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Potential of fascaplysin and palauolide from Fascaplysinopsis cf reticulata to reduce the risk of bacterial infection in fish farming

Potential of fascaplysin and palauolide from Fascaplysinopsis cf reticulata to reduce the risk of... Marine natural products isolated from the sponge Fascaplysinopsis cf reticulata, in French Polynesia, were investigated as an alternative to antibiotics to control pathogens in aquaculture. The overuse of antibiotics in aquaculture is largely considered to be an environmental pollution, because it supports the transfer of antibiotic resistance genes within the aquatic environment. One environmentally friendly alternative to antibiotics is the use of quorum sensing inhibitors (QSIs). Quorum sensing (QS) is a regulatory mechanism in bacteria which control virulence factors through the secretion of autoinducers (AIs), such as acyl-homoserine lactone (AHL) in gram- negative bacteria. Vibrio harveyi QS is controlled through three parallel pathways: HAI-1, AI-2, and CAI-1. Bioassay- guided purification of F. cf reticulata extract was conducted on two bacterial species, i.e., Tenacibaculum maritimum and V. harveyi for antibiotic and QS inhibition bioactivities. Toxicity bioassay of fractions was also evaluated on the freshwater fish Poecilia reticulata and the marine fish Acanthurus triostegus. Cyclohexanic and dichloromethane fractions of F.cf reticulata exhibited QS inhibition on V. harveyi and antibiotic bioactivities on V. harveyi and T. maritimum, respectively. Palauolide (1) and fascaplysin (2) were purified as major molecules from the cyclohexanic and dichloromethane fractions, respectively. Palauolide inhibited QS of V. harveyi through HAI-1 QS pathway at 50 –1 –1 μgml (26 μM), while fascaplysin affected the bacterial growth of V. harveyi (50 μgml ) and T. maritimum (0.25 μg). The toxicity of fascaplysin-enriched fraction (FEF) was evaluated and exhibited a toxic effect against fish at 50 –1 μgml . This study demonstrated for the first time the QSI potential of palauolide (1). Future research may assess the toxicity of both the cyclohexanic fraction of the sponge and palauolide (1) on fish, to confirm their potential as alternative to antibiotics in fish farming. Keywords: Porifera, Marine natural products, Quorum sensing inhibitors, Antibiotic, Fascaplysinopsis cf reticulata Background an urgent need for alternatives to antibiotics (Editorials The overuse of antibiotics in the environment may have 2013; Spellberg and Gilbert 2014). important economic and sanitary outcomes (Martinez In aquaculture antibiotic resistance causes mass mortal- 2009; Hatosy and Martiny 2015). Indeed, the release of ity of cultured species (Karunasagar et al. 1994) which re- antibiotics in natural environments exerts a strong pres- sult in economic loss for farmers (Shrestha et al. 2018). sure on bacteria strains and supports the selection of Aquaculture itself largely contributes to the dissemination resistant bacteria. The recurrent use of antibiotics de- of antibiotics resistance genes in the aquatic environment creases their effectiveness over time (Blair et al. 2015). (WHO, 2006;Shahet al. 2014), which increases the risks To reduce the overuse of antibiotics and minimize the on human health (Aly and Albutti 2014). Policy on antibi- impacts to the environment and human society, there is otics in aquaculture is becoming more strict, and antibi- otics are forbidden in some countries (Lulijwa et al. 2019). * Correspondence: mai.tepoerau@live.fr Finding antibiotic alternatives in this field is the focus of EIO, ILM, IFREMER, IRD, UPF, BP 6570, 98702 FAA’A, Tahiti, French Polynesia the current research (Pérez-Sánchez et al. 2018)due to Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 2 of 11 the promising market they represent. To reduce the of the cytotoxicity of fascaplysin (2) reported in the litera- selective pressure exerted on bacteria strains, novel strat- ture (Hamilton 2014), we also evaluated the toxicity of F.cf egies target natural products that inhibit the expression of reticulata extract on two fish species (Poecilia reticulata virulence genes without exerting a strong bactericide ac- and Acanthurus triostegus) to check the safety of using this tivity (Moloney 2016; Spellberg and Gilbert 2014). Such sponge in fish farming. promising products include inhibitors of quorum sensing (Chen et al. 2018; Pérez-Sánchez et al. 2018) which exhib- Methods ited in vitro and in vivo effectiveness in aquaculture Sponge sampling (Manefield et al. 2000;Brackman et al. 2008; Pande et al. Sponge samples were collected manually using SCUBA, 2013). between 45 and 65 m depth in the Tuamotu Archipelago Quorum sensing (QS) is a cell-to-cell communication (French Polynesia) during the 2011 Tuam expedition process in bacteria based on the secretion and detection aboard the Alis vessel (Debitus 2011), on the outer reef of signal molecules (i.e., autoinducers) by bacteria. Specif- of Anuanuaro Atoll (20°25.394’S, 143°32.930’W). Sam- ically for gram-negative bacteria, autoinducers (AIs) con- ples were frozen immediately at –20 °C on board until sist of small molecules, mainly acyl-homoserine lactone being processed. (AHL) derivatives (Waters and Bassler 2005). Quorum sensing allows the expression of target genes involved in Purification and characterization of secondary biofilm formation, toxin secretion, and bioluminescence metabolites. (Henke and Bassler 2004a). It is influenced by the concen- The sponge collected was freeze dried and grounded to ob- tration in AIs related to the bacterial density and the gen- tain 95 g of dry sponge powder. It was extracted using 100 etic similarity of bacteria neighbors (Schluter et al. 2016). ml of 80% ethanol and then rinsed twice in 100% ethanol. A model species for testing the relevance of antibiotic The solvent was evaporated under reduced pressure, and alternatives in aquaculture is V. harveyi. Vibrio harveyi is the remaining ethanolic extract was dissolved in water and a luminescent bacteria inhabiting the marine environment successively partitioned three times with cyclohexane and and pathogens in aquaculture, specifically when it is asso- three times with dichloromethane. The cyclohexanic frac- ciated with Tenacibaculum maritimum (Reverter et al. tion was subjected to silica gel chromatography (40–60-μm 2016). The QS of V. harveyi is well documented, with mesh) and then eluted with cyclohexane and ethyl acetate three parallel QS systems that are regulated by three cou- mixtures of increasing polarity. Further semi-preparative ples of signal molecules and cognate sensors: V. harveyi HPLC on normal phase column eluted with cyclohexane/ autoinducer-1 (HAI-1) and LuxN sensor; autoinducer-2 ethyl acetate 55/45 vol/vol allowed the isolation of palauo- (AI-2) and LuxPQ sensor; Cholerae autoinducer-1 (CAI- lide (1) (5 mg). The purification of the dichloromethane 1); and CqsS sensor (Henke and Bassler 2004a). Together fraction (called fascaplysin-enriched fraction (FEF)) using these three systems encode bioluminescence and virulence reverse phase HPLC (column: Interchrom Uptisphere strat- factors as biofilm formation, type III secretion, and a egy, 5 μm; solvent: (water/acetonitrile 70:30), TFA 0.1%) led secreted metalloprotease genes (Henke and Bassler 2004a; to the isolation of fascaplysin (2) (17% of FEF, 0.02% dry Henke and Bassler 2004b). sponge weight,19mg).High-performance liquid chroma- Quorum sensing inhibitors (QSIs) of V. harveyi have tography analysis was performed on HPLC (Agilent Tech- already been identified from a variety of marine organisms, nologies 1260 Infinity) with diode array (Agilent G1315C) including bacteria, algae, and sponges (Givskov et al. 1996; and evaporative light-scattering (Agilent G4260C) detec- Peters et al. 2003; Rasch et al. 2004; Teasdale et al. 2009; tion. Yields were calculated using the ratio compound Dobretsov et al. 2011;Natrah et al. 2011; Kalia 2013; Tello weight/freeze-dried sponge weight. Structure elucidation of et al. 2013;Sauravetal. 