The Effects of Nisin-Producing Lactococcus lactis Strain Used as Probiotic on Gilthead Sea Bream (Sparus aurata) Growth, Gut Microbiota, and Transcriptional Response

fmars-08-659519 April 9, 2021 Time: 13:57 # 1 ORIGINAL RESEARCH published: 12 April 2021 doi: 10.3389/fmars.2021.659519 Edited by: Fotini Kokou, Wageningen University and Research, Netherlands Reviewed by: Carlo C. Lazado, Norwegian Institute of Food, Fisheries and Aquaculture Research (Nofima), Norway Yun-Zhang Sun, Jimei University, China *Correspondence: Genciana Terova [email protected] †These authors have contributed equally to this work Specialty section: This article was submitted to Marine Fisheries, Aquaculture and Living Resources, a section of the journal Frontiers in Marine Science Received: 27 January 2021 Accepted: 22 March 2021 Published: 12 April 2021 Citation: Moroni F, Naya-Català F, Piazzon MC, Rimoldi S, Calduch-Giner J, Giardini A, Martínez I, Brambilla F, Pérez-Sánchez J and Terova G (2021) The Effects of Nisin-Producing Lactococcus lactis Strain Used as Probiotic on Gilthead Sea Bream (Sparus aurata) Growth, Gut Microbiota, and Transcriptional Response. Front. Mar. Sci. 8:659519. doi: 10.3389/fmars.2021.659519 The Effects of Nisin-Producing Lactococcus lactis Strain Used as Probiotic on Gilthead Sea Bream (Sparus aurata) Growth, Gut Microbiota, and Transcriptional Response Federico Moroni1†, Fernando Naya-Català2†, M. Carla Piazzon3, Simona Rimoldi1, Josep Calduch-Giner2, Alberto Giardini4, Inés Martínez5, Fabio Brambilla6, Jaume Pérez-Sánchez2and Genciana Terova1* 1Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy, 2Nutrigenomics and Fish Growth Endocrinology, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain, 3Fish Pathology, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain, 4Centro Sperimentale del Latte S.r.l., Zelo Buon Persico, Italy, 5Sacco S.r.l., Cadorago, Italy, 6VRM S.r.l. Naturalleva, Cologna Veneta, Italy The present research tested the effects of dietary nisin-producing Lactococcus lactis on growth performance, feed utilization, intestinal morphology, transcriptional response, and microbiota in gilthead sea bream (Sparus aurata). A feeding trial was conducted with fish weighting 70–90 g. Fish were tagged with passive, integrated transponders and distributed in nine 500 L tanks with 40 fish each. Fish were fed for 12 weeks with either a control (diet A) or experimental diets (diets B and C) in triplicate (3 tanks/diet). Extruded pellets of diets B and C were supplemented with a low (2 ×109CFU/kg) and a high (5 ×109CFU/kg) dose of probiotic, respectively. No significant differences were found between groups for the feed conversion ratio or specific growth rates. However, the final body weight of fish fed diet C was significantly higher than the control group with intermediate values for fish fed diet B. Histological analysis conducted using a semi-quantitative scoring system showed that probiotic did not alter the morphology of the intestine and did not trigger inflammation. With regard to the transcriptomic response, a customized PCR array layout was designed to simultaneously profile a panel of 44 selected genes. Significant differences in the expression of key genes involved in innate and acquired immunity were detected between fish fed probiotic and control diets. To analyze the microbiota associated to the feeds and the gut autochthonous microbial communities, we used the Illumina MiSeq platform for sequencing the 16S rRNA gene and a metagenomics pipeline based on VSEARCH and RDP databases. The analysis of gut microbiota revealed a lack of colonization of the probiotic in the host’s intestinal mucosa. However, probiotic did modulate the fish gut microbiota, confirming that colonization is not always necessary to induce host modification. In fact, diets B and C were enriched with Actinomycetales, as compared to diet A, which instead showed a Frontiers in Marine Science | www.frontiersin.org 1April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 2 Moroni et al. LAB-Fortified Feed for Fish higher percentage of Pseudomonas,Sphyngomonas, and Lactobacillus genera. These results were confirmed by the clear separation of gut bacterial