO particles on reefs could work as slow release agents for Zn(II), being a possible concern for both coral autotrophy (Fig. 1) and heterotrophy (Figure S2).

The observation that histidine was able to keep Zn in a safe form for trypsin-like enzymes led us to verify if other amino acids (Fig. 3) or small peptides (Fig. 4) could regenerate the activity of pre-inhibited enzymes (enzymes loaded with Zn). In fact, only histidine and, to a lesser extent, cysteine, were able to recover some functionality of Zn-laden enzymes (Fig. 3). This is in line with the fact that precisely histidine and cysteine are common Zn-binding sites for important proteins (Fraústo da Silva & Williams, 2001). The reversibility of the inhibition was also achieved by histidine-rich collagen from commercial sources (Fig. 4).

These observations indicate that cheap, ecologically friendly chelators such as histidine or histidine-enriched peptide hydrolysates might be useful as (i) decontaminating agents in episodes of Zn emission (performing better than EDTA), and (ii) as biocarriers for mineral supplementation of Zn to ex-situ corals (in aquarism or in nurseries for coral repopulation efforts), since zinc histidinate may be absorbed by Symbiodiniaceae while not affecting trypsin-like enzymes.

The chemical nature of zinc led to distinct effects on the growth and metal load of several Symbiodiniaceae, and on the proteolytic activity of M. alcicornis extracts. Therefore, metal speciation needs to be taken into account when assessing the potential risks of seawater contaminants such as ZnO-NPs present in sunscreen formulations. [Zn(His)₂] was an useful biocarrier of essential Zn to the dinoflagellates, while not interfering with the activity of trypsin-like enzymes from M. alcicornis. The enzymatic inhibition by zinc was reversed by the presence of histidine. The association of zinc with histidine might provide an interesting means to both nurture the coral holobiont and to decontaminate affected coral reefs in future environmental researches. Further investigations are needed to assess the response of the holobiont to different zinc chemical species both in vitro and in situ.

Author Contribution

LLFN performed the zooxanthellae growth and zinc-loading experiments, and co-wrote the main manuscript. RFBS procured the coral samples. RFBS and RSB developed the enzyme tests. MAS performed enzyme tests. FSC kept and supplied the zooxanthellae. BPE procured funding, co-wrote the main manuscript and supervised the project. All authors reviewed the manuscript.

ACKNOWLEDGEMENTS

The authors thank the São Paulo Research Foundation (FAPESP) (Projects 2021/07153-3 and 2021/10894-5) and the National Council of Scientific and Technological Development (CNPq) (Project 372742/2022-0) for financial support.

COMPETING INTERESTS

The authors have no competing interests to disclose.

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No competing interests reported.

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Zinc speciation promotes distinct effects on dinoflagellate growth and coral trypsin-like enzyme activity

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Journal Publication

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

i. Microalgae

ii. Coral

b. Zinc compounds

c. Microalgae exposure to zinc

RESULTS

a. Effect of zinc species over the growth and metal load of Symbiodiniaceae

DISCUSSION

a. Effect of zinc species over the growth and metal load of symbiodiniaceae

CONCLUSION

Declarations

Author Contribution

ACKNOWLEDGEMENTS

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1