2017). Marine sponges are promis- the two known compounds was performed on the basis of 1 13 ing sources of antibiotic alternatives because (i) they are Hand C NMR and mass spectra. known to be a reservoir of diverse microbial communities (Thomas et al. 2016) and (ii) as primitive sessile organisms Fish toxicity bioassay featured with asimplemulticellular structure, their main The toxicity effect of F.cf reticulata’s FEF on fish was defense against pathogen rely on the production of second- evaluated on two fish species that can be easily found in ary metabolites with antibiotic and antibiofilm (Feng et al. French Polynesia and reared in the laboratory: P. reticu- 2013), and QS inhibition activities against pathogens (Blunt lata (the guppy or mosquito fish) and A. triostegus (the et al. 2005; Müller et al. 2013;Quévrainet al. 2014). In this convict tang fish). Poecilia reticulata specimens (5–8cm study, Vibrio harveyi and T. maritimum were used as length) were collected from a freshwater pool at Tahiti model species to test antibiotic and QS inhibition bioactiv- at night. Poecilia reticulata specimens were appealed ities of two compounds isolated from the sponge Fascaply- with a flashlight and then caught with a landing net (5- sinopsis cf reticulata collected in French Polynesia. Because mm mesh size) and kept in 3 L plastic jar containing Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 3 of 11 freshwater. Young settlers (or recruits, 1,5, 2 cm length) et al. 1997), and three derived mutants, JAF 375 (Freeman and juveniles (3–7 cm length) of A. triostegus (at the two and Bassler 1999), JMH 597, and JMH 612 (Henke and distinct developmental stages) were caught during full Bassler 2004a). All strains were obtained from Bassler la- moon nights on the foreshore puddles and on the reef boratory (Bassler et al. 1997; Freeman and Bassler 1999; crest using a net of the northeast coast of Moorea Island Henke and Bassler 2004a). Each mutant only expressed (17°29'52.19"S, 149°45'13.55”W). Acanthurus triostegus one of the three QS systems of V. harveyi: JAF 375 (CAI-1 recruits (fish larvae undergoing metamorphosis) were activated), JMH 597 (AI-2 activated), or JMH 612 (HAI-1 transparent at the time of capture, demonstrating that activated) (Freeman and Bassler 1999;Henke andBassler they had just entered the reef following their pelagic lar- 2004a). Quorum sensing inhibition bioassay was per- val stage, while the juveniles (old settlers, already meta- formed by combining simultaneously luminescence kinet- morphosed and settled when captured) were already ics (in relative luminescence units, RLU) and absorbance fully pigmented when caught, demonstrating that they kinetics (at λ = 600 nm) (Givskov et al. 1996;Brackman had already settled in this reef area for at least a week et al. 2008; Steenackers et al. 2010). Absorbance kinetics (Lecchini et al. 2004). was used to measure the growth of V. harveyi with any A preliminary assay was performed on P. reticulata by tested compound or controls. Data was obtained using a balneation, as described previously for environmental Fluostar Omega spectrophoto-luminometer (BMG Lab- toxicity studies of acetylcholinesterase (AChE) inhibitor tech Fluostar OPTIMA, Ortenberg, Germany). pesticides (Wester and Vos 1994; Bocquené and Galgani The quorum sensing inhibition bioassay was modified 2004; El-Demerdash et al. 2018). Fascaplysin-enriched from Mai et al. (2015). A V. harveyi colony was grown on fraction ethanolic solution was further tested in dupli- Zobell agar plates (BD Bacto™ peptone, 5 g; BD BBL™ yeast –1 cate at 1 and 5 μgml during 72 h (chronic toxicity) extract, 1 g; BD Bacto™ agar, 17 g; sterilized sea water, 1 L) –1 and at 50 μgml during 1 h (acute toxicity) in 2-L for 24 h. The plates were then suspended in liquid Lennox tanks, each containing five fishes. Solvent controls were L broth base medium (Invitrogen, Carlsbad, CA, USA) run for each experiment. For the 72-h experiment, which was supplemented with artificial sea salts (Sigma Al- –1 water, FEF, and EtOH were renewed, and fishes were fed drich Co., St Louis, MO, USA) at 40 g l and was then in- once a day with commercial flakes. Abnormal behavior cubated for 16 h under constant orbital stirring at 27 °C. of fishes after exposure to FEF was evaluated qualita- This suspension (50 μl) was then diluted in Marine Broth tively, such as swimming difficulties (i.e., irregularity of (CONDA®, Madrid, Spain) (10 ml) and was incubated for swim velocity, asymmetric pectorals fins movements, 30 min while stirring at 27 °C. Compounds were dissolved upside down swimming, and quick jumps) and loss of in absolute ethanol, deposited in sterile 96-μClear® bottom appetite. wells microplates (Greiner Bio-One, Germany) that were Since the preliminary assay highlighted a modification dried at room temperature under a laminar flow hood. of P. reticulata behavior by FEF (see results section), a sec- Each sample was tested in triplicate for each concentration –1 ond toxicity assay was performed on A. triostegus focusing of purified compound tested (1, 5, 10, and 50 μgml ). on feeding behavior by using a quantitative method. The Compounds were then dissolved in Marine Broth (100 μl) effect on FEF exposition on A. triostegus feeding behavior by sonication at 50/60 Hz for 30 min, and a bacterial was assessed on two distinct developmental stages in suspension (100 μl) was added in the appropriate wells. order to compare the activity of FEF at both stages of de- The 96 wells plates were incubated at 27 °C for 12 h in a velopment. The bioassays on A. triostegus were performed microplate incubator reader, with luminescence and ab- in 3-L tanks. Fishes (young settlers or juveniles) were ex- sorbance reading conducted every 10 min, after 1 min of –1 posed to FEF at 1 μgml in groups of four or five individ- double orbital stirring. The sterility of the culture medium uals during 24, 48, and 72 h. Rubble with encrusting turf was checked throughout the experiment, as well as the ab- algae were placed in the tank for fish to feed on 1 h per sorbance of each tested compound. Luminescence and ab- day during 3 days. The feeding behavior was assessed by sorbance data at the N-cycle reading (L and A ) N-cycle N-cycle counting the number of bites on the algae encrusted rub- were respectively obtained after subtracting the mean of ble in each aquarium. Six video sequences of 5 or 10 min the first ten cycles of the luminescence and absorbance (L per aquarium per day were analyzed. Results are expressed and A ) from the raw data mean first 10 cycles mean first 10 cycles in number of bites per fish per hour. (L and A )(Eqs. 1 and 2). N-cycle raw data N-cycle raw data L ¼ L −L ð1Þ N−cycle N−cycle raw data mean first 10 cycles Antibacterial and quorum sensing inhibition bioassays on Vibrio harveyi A ¼ A −A ð2Þ N−cycle N−cycle raw data mean first 10 cycles Every purified compound was tested in triplicate at four –1 concentrations, 1, 5, 10, and 50 μgml against the wild The kinetic curves obtained were sigmoidal. Any delay strain V. harveyi BB120 (Johnson and Shunk 1936;Bassler or inhibition of both growth and luminescence curves Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 4 of 11 compared to the control curves (which mean an inhib- bacterial concentration (i.e., fixed absorbance A = 0.055, ition of growth rate) is translated to an antibiotic effect which corresponded to half the maximum absorbance of the compound. By contrast, no change in bacterial A of control). max growth between tested and control curves associated For all parameters involved in QS activity (k and inflec- with a delay of luminescence between tested and control tion points) as well as in toxicity (number of bites per unit curves translated to a QSI effect of the compound. of time per fish), differences between concentration were tested using the non-parametric Kruskal-Wallis test (func- Antibacterial activity on Tenacibaculum maritimum tion kruskal.test of pgirmess package in R.3.1.0) and a Antibiotic activity on T. maritimum could not be per- multiple comparison test after Kruskal-Wallis (function formed through the absorbance kinetics method as previ- kruskalmc), suitable for small samples. A QSI activity was ously described for V. harveyi strains, because T. evidenced when (1) V. harveyi population growth rate (k, maritimum precipitated at the beginning of the experiment see Eq. 3) was not significantly lower with compound (or which prevented measuring absorbance. Antibiotic activity extract) compared to control (Kruskal-Wallis test and on T. maritimum was tested using the disk diffusion multiple comparison test after Kruskal-Wallis, α =0.05) method on solid agar medium (Bauer et al. 1966). This bio- and (2) the inflection point of luminescence is significantly assay was performed on a strain of the marine bacteria higher with compound (or extract) compared to control named TFA4 (Reverter et al. 2016). Pure compounds were (Kruskal-Wallis test and multiple comparison test after dissolved in 100% ethanol to obtain impregnated disks (cel- Kruskal-Wallis, α =0.05). lulose disks, 6 mm diameter) with 0.5, 0.25, 0.125, and 0.0625 μg of compound. Disks were air-dried in a laminar Results flow cabinet and then deposited on Zobell agar plates, pre- Purification of the Fascaplysinopsis cf reticulata extract viously seeded with TFA4 strain. Petri dishes were incu- The hydro-alcoholic extraction of sponge powder (95 g) bated at 27 °C for 2 days. provided 2.8 g of extract. The partitioning of this extract led to cyclohexanic (1.46 g, yield 1.54% w/w) and dichlo- Statistical analyses romethane (0.112 g, yield 0.11% w/w) fractions. The Absorbance was modeled as a logistic function of time