community of fish fed with the probiotic from the bacterial community of control fish group in the beta-diversity and PLS-DA (supervised partial least-squares discriminant analysis) analyses. Keywords: aquaculture, gilthead sea bream, probiotic, Lactococcus lactis, gut microbiota, transcriptomic INTRODUCTION The definition of “Probiotics” has changed many times during this century. However, according to (Food and Agriculture Organization of the United [FAO] and World Health Organisation [WHO], 2001) probiotics are “live microorganisms that confer a health benefit on the host when administered in adequate amounts.” The most commonly used probiotics are bacteria belonging to Lactobacillus,Bifidobacterium,Bacillus, and Enterococcus genera (European Medicines Agency [EMA], and European Food Safety Authority [EFSA], 2017;EFSA FEEDAP [EFSA Panel on Additives and Products or Substances used in Animal Feed] et al., 2018), but some fungal genera have also been reported as novel probiotics. In the last 25–30 years, the use of probiotics in animal production has increased (Chaucheyras-Durand and Durand, 2010;Ezema, 2013). Indeed, several publications have reported numerous beneficial effects associated with the supplementation of live yeast or bacteria (mostly Lactobacillus) in the diet of terrestrial animals, including amelioration of resistance to pathogens, improvement in growth parameters (in swine and poultry), increase in productivity and quality of eggs in laying hens, and enhancement of milk production in cattle (Gallazzi et al., 2008;Shabani et al., 2012;Puphan et al., 2015;Uyeno et al., 2015;De Cesare et al., 2017;Wang et al., 2017;Dowarah et al., 2018;Forte et al., 2018). In aquaculture, a great number of bacterial species are currently used as probiotics (for a review, please see NewajFyzul et al., 2014). These microorganisms can be administered as multi-species (multi-strain) or single-species (single-strain) (Food and Agriculture Organization of the United [FAO], 2016) and provided either as a suspension in water, or added to the feed. However, use in feed is considered the best option; therefore, this approach is employed most frequently (Nayak, 2010;Jahangiri and Esteban, 2018). In the European Union (EU), probiotic strains, must obtain a market authorization by the EFSA (European Safety Food Authority)1, which grants a QPS (Qualified Presumption of Safety) status. The QPS is based on reasonable evidence. No microorganism belonging to a QPS status group needs to undergo a full safety assessment, but microorganisms that pose a safety concern to humans, animals, or environment are not considered suitable for QPS status and must undergo a full safety assessment. The QPS assessment requires: (1) the identity of the strain to be conclusively established, and (2) absence of resistance to antibiotics (for bacteria) or antimycotics (for yeasts) used in 1https://www.efsa.europa.eu/en human and veterinary medicine (EFSA Panel on Biological Hazards (BIOHAZ) et al., 2020). The increase in the use of probiotics in aquaculture is mostly related to the need to decrease or even avoid the use of antibiotics, increasing at the same time the sustainability of the aquaculture industry. The negative effects of antibiotics overuse include the accumulation of residue in the aquatic environment, particularly in the marine sediments where antibiotics can persist for months, favoring the selection of multi-antibiotic-resistant bacterial strains. Indeed, there is an increasing risk that antibiotic-resistant bacteria, initially derived from food-producing animals, could render the latest generation of antibiotics virtually ineffective for humans (Cabello, 2006;World Health Organisation [WHO] et al., 2006). Another negative outcome of antibiotics being used as growth promoters in cultured fish is the reduction of biodiversity and quantity of indigenous gut microbiota, which can impair fish immune responses (Borch et al., 2015). For these reasons, the use of antibiotics as growth promoters in animal production has been fully banned in the EU since 2006 (Casewell et al., 2003;European Parliament and the Council of the European Union, 2003, 2019;European Medicines Agency [EMA], and European Food Safety Authority [EFSA], 2017) and many research efforts have been undertaken to replace them with probiotics for animal health management (Ezema, 2013). Several studies have demonstrated that probiotics can reduce pathogenic bacteria due to direct competition-colonizing dynamics, through which microorganisms can partition spatial niche habitats in the intestinal mucosa (Balcázar et al., 2007b; Sugimura et al., 2011). Probiotics can also produce inhibitory molecules, such as bacteriocins, siderophores, enzymes, and hydrogen peroxide, or inhibit pathogenic bacteria by decreasing the intestinal pH through the release of organic acids (Ringø, 2008;Zhou X. et al., 2010;Ustyugova et al., 2012;Perez et al., 2014; Dahiya et al., 2020). In addition, probiotics enhance the host immune system by generating systemic and/or local responses (Balcázar et al., 2006b;Salinas et al., 2008) that include activation of various antioxidant pathways and an increase in several innate immune parameters, such as phagocytosis, lysozyme levels, respiratory burst peroxidase and antiprotease activity, cytokine production, and white blood cell count (Nayak, 2010;Lazado and Caipang, 2014;Newaj-Fyzul et al., 2014;Simó-Mirabet et al., 2017). In cultured fish, probiotics improve fish growth and feed conversion rates, too, due to an increase in feed digestibility and absorption of nutrients (Dimitroglou et al., 2011;Martínez Cruz et al., 2012). These effects stem from the capacity of probiotics to secrete enzymes, such as proteases, amylases, and lipases that hydrolyze molecules, which the fish intestine cannot Frontiers in Marine Science | www.frontiersin.org 2April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 3 Moroni et al. LAB-Fortified Feed for Fish otherwise digest (Balcázar et al., 2006b;Abd El-Rhman et al., 2009). Furthermore, the use of probiotics can restore the eubiotic state of the intestinal microbiota after antibiotic treatment or a pathogenic insult or can help maintain gut microbiota homeostasis, even in larval stages, when vaccination is difficult (Abdelhamid et al., 2009;Borch et al., 2015). Hence, positive effects of different probiotics have been reported in several fish species, such as Nile tilapia (Oreochromis niloticus) (Ridha and Azad, 2012), common carp (Cyprinus carpio) (Feng et al., 2019), African catfish (Clarias gariepinus) (Al-Dohail et al., 2009), olive flounder (Paralichthys olivaceus) (Heo et al., 2013), Asian sea bass (Lates calcarifer) (Ringø, 2008; Lin et al., 2017), red drum (Sciaenops ocellatus) (Zhou Q.C. et al., 2010), European sea bass (Dicentrarchus labrax) (Carnevali et al., 2006;Mahdhi, 2012), common dentex (Dentex dentex) (Hidalgo et al., 2006), gilthead sea bream (Sparus aurata) (Suzer et al., 2008;Varela et al., 2010), rainbow trout (Oncorhynchus mykiss) (Merrifield et al., 2010), and abalone (Haliotis midae) (Macey and Coyne, 2005), and in crustaceans, such as white shrimp (Litopenaeus vannamei) (Lin et al., 2004). According to the above findings, the aim of the present research was to evaluate the effects of the lactic acid bacteria Lactococcus lactis subsp. lactis SL242, used as feed additive, on growth performance, feed utilization, intestinal morphology, transcriptional response, and microbiota in gilthead sea bream (Sparus aurata). The probiotic strain L. lactis subsp. lactis SL242 was selected due to important characteristics of Lc. lactis in general and SL242 in particular. Lc. lactis are mesophilic lactic acid bacteria that are present in the intestinal microbiota of fish (Tarnecki et al., 2017;Ringø et al., 2020) and can adapt to the water temperature of many reared fish species. Lactococci are proteolytic bacteria (Samaržija et al., 2001) that are potentially useful for improving the digestion of proteins contained in fish feed. The proteolytic system of lactococci includes a cell wall-associated proteinase and an extracellular peptidase (Samaržija et al., 2001). Furthermore, SL242 produces the antibiotic nisin A (Malvisi et al., 2016), which can inhibit or kill vegetative cells and bacterial spores (European Safety Food Authority [EFSA], 2005). Due to its antibacterial activity, nisin is of