purification of the cyclohexanic fraction conducted to (t) (Kingsland 1982) according to Equation 3, where the known palauolide (1) (0.005 g, yield 0.005% w/w) A is the maximum or asymptotic value of absorbance, and the dichloromethylenic fraction to the alkaloid fas- max k is the steepness of the curve, and t is the x value of caplysin (2) (0.019 g, yield 0.02% w/w) (Fig. 1). the sigmoid’s midpoint. Quorum sensing inhibition Effect of palauolide max Absorbance and luminescence kinetics of the V. harveyi Y ¼ ð3Þ 1 þ expðÞ −ktðÞ −t wild strain (Fig. 2 a and b) highlighted a dose-dependent effect of palauolide (1) on BB120 bacterial growth. Dur- max Y ¼ ð4Þ ing the growth of V. harveyi bacterial strains, the growth 1 þ aexpðÞ −ktðÞ −t Luminescence was also modeled as a logistic function, following Equation 4, where L is the maximum or max asymptotic value of luminescence. Equation 4 includes an a parameter to adequately model the high steepness found for luminescence curves. For each compound and concentration tested, the parameters of the logistic curve were fitted using the function “nls” of the package “stat” in R.3.1.0. The effect of compounds on the growth and the bioluminescence of V. harveyi populations were eval- uated by comparison of the growth rate (assimilated to the parameter k) and the curve inflection points. For the absorbance kinetics, the inflection point was equal to t . For the luminescence kinetics, the derivative (Y’) of the sigmoid function was calculated, and the inflection point was identified as the time for which Y’ was maximal. Furthermore, to provide comparable values of biolumin- Fig. 1 Chemical structure of palauolide (1) and fascaplysin (2) escence, luminescence values were compared at a fixed Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 5 of 11 Fig. 2 Effect of palauolide (1) on BB120 strain. (a) Absorbance kinetics, (b) luminescence kinetics (RLU), (c) steepness data (k) of absorbance kinetic, (d) luminescence value (RLU) measured for absorbance at 0.055 (represented on (A) by a dashed line, corresponding to the absorbance –1 –1 –1 value at the inflection point of control) without palauolide (black, control), with palauolide 1 μgml (blue, C4), 5 μgml (green, C3), 10 μgml –1 (orange, C2), and 50 μgml (red, C1). Data are reported as means ± SD from three technical replicates (*significant Kruskall-Wallis p value < 0.05 by comparing with control) rate (k parameter) of absorbance increased as the con- 50 min in average. These results indicate that palauolide centration of palauolide (1) increased (Table 1, Fig. 2c). (1) boosted bacterial growth and inhibited V. harveyi QS As a consequence, the sigmoid midpoint (t ) decreased through HAI-1 QS pathway. as concentration of palauolide (1) increased (data not –1 shown). At 50 μgml of palauolide (1), the growth rate Effect of fascaplysin of absorbance (k = 0.0127 ± 0.0005) reached values sig- Vibrio harveyi BB120 population growth rate (k, see Eq. nificantly higher than for controls (k = 0.0086 ± 0.0008; 3) was significantly lower with fascaplysin (2)at50 μgml multiple comparison test after Kruskal-Wallis; p < 0.05). (k = 0.0021) compared to control (k = 0.0121; p value < Also not significant due to the lack of statistical power, 0.05). Similar results were obtained for mutant JAF 375, similar trends were obtained for the three derived QS with lower growth rate (k = 0.0036) and with fascaplysin –1 mutants (Table 1). Despite the stimulating effect of (2)at 50 μgml compared to control (k = 0.0119). Strong palauolide (1)on V. harveyi growth, a delay in lumines- decreases of population growth rate were also obtained cence activation of approximately 17 min was observed for mutants JMH 597 and JMH 612 with fascaplysin (2)at –1 –1 for the highest concentrations tested 50 μgml , com- 50 μgml compared to control. For several replicates in- pared to the luminescence curve of the control (Fig. 2b, volving the two last mutants, population growth was null –1 red and black curves, respectively). At the same growth or negative with fascaplysin (2)at 50 μgml , which pre- stage (A = 0.055), a decrease in RLU was observed for vented the growth model to be fitted and k estimates to the highest concentration of palauolide (1) compared to be provided (Table 2; Additional file 1). This suggests an control. Such decrease was found for the BB120 wild antibiotic effect of fascaplysin (2)on V. harveyi and pre- strain (RLU respectively at 106 210 ± 24 385 at 50 μg vents concluding on a QS inhibition effect. –1 ml (26 μM) of palauolide (1) compared to 172 416 (± 2 489) for control; Table 1; Fig. 2d) and the JMH 612 Antibiotic bioassay mutant only (RLU respectively at 99 806 ± 18 002 at 50 Palauolide did not display any antibiotic activity against –1 μgml (26 μM) of palauolide (1) compared to 189 392 the marine pathogen T. maritimum. By contrast fasca- ± 2 609 for control; Table 1; Fig. 2d). For the JMH 612 plysin (2) displayed antibiotic activity at 0.25 μg per disk mutant, the delay between the luminescence kinetics at (11 mm) and 0.5 μg per disk (18 mm) against T. mariti- –1 50 μgml and the luminescence kinetics of control was mum (TFA4) (disk diffusion bioassay). Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 6 of 11 Table 1 Steepness of absorbance kinetic (k) and luminescence value measured for absorbance at 0.055 (RLU) estimated for various concentration of palauolide (1) and Vibrio harveyi strains Strain Concentration of palauolide Replicate k RLU BB120 Control 1 0.0093 173985 2 0.0088 173717 3 0.0077 169546 All 0.0086 ( ± 0.0008) 172416 ( ± 2489) C4 1 0.0100 173727 2 0.0104 174212 3 0.0093 172441 All 0.0099 ( ± 0.0006) 173460 ( ± 915) C3 1 0.0105 178786 2 0.0116 179795 3 0.0107 179754 All 0.0110 ( ± 0.0006) 179445 ( ± 571) C2 1 0.0119 174835 2 0.0116 178950 3 0.0117 178843 All 0.0117 ( ± 0.0001) 177542 ( ± 2345) C1 1 0.0122 107211 2 0.0131 81339 3 0.0129 130080 All 0.0127 ( ± 0.0005) 106210 ( ± 24385) JAF 375 Control 1 0.0144 131953 2 0.0140 134519 3 0.0135 131708 All 0.0139 ( ± 0.0005) 132727 ( ± 1557) C4 1 0.0160 129253 2 0.0168 131105 3 0.0183 130501 All 0.0170 ( ± 0.0012) 130286 ( ± 944) C3 1 0.0151 146894 2 0.0154 146197 3 0.0171 145802 All 0.0159 ( ± 0.0011) 146298 ( ± 553) C2 1 0.0172 140852 2 0.0163 141282 3 0.0160 140905 All 0.0165 ( ± 0.0006) 141014 ( ± 235) C1 1 0.0170 159632 2 0.0179 158643 3 0.0165 158767 All 0.0171 ( ± 0.0007) 159014 ( ± 539) JMH 597 Control 1 0.0094 147880 2 0.0081 146686 3 0.0100 147955 Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 7 of 11 Table 1 Steepness of absorbance kinetic (k) and luminescence value measured for absorbance at 0.055 (RLU) estimated for various concentration of palauolide (1) and Vibrio harveyi strains (Continued) Strain Concentration of palauolide Replicate k RLU All 0.0091 ( ± 0.0010) 147507 (± 712) C4 1 0.0120 150354 2 0.0118 147321 3 0.0121 147103 All 0.0119 ( ± 0.0001) 148259 ( ± 1818) C3 1 0.0132 154515 2 0.0138 152906 3 0.0140 153875 All 0.0137 ( ± 0.0004) 153765 ( ± 810) C2 1 0.0129 152211 2 0.0123 151867 3 0.0122 152261 All 0.0125 ( ± 0.0003) 152113 ( ± 214) C1 1 0.0159 150802 2 0.0183 161802 3 0.0145 157737 All 0.0162 ( ± 0.0019) 156780 ( ± 5562) JMH 612 Control 1 0.0057 186864 2 0.0083 192076 3 0.0087 189235 All 0.0076 ( ± 0.0017) 189392 ( ± 2609) C4 1 0.0074 186349 2 0.0076 189886 3 0.0087 189302 All 0.0079 ( ± 0.0007) 188512 ( ± 1896) C3 1 0.0088 187819 2 0.0076 174498 3 0.0078 184672 All 0.0081 ( ± 0.0007) 182330 ( ± 6963) C2 1 0.0089 190911 2 0.0086 188991 3 0.0089 188746 All 0.0088 ( ± 0.0002) 189549 ( ± 1186) C1 1 0.0106 120398 2 0.0097 91967 3 0.0091 87053 All 0.0098 ( ± 0.0008) 99806 ( ± 18002) –1 Control, C4, C3, C2, and C1, respectively, refer to palauolide (1) concentration at 0, 1, 5, 10, and 50 μgml . Estimated values of k and RLU are provided for each replicate (n = 3) and for all replicates (mean ± standard deviation over the three replicated). Values significantly different from controls (α = 0.05) are marked with a star (*) Fish toxicity assay periods) within the first hour of treatment. None motility –1 –1 At 50 μgml of FEF, P. reticulata exhibited signs of disarrangement was observed at 1 μgml FEF solutions, hyperventilation as well as motility disarrangement (i.e., but changes to the feeding behavior were noticed for P. jerky movements with sudden accelerations or motionless reticulata,i.e., P. reticulata tasted the food flakes but did Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 8 of 11 Table 2 Steepness of absorbance kinetic (k) estimated with time. Palauolide (1) revealed a potential as QSI by inhi- –1 fascaplysin (2) at 50 μgml (C1), and without fascaplysin biting V. harveyi luminescence at 26 μM. In quantitative (control), for the various Vibrio harveyi strains analysis, palauolide (1) delayed the activation of bio- Strain Dose Replicate k luminescence expression to 50 min of V. harveyi BB120. BB120 Control 1 0.0118 The V. harveyi growth rate was also significantly in- creased (p value < 0.05). The boosted growth rate of V. 2 0.0123 harveyi with palauolide (1) can be interpreted as a con- All 0.0121 sequence of QS inhibition, because the expression of C1 1 0.0024 bioluminescence slows down bacterial growth rate to 2 0.0017 save energy (Nackerdien et al. 2008). The present data All 0.0021 corroborates well with the result obtained previously on JAF 375 Control 1 0.0117 the QSI at 23 μM of isonaamidine A isolated from the sponge Leucetta chagosensis (Mai et al. 2015). Other 2 0.0120 studies compared bioluminescence data at a time t,to All 0.0119 determine the inhibition of QS (Brackman et al. 2008; C1 1 0.0051 Teasdale et al. 2009; Natrah et al. 2011). For example, 2 0.0021 Brackman et al. (2008) showed inhibition of V. harveyi All 0.0036 bioluminescence with cinnamaldehyde and derivatives at JMH 597 Control 1 0.0108 100 μM, 6 h after the addition of compounds (Brackman et al. 2008). Skindersoe et al. (2008) found that manoa- 2 0.0118 lide, a compound of similar structure to palauolide (1), All 0.0113 inhibits QS at IC = 0.66 μM. The better bioactivity of C1 1 0.0019 manoalide compared to palauolide (1) could be ex- 2<0 plained from the sensitivity of the intracellular bioassay All - used by authors. JMH 612 Control 1 0.0137 Themodeofaction of palauolide (1) on the inhibition of QS has potential as an antibiotic alternative in aquaculture 2 0.0119 for Vibrio species. Our bioassay on V. harveyi double mu- All 0.0128 tants JAF 375, JMH 597, and JMH 612 highlighted interfer- C1 1 < 0 ence of palauolide (1)on V. harveyi QS, specifically with 2<0 the acyl-homoserine lactone: HAI-1. Quorum sensing reg- All - ulates bioluminescence and virulence factors of bacteria through autoinducers (Henke and Bassler 2004a)suchas –1 not ingest them. At 5 μgml of FEF, all of the P. reticu- HAI-1 used for intraspecies communication (Waters and lata died within 12 h. Bassler 2005;Yang et al. 2011). Acyl-homoserine lactone The experiment on A. triostegus was only performed molecules are found in the family Vibrionaceae (Yang –1 at 1 μgml of FEF. For each time of incubation (24, 48, et al. 2011). Palauolide (1) can therefore interfere with Vib- and 72 h), the number of bites of A. triostegus (both re- rio species QS through HAI-1 pathway and then be used cruits and juveniles) decreased significantly compared to as an antivirulent against Vibrio species as antagonist of control A. triostegus (Fig 3). After 24 h of incubation AIs. Most of antagonists of QS sensors are small molecules –1 with 1 μgml FEF solution, the number of bites de- (Swem et al. 2008; Gamby et al. 2012) with structural simi- creased by 91.3% (± 1. 6%, p value < 0.01) for recruits larities to AIs such as brominated furanone derivatives and by 95.9% (±0.8%, p value < 0.001) for juveniles com- (Givskov et al. 1996; Rasch et al. 2004; Steenackers et al. pared to the control A. triostegus (Fig 3). This trend was 2010). Palauolide (1) is a sesterterpene composed by a δ- confirmed for others times of exposition. hydroxybutenolide moiety and a carbon skeleton. The po- tential of palauolide as a competitor of HAI-1 is most likely Discussion due to its small structure and the moderate polarity of its The isolation of palauolide (1) and the major compound chemical structure. This enables palauolide (1)tocross fascaplysin (2) from the French Polynesian F. cf reticulata over the external membrane lipid of bacteria and to bind extracts is similar to those results obtained by Sullivan on the periplasmic sensors Lux N (Swem et al. 2008). and Faulkner (1982) on Palauan sponges. Further research would indicate if there is an antagonist The QSI potential of the French Polynesian sponge F. effect of palauolide (1) on the HAI-1 sensor, such as cf reticulata against the QS-dependent phenotypic ex- testing against additional V. harveyi mutants (Swem pression in V. harveyi was demonstrated for the first et al. 2008; Blair and Doucette 2013). Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 9 of 11 Fig. 3 Number of bites on coral pieces of Acanthurus triostegus (a) juveniles and (b) recruits per hour without FEF, fascaplysin-enriched fraction –1 (C), with ethanolic solvent (S), with fascaplysin-enriched fraction powder (FEF) at 1 μgml . Error bars represent standard deviation of the mean (N = 6) (**p value < 0.01 significant, *** p value < 0.001 very significant compare to control without fascaplysin-enriched fraction (C)s) Fascaplysin (2) supplies a broad range of biological activ- QSIs help and increase antibioticactiononbiofilm forma- ity within F. cf reticulata.First, asother β-carboline alka- tion (Brackman et al. 2011). However, the toxicity on fish loids as dysideanin (20 μg) and didemnolines A-D (100 of the major compound of F.cf reticulata fascaplysin (2) μg), fascaplysin is a strong antibiotic (0.25 μg) (Charan (yield 0.02% w/w) prevents the use of the sponge extract in et al. 2002; Hamilton 2014). In the sponge, fascaplysin (2) fish farming context. We recommend in future research to is the major compound which represents 0.02% of the ly- test toxicity of the cyclohexanic fraction of the sponge and ophilized sponge weigh. It exhibits many biological activ- palauolide (1) on fish before concluding on the potential of ities including cytotoxicity against tumoral cells (Segraves the cyclohexanic fraction and palauolide (1)asanalterna- et al. 2004;Shafiqetal. 2012; Hamilton 2014; Cells et al. tive to antibiotics in fish farming. 2015; Kumar et al. 2015), antimicrobial activities (Roll et al. Acknowledgements 1988), and the inhibition of acetylcholinesterase (Bharate This work is part of TM’s PhD thesis. We acknowledge ANR, Netbiome, et al. 2012; Manda et al. 2016). For microbial disease treat- French Polynesian authorities, and Délégation à la Recherche-France in Tahiti for their grant, help, and support, IRD and GOPS for funding the field trips ments in aquaculture, fascaplysin (2) is not ideal. Despite aboard R/V ALIS, her crew and IRD’s diving team for their on-field help, and its antibiotic activity against marine pathogens V. harveyi Laboratoire d’Excellence LabEx CORAIL, 66100 Perpignan, France. We would (Table 2)and T. maritimum, fascaplysin (2)istoxic toward also like to thank Johanna Loacker and Christina Piotrowski from Calacademy for their help with the loan of some comparative material. both fresh and saltwater fish, P. reticulata and A. triostegus, respectively. Indeed, fascaplysin (2) modified fish behavior Author’s contribution and displayed an anorexic effect. The AchE inhibition CD supervised sponge sampling and TM’s PhD thesis. SP supervised the setup of the extract library for biological screening. ME, KH, and DE identified properties of fascaplysin (Bharate et al. 2012) could explain sponge samples. TM and MAB carried out chemical experiments. JT, CD, MB, both its toxicity (Bocquené and Galgani 2004;Modesto and DL carried out fish toxicity bioassay, and TM, EA, DS, and CD carried out and Martinez 2010; Assis et al. 2012) and its effect on loss antibiotic and quorum sensing bioassays. SVW was in charge of statistical analyses. TM wrote the manuscript. All authors reviewed and approved the of appetite of fish (Schneider 2000). manuscript. The toxicity of palauolide (1) on fish was not tested in this study because previous work highlighted a weaker Funding This research was supported by the research grant ANR-11-EBIM-006-1 cytotoxic activity of palauolide (1) compared to fascaply- (Project: POMARE) from ANR and Netbiome, and the grant 113-163 (Project: sin (2) (Charan et al. 2002; Hamilton 2014). However, Biopolyval) from French Polynesian authorities and Délégation à la we recommend performing additional toxicity bioassays Recherche-France in Tahiti. of palauolide (1) on fish before using it as alternative of Availability of data and materials antibiotic in fish farming. Not applicable. Ethics approval and consent to participate Conclusion Not applicable. In conclusion, the presence of palauolide (1) and fascaply- sin (2)in F.cf reticulata, with QS inhibition and antibiotic Consent for publication properties, respectively, could act as complementary where Not applicable. Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 10 of 11 Competing interests Debitus C. TUAM 2011. 2011. http://dx.doi.org/https://doi.org/10.17600/11100010. The authors declare that they have no competing of interest. Dobretsov S, Teplitski M, Bayer M, Gunasekera S, Proksch P, Paul VJ. Inhibition of marine biofouling by bacterial quorum sensing inhibitors. Biofouling. 2011; Author details 27(8):893–905. https://doi.org/10.1080/08927014.2011.609616. EIO, ILM, IFREMER, IRD, UPF, BP 6570, 98702 FAA’A, Tahiti, French Polynesia. Editorials. The antibiotic alarm. Nature. 2013;495:141. 2 3 IRD, Univ. Brest, CNRS, IFREMER, LEMAR, F-29280 Plouzane, France. 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Potential of fascaplysin and palauolide from Fascaplysinopsis cf reticulata to reduce the risk of bacterial infection in fish farming

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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

Marine natural products isolated from the sponge Fascaplysinopsis cf reticulata, in French Polynesia, were investigated as an alternative to antibiotics to control pathogens in aquaculture. The overuse of antibiotics in aquaculture is largely considered to be an environmental pollution, because it supports the transfer of antibiotic resistance genes within the aquatic environment. One environmentally friendly alternative to antibiotics is the use of quorum sensing inhibitors (QSIs). Quorum sensing (QS) is a regulatory mechanism in bacteria which control virulence factors through the secretion of autoinducers (AIs), such as acyl-homoserine lactone (AHL) in gram- negative bacteria. Vibrio harveyi QS is controlled through three parallel pathways: HAI-1, AI-2, and CAI-1. Bioassay- guided purification of F. cf reticulata extract was conducted on two bacterial species, i.e., Tenacibaculum maritimum and V. harveyi for antibiotic and QS inhibition bioactivities. Toxicity bioassay of fractions was also evaluated on the freshwater fish Poecilia reticulata and the marine fish Acanthurus triostegus. Cyclohexanic and dichloromethane fractions of F.cf reticulata exhibited QS inhibition on V. harveyi and antibiotic bioactivities on V. harveyi and T. maritimum, respectively. Palauolide (1) and fascaplysin (2) were purified as major molecules from the cyclohexanic and dichloromethane fractions, respectively. Palauolide inhibited QS of V. harveyi through HAI-1 QS pathway at 50 –1 –1 μgml (26 μM), while fascaplysin affected the bacterial growth of V. harveyi (50 μgml ) and T. maritimum (0.25 μg). The toxicity of fascaplysin-enriched fraction (FEF) was evaluated and exhibited a toxic effect against fish at 50 –1 μgml . This study demonstrated for the first time the QSI potential of palauolide (1). Future research may assess the toxicity of both the cyclohexanic fraction of the sponge and palauolide (1) on fish, to confirm their potential as alternative to antibiotics in fish farming. Keywords: Porifera, Marine natural products, Quorum sensing inhibitors, Antibiotic, Fascaplysinopsis cf reticulata Background an urgent need for alternatives to antibiotics (Editorials The overuse of antibiotics in the environment may have 2013; Spellberg and Gilbert 2014). important economic and sanitary outcomes (Martinez In aquaculture antibiotic resistance causes mass mortal- 2009; Hatosy and Martiny 2015). Indeed, the release of ity of cultured species (Karunasagar et al. 1994) which re- antibiotics in natural environments exerts a strong pres- sult in economic loss for farmers (Shrestha et al. 2018). sure on bacteria strains and supports the selection of Aquaculture itself largely contributes to the dissemination resistant bacteria. The recurrent use of antibiotics de- of antibiotics resistance genes in the aquatic environment creases their effectiveness over time (Blair et al. 2015). (WHO, 2006;Shahet al. 2014), which increases the risks To reduce the overuse of antibiotics and minimize the on human health (Aly and Albutti 2014). Policy on antibi- impacts to the environment and human society, there is otics in aquaculture is becoming more strict, and antibi- otics are forbidden in some countries (Lulijwa et al. 2019). * Correspondence: mai.tepoerau@live.fr Finding antibiotic alternatives in this field is the focus of EIO, ILM, IFREMER, IRD, UPF, BP 6570, 98702 FAA’A, Tahiti, French Polynesia the current research (Pérez-Sánchez et al. 2018)due to Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 2 of 11 the promising market they represent. To reduce the of the cytotoxicity of fascaplysin (2) reported in the litera- selective pressure exerted on bacteria strains, novel strat- ture (Hamilton 2014), we also evaluated the toxicity of F.cf egies target natural products that inhibit the expression of reticulata extract on two fish species (Poecilia reticulata virulence genes without exerting a strong bactericide ac- and Acanthurus triostegus) to check the safety of using this tivity (Moloney 2016; Spellberg and Gilbert 2014). Such sponge in fish farming. promising products include inhibitors of quorum sensing (Chen et al. 2018; Pérez-Sánchez et al. 2018) which exhib- Methods ited in vitro and in vivo effectiveness in aquaculture Sponge sampling (Manefield et al. 2000;Brackman et al. 2008; Pande et al. Sponge samples were collected manually using SCUBA, 2013). between 45 and 65 m depth in the Tuamotu Archipelago Quorum sensing (QS) is a cell-to-cell communication (French Polynesia) during the 2011 Tuam expedition process in bacteria based on the secretion and detection aboard the Alis vessel (Debitus 2011), on the outer reef of signal molecules (i.e., autoinducers) by bacteria. Specif- of Anuanuaro Atoll (20°25.394’S, 143°32.930’W). Sam- ically for gram-negative bacteria, autoinducers (AIs) con- ples were frozen immediately at –20 °C on board until sist of small molecules, mainly acyl-homoserine lactone being processed. (AHL) derivatives (Waters and Bassler 2005). Quorum sensing allows the expression of target genes involved in Purification and characterization of secondary biofilm formation, toxin secretion, and bioluminescence metabolites. (Henke and Bassler 2004a). It is influenced by the concen- The sponge collected was freeze dried and grounded to ob- tration in AIs related to the bacterial density and the gen- tain 95 g of dry sponge powder. It was extracted using 100 etic similarity of bacteria neighbors (Schluter et al. 2016). ml of 80% ethanol and then rinsed twice in 100% ethanol. A model species for testing the relevance of antibiotic The solvent was evaporated under reduced pressure, and alternatives in aquaculture is V. harveyi. Vibrio harveyi is the remaining ethanolic extract was dissolved in water and a luminescent bacteria inhabiting the marine environment successively partitioned three times with cyclohexane and and pathogens in aquaculture, specifically when it is asso- three times with dichloromethane. The cyclohexanic frac- ciated with Tenacibaculum maritimum (Reverter et al. tion was subjected to silica gel chromatography (40–60-μm 2016). The QS of V. harveyi is well documented, with mesh) and then eluted with cyclohexane and ethyl acetate three parallel QS systems that are regulated by three cou- mixtures of increasing polarity. Further semi-preparative ples of signal molecules and cognate sensors: V. harveyi HPLC on normal phase column eluted with cyclohexane/ autoinducer-1 (HAI-1) and LuxN sensor; autoinducer-2 ethyl acetate 55/45 vol/vol allowed the isolation of palauo- (AI-2) and LuxPQ sensor; Cholerae autoinducer-1 (CAI- lide (1) (5 mg). The purification of the dichloromethane 1); and CqsS sensor (Henke and Bassler 2004a). Together fraction (called fascaplysin-enriched fraction (FEF)) using these three systems encode bioluminescence and virulence reverse phase HPLC (column: Interchrom Uptisphere strat- factors as biofilm formation, type III secretion, and a egy, 5 μm; solvent: (water/acetonitrile 70:30), TFA 0.1%) led secreted metalloprotease genes (Henke and Bassler 2004a; to the isolation of fascaplysin (2) (17% of FEF, 0.02% dry Henke and Bassler 2004b). sponge weight,19mg).High-performance liquid chroma- Quorum sensing inhibitors (QSIs) of V. harveyi have tography analysis was performed on HPLC (Agilent Tech- already been identified from a variety of marine organisms, nologies 1260 Infinity) with diode array (Agilent G1315C) including bacteria, algae, and sponges (Givskov et al. 1996; and evaporative light-scattering (Agilent G4260C) detec- Peters et al. 2003; Rasch et al. 2004; Teasdale et al. 2009; tion. Yields were calculated using the ratio compound Dobretsov et al. 2011;Natrah et al. 2011; Kalia 2013; Tello weight/freeze-dried sponge weight. Structure elucidation of et al. 2013;Sauravetal. 2017). Marine sponges are promis- the two known compounds was performed on the basis of 1 13 ing sources of antibiotic alternatives because (i) they are Hand C NMR and mass spectra. known to be a reservoir of diverse microbial communities (Thomas et al. 2016) and (ii) as primitive sessile organisms Fish toxicity bioassay featured with asimplemulticellular structure, their main The toxicity effect of F.cf reticulata’s FEF on fish was defense against pathogen rely on the production of second- evaluated on two fish species that can be easily found in ary metabolites with antibiotic and antibiofilm (Feng et al. French Polynesia and reared in the laboratory: P. reticu- 2013), and QS inhibition activities against pathogens (Blunt lata (the guppy or mosquito fish) and A. triostegus (the et al. 2005; Müller et al. 2013;Quévrainet al. 2014). In this convict tang fish). Poecilia reticulata specimens (5–8cm study, Vibrio harveyi and T. maritimum were used as length) were collected from a freshwater pool at Tahiti model species to test antibiotic and QS inhibition bioactiv- at night. Poecilia reticulata specimens were appealed ities of two compounds isolated from the sponge Fascaply- with a flashlight and then caught with a landing net (5- sinopsis cf reticulata collected in French Polynesia. Because mm mesh size) and kept in 3 L plastic jar containing Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 3 of 11 freshwater. Young settlers (or recruits, 1,5, 2 cm length) et al. 1997), and three derived mutants, JAF 375 (Freeman and juveniles (3–7 cm length) of A. triostegus (at the two and Bassler 1999), JMH 597, and JMH 612 (Henke and distinct developmental stages) were caught during full Bassler 2004a). All strains were obtained from Bassler la- moon nights on the foreshore puddles and on the reef boratory (Bassler et al. 1997; Freeman and Bassler 1999; crest using a net of the northeast coast of Moorea Island Henke and Bassler 2004a). Each mutant only expressed (17°29'52.19"S, 149°45'13.55”W). Acanthurus triostegus one of the three QS systems of V. harveyi: JAF 375 (CAI-1 recruits (fish larvae undergoing metamorphosis) were activated), JMH 597 (AI-2 activated), or JMH 612 (HAI-1 transparent at the time of capture, demonstrating that activated) (Freeman and Bassler 1999;Henke andBassler they had just entered the reef following their pelagic lar- 2004a). Quorum sensing inhibition bioassay was per- val stage, while the juveniles (old settlers, already meta- formed by combining simultaneously luminescence kinet- morphosed and settled when captured) were already ics (in relative luminescence units, RLU) and absorbance fully pigmented when caught, demonstrating that they kinetics (at λ = 600 nm) (Givskov et al. 1996;Brackman had already settled in this reef area for at least a week et al. 2008; Steenackers et al. 2010). Absorbance kinetics (Lecchini et al. 2004). was used to measure the growth of V. harveyi with any A preliminary assay was performed on P. reticulata by tested compound or controls. Data was obtained using a balneation, as described previously for environmental Fluostar Omega spectrophoto-luminometer (BMG Lab- toxicity studies of acetylcholinesterase (AChE) inhibitor tech Fluostar OPTIMA, Ortenberg, Germany). pesticides (Wester and Vos 1994; Bocquené and Galgani The quorum sensing inhibition bioassay was modified 2004; El-Demerdash et al. 2018). Fascaplysin-enriched from Mai et al. (2015). A V. harveyi colony was grown on fraction ethanolic solution was further tested in dupli- Zobell agar plates (BD Bacto™ peptone, 5 g; BD BBL™ yeast –1 cate at 1 and 5 μgml during 72 h (chronic toxicity) extract, 1 g; BD Bacto™ agar, 17 g; sterilized sea water, 1 L) –1 and at 50 μgml during 1 h (acute toxicity) in 2-L for 24 h. The plates were then suspended in liquid Lennox tanks, each containing five fishes. Solvent controls were L broth base medium (Invitrogen, Carlsbad, CA, USA) run for each experiment. For the 72-h experiment, which was supplemented with artificial sea salts (Sigma Al- –1 water, FEF, and EtOH were renewed, and fishes were fed drich Co., St Louis, MO, USA) at 40 g l and was then in- once a day with commercial flakes. Abnormal behavior cubated for 16 h under constant orbital stirring at 27 °C. of fishes after exposure to FEF was evaluated qualita- This suspension (50 μl) was then diluted in Marine Broth tively, such as swimming difficulties (i.e., irregularity of (CONDA®, Madrid, Spain) (10 ml) and was incubated for swim velocity, asymmetric pectorals fins movements, 30 min while stirring at 27 °C. Compounds were dissolved upside down swimming, and quick jumps) and loss of in absolute ethanol, deposited in sterile 96-μClear® bottom appetite. wells microplates (Greiner Bio-One, Germany) that were Since the preliminary assay highlighted a modification dried at room temperature under a laminar flow hood. of P. reticulata behavior by FEF (see results section), a sec- Each sample was tested in triplicate for each concentration –1 ond toxicity assay was performed on A. triostegus focusing of purified compound tested (1, 5, 10, and 50 μgml ). on feeding behavior by using a quantitative method. The Compounds were then dissolved in Marine Broth (100 μl) effect on FEF exposition on A. triostegus feeding behavior by sonication at 50/60 Hz for 30 min, and a bacterial was assessed on two distinct developmental stages in suspension (100 μl) was added in the appropriate wells. order to compare the activity of FEF at both stages of de- The 96 wells plates were incubated at 27 °C for 12 h in a velopment. The bioassays on A. triostegus were performed microplate incubator reader, with luminescence and ab- in 3-L tanks. Fishes (young settlers or juveniles) were ex- sorbance reading conducted every 10 min, after 1 min of –1 posed to FEF at 1 μgml in groups of four or five individ- double orbital stirring. The sterility of the culture medium uals during 24, 48, and 72 h. Rubble with encrusting turf was checked throughout the experiment, as well as the ab- algae were placed in the tank for fish to feed on 1 h per sorbance of each tested compound. Luminescence and ab- day during 3 days. The feeding behavior was assessed by sorbance data at the N-cycle reading (L and A ) N-cycle N-cycle counting the number of bites on the algae encrusted rub- were respectively obtained after subtracting the mean of ble in each aquarium. Six video sequences of 5 or 10 min the first ten cycles of the luminescence and absorbance (L per aquarium per day were analyzed. Results are expressed and A ) from the raw data mean first 10 cycles mean first 10 cycles in number of bites per fish per hour. (L and A )(Eqs. 1 and 2). N-cycle raw data N-cycle raw data L ¼ L −L ð1Þ N−cycle N−cycle raw data mean first 10 cycles Antibacterial and quorum sensing inhibition bioassays on Vibrio harveyi A ¼ A −A ð2Þ N−cycle N−cycle raw data mean first 10 cycles Every purified compound was tested in triplicate at four –1 concentrations, 1, 5, 10, and 50 μgml against the wild The kinetic curves obtained were sigmoidal. Any delay strain V. harveyi BB120 (Johnson and Shunk 1936;Bassler or inhibition of both growth and luminescence curves Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 4 of 11 compared to the control curves (which mean an inhib- bacterial concentration (i.e., fixed absorbance A = 0.055, ition of growth rate) is translated to an antibiotic effect which corresponded to half the maximum absorbance of the compound. By contrast, no change in bacterial A of control). max growth between tested and control curves associated For all parameters involved in QS activity (k and inflec- with a delay of luminescence between tested and control tion points) as well as in toxicity (number of bites per unit curves translated to a QSI effect of the compound. of time per fish), differences between concentration were tested using the non-parametric Kruskal-Wallis test (func- Antibacterial activity on Tenacibaculum maritimum tion kruskal.test of pgirmess package in R.3.1.0) and a Antibiotic activity on T. maritimum could not be per- multiple comparison test after Kruskal-Wallis (function formed through the absorbance kinetics method as previ- kruskalmc), suitable for small samples. A QSI activity was ously described for V. harveyi strains, because T. evidenced when (1) V. harveyi population growth rate (k, maritimum precipitated at the beginning of the experiment see Eq. 3) was not significantly lower with compound (or which prevented measuring absorbance. Antibiotic activity extract) compared to control (Kruskal-Wallis test and on T. maritimum was tested using the disk diffusion multiple comparison test after Kruskal-Wallis, α =0.05) method on solid agar medium (Bauer et al. 1966). This bio- and (2) the inflection point of luminescence is significantly assay was performed on a strain of the marine bacteria higher with compound (or extract) compared to control named TFA4 (Reverter et al. 2016). Pure compounds were (Kruskal-Wallis test and multiple comparison test after dissolved in 100% ethanol to obtain impregnated disks (cel- Kruskal-Wallis, α =0.05). lulose disks, 6 mm diameter) with 0.5, 0.25, 0.125, and 0.0625 μg of compound. Disks were air-dried in a laminar Results flow cabinet and then deposited on Zobell agar plates, pre- Purification of the Fascaplysinopsis cf reticulata extract viously seeded with TFA4 strain. Petri dishes were incu- The hydro-alcoholic extraction of sponge powder (95 g) bated at 27 °C for 2 days. provided 2.8 g of extract. The partitioning of this extract led to cyclohexanic (1.46 g, yield 1.54% w/w) and dichlo- Statistical analyses romethane (0.112 g, yield 0.11% w/w) fractions. The Absorbance was modeled as a logistic function of time purification of the cyclohexanic fraction conducted to (t) (Kingsland 1982) according to Equation 3, where the known palauolide (1) (0.005 g, yield 0.005% w/w) A is the maximum or asymptotic value of absorbance, and the dichloromethylenic fraction to the alkaloid fas- max k is the steepness of the curve, and t is the x value of caplysin (2) (0.019 g, yield 0.02% w/w) (Fig. 1). the sigmoid’s midpoint. Quorum sensing inhibition Effect of palauolide max Absorbance and luminescence kinetics of the V. harveyi Y ¼ ð3Þ 1 þ expðÞ −ktðÞ −t wild strain (Fig. 2 a and b) highlighted a dose-dependent effect of palauolide (1) on BB120 bacterial growth. Dur- max Y ¼ ð4Þ ing the growth of V. harveyi bacterial strains, the growth 1 þ aexpðÞ −ktðÞ −t Luminescence was also modeled as a logistic function, following Equation 4, where L is the maximum or max asymptotic value of luminescence. Equation 4 includes an a parameter to adequately model the high steepness found for luminescence curves. For each compound and concentration tested, the parameters of the logistic curve were fitted using the function “nls” of the package “stat” in R.3.1.0. The effect of compounds on the growth and the bioluminescence of V. harveyi populations were eval- uated by comparison of the growth rate (assimilated to the parameter k) and the curve inflection points. For the absorbance kinetics, the inflection point was equal to t . For the luminescence kinetics, the derivative (Y’) of the sigmoid function was calculated, and the inflection point was identified as the time for which Y’ was maximal. Furthermore, to provide comparable values of biolumin- Fig. 1 Chemical structure of palauolide (1) and fascaplysin (2) escence, luminescence values were compared at a fixed Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 5 of 11 Fig. 2 Effect of palauolide (1) on BB120 strain. (a) Absorbance kinetics, (b) luminescence kinetics (RLU), (c) steepness data (k) of absorbance kinetic, (d) luminescence value (RLU) measured for absorbance at 0.055 (represented on (A) by a dashed line, corresponding to the absorbance –1 –1 –1 value at the inflection point of control) without palauolide (black, control), with palauolide 1 μgml (blue, C4), 5 μgml (green, C3), 10 μgml –1 (orange, C2), and 50 μgml (red, C1). Data are reported as means ± SD from three technical replicates (*significant Kruskall-Wallis p value < 0.05 by comparing with control) rate (k parameter) of absorbance increased as the con- 50 min in average. These results indicate that palauolide centration of palauolide (1) increased (Table 1, Fig. 2c). (1) boosted bacterial growth and inhibited V. harveyi QS As a consequence, the sigmoid midpoint (t ) decreased through HAI-1 QS pathway. as concentration of palauolide (1) increased (data not –1 shown). At 50 μgml of palauolide (1), the growth rate Effect of fascaplysin of absorbance (k = 0.0127 ± 0.0005) reached values sig- Vibrio harveyi BB120 population growth rate (k, see Eq. nificantly higher than for controls (k = 0.0086 ± 0.0008; 3) was significantly lower with fascaplysin (2)at50 μgml multiple comparison test after Kruskal-Wallis; p < 0.05). (k = 0.0021) compared to control (k = 0.0121; p value < Also not significant due to the lack of statistical power, 0.05). Similar results were obtained for mutant JAF 375, similar trends were obtained for the three derived QS with lower growth rate (k = 0.0036) and with fascaplysin –1 mutants (Table 1). Despite the stimulating effect of (2)at 50 μgml compared to control (k = 0.0119). Strong palauolide (1)on V. harveyi growth, a delay in lumines- decreases of population growth rate were also obtained cence activation of approximately 17 min was observed for mutants JMH 597 and JMH 612 with fascaplysin (2)at –1 –1 for the highest concentrations tested 50 μgml , com- 50 μgml compared to control. For several replicates in- pared to the luminescence curve of the control (Fig. 2b, volving the two last mutants, population growth was null –1 red and black curves, respectively). At the same growth or negative with fascaplysin (2)at 50 μgml , which pre- stage (A = 0.055), a decrease in RLU was observed for vented the growth model to be fitted and k estimates to the highest concentration of palauolide (1) compared to be provided (Table 2; Additional file 1). This suggests an control. Such decrease was found for the BB120 wild antibiotic effect of fascaplysin (2)on V. harveyi and pre- strain (RLU respectively at 106 210 ± 24 385 at 50 μg vents concluding on a QS inhibition effect. –1 ml (26 μM) of palauolide (1) compared to 172 416 (± 2 489) for control; Table 1; Fig. 2d) and the JMH 612 Antibiotic bioassay mutant only (RLU respectively at 99 806 ± 18 002 at 50 Palauolide did not display any antibiotic activity against –1 μgml (26 μM) of palauolide (1) compared to 189 392 the marine pathogen T. maritimum. By contrast fasca- ± 2 609 for control; Table 1; Fig. 2d). For the JMH 612 plysin (2) displayed antibiotic activity at 0.25 μg per disk mutant, the delay between the luminescence kinetics at (11 mm) and 0.5 μg per disk (18 mm) against T. mariti- –1 50 μgml and the luminescence kinetics of control was mum (TFA4) (disk diffusion bioassay). Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 6 of 11 Table 1 Steepness of absorbance kinetic (k) and luminescence value measured for absorbance at 0.055 (RLU) estimated for various concentration of palauolide (1) and Vibrio harveyi strains Strain Concentration of palauolide Replicate k RLU BB120 Control 1 0.0093 173985 2 0.0088 173717 3 0.0077 169546 All 0.0086 ( ± 0.0008) 172416 ( ± 2489) C4 1 0.0100 173727 2 0.0104 174212 3 0.0093 172441 All 0.0099 ( ± 0.0006) 173460 ( ± 915) C3 1 0.0105 178786 2 0.0116 179795 3 0.0107 179754 All 0.0110 ( ± 0.0006) 179445 ( ± 571) C2 1 0.0119 174835 2 0.0116 178950 3 0.0117 178843 All 0.0117 ( ± 0.0001) 177542 ( ± 2345) C1 1 0.0122 107211 2 0.0131 81339 3 0.0129 130080 All 0.0127 ( ± 0.0005) 106210 ( ± 24385) JAF 375 Control 1 0.0144 131953 2 0.0140 134519 3 0.0135 131708 All 0.0139 ( ± 0.0005) 132727 ( ± 1557) C4 1 0.0160 129253 2 0.0168 131105 3 0.0183 130501 All 0.0170 ( ± 0.0012) 130286 ( ± 944) C3 1 0.0151 146894 2 0.0154 146197 3 0.0171 145802 All 0.0159 ( ± 0.0011) 146298 ( ± 553) C2 1 0.0172 140852 2 0.0163 141282 3 0.0160 140905 All 0.0165 ( ± 0.0006) 141014 ( ± 235) C1 1 0.0170 159632 2 0.0179 158643 3 0.0165 158767 All 0.0171 ( ± 0.0007) 159014 ( ± 539) JMH 597 Control 1 0.0094 147880 2 0.0081 146686 3 0.0100 147955 Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 7 of 11 Table 1 Steepness of absorbance kinetic (k) and luminescence value measured for absorbance at 0.055 (RLU) estimated for various concentration of palauolide (1) and Vibrio harveyi strains (Continued) Strain Concentration of palauolide Replicate k RLU All 0.0091 ( ± 0.0010) 147507 (± 712) C4 1 0.0120 150354 2 0.0118 147321 3 0.0121 147103 All 0.0119 ( ± 0.0001) 148259 ( ± 1818) C3 1 0.0132 154515 2 0.0138 152906 3 0.0140 153875 All 0.0137 ( ± 0.0004) 153765 ( ± 810) C2 1 0.0129 152211 2 0.0123 151867 3 0.0122 152261 All 0.0125 ( ± 0.0003) 152113 ( ± 214) C1 1 0.0159 150802 2 0.0183 161802 3 0.0145 157737 All 0.0162 ( ± 0.0019) 156780 ( ± 5562) JMH 612 Control 1 0.0057 186864 2 0.0083 192076 3 0.0087 189235 All 0.0076 ( ± 0.0017) 189392 ( ± 2609) C4 1 0.0074 186349 2 0.0076 189886 3 0.0087 189302 All 0.0079 ( ± 0.0007) 188512 ( ± 1896) C3 1 0.0088 187819 2 0.0076 174498 3 0.0078 184672 All 0.0081 ( ± 0.0007) 182330 ( ± 6963) C2 1 0.0089 190911 2 0.0086 188991 3 0.0089 188746 All 0.0088 ( ± 0.0002) 189549 ( ± 1186) C1 1 0.0106 120398 2 0.0097 91967 3 0.0091 87053 All 0.0098 ( ± 0.0008) 99806 ( ± 18002) –1 Control, C4, C3, C2, and C1, respectively, refer to palauolide (1) concentration at 0, 1, 5, 10, and 50 μgml . Estimated values of k and RLU are provided for each replicate (n = 3) and for all replicates (mean ± standard deviation over the three replicated). Values significantly different from controls (α = 0.05) are marked with a star (*) Fish toxicity assay periods) within the first hour of treatment. None motility –1 –1 At 50 μgml of FEF, P. reticulata exhibited signs of disarrangement was observed at 1 μgml FEF solutions, hyperventilation as well as motility disarrangement (i.e., but changes to the feeding behavior were noticed for P. jerky movements with sudden accelerations or motionless reticulata,i.e., P. reticulata tasted the food flakes but did Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 8 of 11 Table 2 Steepness of absorbance kinetic (k) estimated with time. Palauolide (1) revealed a potential as QSI by inhi- –1 fascaplysin (2) at 50 μgml (C1), and without fascaplysin biting V. harveyi luminescence at 26 μM. In quantitative (control), for the various Vibrio harveyi strains analysis, palauolide (1) delayed the activation of bio- Strain Dose Replicate k luminescence expression to 50 min of V. harveyi BB120. BB120 Control 1 0.0118 The V. harveyi growth rate was also significantly in- creased (p value < 0.05). The boosted growth rate of V. 2 0.0123 harveyi with palauolide (1) can be interpreted as a con- All 0.0121 sequence of QS inhibition, because the expression of C1 1 0.0024 bioluminescence slows down bacterial growth rate to 2 0.0017 save energy (Nackerdien et al. 2008). The present data All 0.0021 corroborates well with the result obtained previously on JAF 375 Control 1 0.0117 the QSI at 23 μM of isonaamidine A isolated from the sponge Leucetta chagosensis (Mai et al. 2015). Other 2 0.0120 studies compared bioluminescence data at a time t,to All 0.0119 determine the inhibition of QS (Brackman et al. 2008; C1 1 0.0051 Teasdale et al. 2009; Natrah et al. 2011). For example, 2 0.0021 Brackman et al. (2008) showed inhibition of V. harveyi All 0.0036 bioluminescence with cinnamaldehyde and derivatives at JMH 597 Control 1 0.0108 100 μM, 6 h after the addition of compounds (Brackman et al. 2008). Skindersoe et al. (2008) found that manoa- 2 0.0118 lide, a compound of similar structure to palauolide (1), All 0.0113 inhibits QS at IC = 0.66 μM. The better bioactivity of C1 1 0.0019 manoalide compared to palauolide (1) could be ex- 2<0 plained from the sensitivity of the intracellular bioassay All - used by authors. JMH 612 Control 1 0.0137 Themodeofaction of palauolide (1) on the inhibition of QS has potential as an antibiotic alternative in aquaculture 2 0.0119 for Vibrio species. Our bioassay on V. harveyi double mu- All 0.0128 tants JAF 375, JMH 597, and JMH 612 highlighted interfer- C1 1 < 0 ence of palauolide (1)on V. harveyi QS, specifically with 2<0 the acyl-homoserine lactone: HAI-1. Quorum sensing reg- All - ulates bioluminescence and virulence factors of bacteria through autoinducers (Henke and Bassler 2004a)suchas –1 not ingest them. At 5 μgml of FEF, all of the P. reticu- HAI-1 used for intraspecies communication (Waters and lata died within 12 h. Bassler 2005;Yang et al. 2011). Acyl-homoserine lactone The experiment on A. triostegus was only performed molecules are found in the family Vibrionaceae (Yang –1 at 1 μgml of FEF. For each time of incubation (24, 48, et al. 2011). Palauolide (1) can therefore interfere with Vib- and 72 h), the number of bites of A. triostegus (both re- rio species QS through HAI-1 pathway and then be used cruits and juveniles) decreased significantly compared to as an antivirulent against Vibrio species as antagonist of control A. triostegus (Fig 3). After 24 h of incubation AIs. Most of antagonists of QS sensors are small molecules –1 with 1 μgml FEF solution, the number of bites de- (Swem et al. 2008; Gamby et al. 2012) with structural simi- creased by 91.3% (± 1. 6%, p value < 0.01) for recruits larities to AIs such as brominated furanone derivatives and by 95.9% (±0.8%, p value < 0.001) for juveniles com- (Givskov et al. 1996; Rasch et al. 2004; Steenackers et al. pared to the control A. triostegus (Fig 3). This trend was 2010). Palauolide (1) is a sesterterpene composed by a δ- confirmed for others times of exposition. hydroxybutenolide moiety and a carbon skeleton. The po- tential of palauolide as a competitor of HAI-1 is most likely Discussion due to its small structure and the moderate polarity of its The isolation of palauolide (1) and the major compound chemical structure. This enables palauolide (1)tocross fascaplysin (2) from the French Polynesian F. cf reticulata over the external membrane lipid of bacteria and to bind extracts is similar to those results obtained by Sullivan on the periplasmic sensors Lux N (Swem et al. 2008). and Faulkner (1982) on Palauan sponges. Further research would indicate if there is an antagonist The QSI potential of the French Polynesian sponge F. effect of palauolide (1) on the HAI-1 sensor, such as cf reticulata against the QS-dependent phenotypic ex- testing against additional V. harveyi mutants (Swem pression in V. harveyi was demonstrated for the first et al. 2008; Blair and Doucette 2013). Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 9 of 11 Fig. 3 Number of bites on coral pieces of Acanthurus triostegus (a) juveniles and (b) recruits per hour without FEF, fascaplysin-enriched fraction –1 (C), with ethanolic solvent (S), with fascaplysin-enriched fraction powder (FEF) at 1 μgml . Error bars represent standard deviation of the mean (N = 6) (**p value < 0.01 significant, *** p value < 0.001 very significant compare to control without fascaplysin-enriched fraction (C)s) Fascaplysin (2) supplies a broad range of biological activ- QSIs help and increase antibioticactiononbiofilm forma- ity within F. cf reticulata.First, asother β-carboline alka- tion (Brackman et al. 2011). However, the toxicity on fish loids as dysideanin (20 μg) and didemnolines A-D (100 of the major compound of F.cf reticulata fascaplysin (2) μg), fascaplysin is a strong antibiotic (0.25 μg) (Charan (yield 0.02% w/w) prevents the use of the sponge extract in et al. 2002; Hamilton 2014). In the sponge, fascaplysin (2) fish farming context. We recommend in future research to is the major compound which represents 0.02% of the ly- test toxicity of the cyclohexanic fraction of the sponge and ophilized sponge weigh. It exhibits many biological activ- palauolide (1) on fish before concluding on the potential of ities including cytotoxicity against tumoral cells (Segraves the cyclohexanic fraction and palauolide (1)asanalterna- et al. 2004;Shafiqetal. 2012; Hamilton 2014; Cells et al. tive to antibiotics in fish farming. 2015; Kumar et al. 2015), antimicrobial activities (Roll et al. Acknowledgements 1988), and the inhibition of acetylcholinesterase (Bharate This work is part of TM’s PhD thesis. We acknowledge ANR, Netbiome, et al. 2012; Manda et al. 2016). For microbial disease treat- French Polynesian authorities, and Délégation à la Recherche-France in Tahiti for their grant, help, and support, IRD and GOPS for funding the field trips ments in aquaculture, fascaplysin (2) is not ideal. Despite aboard R/V ALIS, her crew and IRD’s diving team for their on-field help, and its antibiotic activity against marine pathogens V. harveyi Laboratoire d’Excellence LabEx CORAIL, 66100 Perpignan, France. We would (Table 2)and T. maritimum, fascaplysin (2)istoxic toward also like to thank Johanna Loacker and Christina Piotrowski from Calacademy for their help with the loan of some comparative material. both fresh and saltwater fish, P. reticulata and A. triostegus, respectively. Indeed, fascaplysin (2) modified fish behavior Author’s contribution and displayed an anorexic effect. The AchE inhibition CD supervised sponge sampling and TM’s PhD thesis. SP supervised the setup of the extract library for biological screening. ME, KH, and DE identified properties of fascaplysin (Bharate et al. 2012) could explain sponge samples. TM and MAB carried out chemical experiments. JT, CD, MB, both its toxicity (Bocquené and Galgani 2004;Modesto and DL carried out fish toxicity bioassay, and TM, EA, DS, and CD carried out and Martinez 2010; Assis et al. 2012) and its effect on loss antibiotic and quorum sensing bioassays. SVW was in charge of statistical analyses. TM wrote the manuscript. All authors reviewed and approved the of appetite of fish (Schneider 2000). manuscript. The toxicity of palauolide (1) on fish was not tested in this study because previous work highlighted a weaker Funding This research was supported by the research grant ANR-11-EBIM-006-1 cytotoxic activity of palauolide (1) compared to fascaply- (Project: POMARE) from ANR and Netbiome, and the grant 113-163 (Project: sin (2) (Charan et al. 2002; Hamilton 2014). However, Biopolyval) from French Polynesian authorities and Délégation à la we recommend performing additional toxicity bioassays Recherche-France in Tahiti. of palauolide (1) on fish before using it as alternative of Availability of data and materials antibiotic in fish farming. Not applicable. Ethics approval and consent to participate Conclusion Not applicable. In conclusion, the presence of palauolide (1) and fascaply- sin (2)in F.cf reticulata, with QS inhibition and antibiotic Consent for publication properties, respectively, could act as complementary where Not applicable. Mai et al. Fisheries and Aquatic Sciences (2019) 22:30 Page 10 of 11 Competing interests Debitus C. TUAM 2011. 2011. http://dx.doi.org/https://doi.org/10.17600/11100010. The authors declare that they have no competing of interest. Dobretsov S, Teplitski M, Bayer M, Gunasekera S, Proksch P, Paul VJ. Inhibition of marine biofouling by bacterial quorum sensing inhibitors. Biofouling. 2011; Author details 27(8):893–905. https://doi.org/10.1080/08927014.2011.609616. EIO, ILM, IFREMER, IRD, UPF, BP 6570, 98702 FAA’A, Tahiti, French Polynesia. Editorials. The antibiotic alarm. Nature. 2013;495:141. 2 3 IRD, Univ. Brest, CNRS, IFREMER, LEMAR, F-29280 Plouzane, France. 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