great interest in aquaculture. Nisin-susceptible bacterial species are found among Bacillus,Clostridium,Listeria, Staphylococcus,Streptococcus, and Vibrio genera (European Safety Food Authority [EFSA], 2005;Malvisi et al., 2016;Hamid et al., 2020), including known aquatic pathogens, such as V. parahaemolyticus, and V. alginotlyticus (Hamid et al., 2020). Lc. lactis probiotics have also shown inhibitory action against Yersinia rukeri and Aeromonas salmonicida, which can affect fish growth (Balcázar et al., 2007a, 2006b). Furthermore, Lc. lactis probiotic has been effective against Aeromonas hydrophila in Oreochromis niloticus (Zhou X. et al., 2010). MATERIALS AND METHODS Ethics Statement Procedures for fish manipulation and tissue collection were carried out according to the Spanish (Royal Decree RD53/2013) and the current EU legislation (2010/63/EU) for handling of experimental fish. All procedures were approved by the Ethics and Animal Welfare Committees of Institute of Aquaculture Torre de la Sal (IATS-CSIC, Castellón, Spain) (Permit number 824/2019) and “Generalitat Valenciana” (permit number 2019/VSC/PEA/0197). Animals On June 2019, juveniles of gilthead sea bream were purchased from a Mediterranean hatchery (Piscimar, Burriana, Spain) and adapted for more than 2 months to the indoor experimental facilities of IATS-CSIC, under natural photoperiod and temperature conditions (40◦50N; 0◦100E). Seawater was pumped ashore (open system); oxygen content of water effluents was always above 85% saturation, and unionized ammonia remained below 0.02 mg/L. During the acclimation and experimental period, water temperature increased from 20–22◦C in June to 28◦C in August, decreasing thereafter from 24–25◦C in mid-September to 13–16◦C in December. Diets Extruded pellets of a control (diet A) and two experimental diets (diets B and C) were manufactured by VRM Srl Naturalleva (Verona, Italy), mimicking commercial fish feed formulations with traditional vegetable proteins and oils as the main replacers of fishmeal and fish oil, respectively (Table 1). The mash of each diet was extruded using a single-screw extruder (X-165, Wenger United States). To ensure product stability, the probiotic was homogenized with the dietary oil and included by vacuum coating (La Meccanica vacuum coater, Italy) during the postextrusion process. During the vacuum process, only dry basal extruded pellets of diets B and C were supplemented with 2.5 and 6.2 g/100 kg of L. lactis subsp. lactis SL242, corresponding to a probiotic dosage of 2 ×109CFU/kg (low dose) and 5×109CFU/Kg (high dose), respectively. Sacco S.r.l [Cadorago (Co), Italy] provided the probiotic strain. The two doses were chosen on the basis of our experience and literature data (Villamil et al., 2002;Adel et al., 2017) in order to verify the most effective one. They are also in line with dosages that could be used commercially in a cost-effective manner. The final feeds were stored in a refrigerated room (6–7◦C) for the entire duration of the feeding trial. A preliminary stability study of SL242 in the feed supplemented with probiotic was conducted for 12 weeks (the duration of the experiment), at 6◦C. At the end of this period, the average loss of viability determined by plate count resulted about 50%, consistent with our expectations. Although further improvement may be warranted for a commercial probiotic product, at this stage of the process, the observed stability is considered acceptable. Feeding Trial In September 2019, fish weighing 70–90 g were randomly distributed in nine 500 L tanks to establish triplicate groups of 40 fish each (initial rearing density, 6.6–6.7 kg/m3). All fish were tagged with PIT (passive integrated transponders) (ID-100A 1.25 Nano Transponder, Trovan) in the dorsal skeletal muscle. Fish were individually weighed and measured at initial, intermediate, Frontiers in Marine Science | www.frontiersin.org 3April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 4 Moroni et al. LAB-Fortified Feed for Fish TABLE 1 | Ingredients and chemical composition (%) of control diet (Diet A) used in the trial. Ingredients Diet A Fishmeal 10.1 Corn gluten 24.3 Guar germ meal 10.0 Soybean meal 13.1 Soya protein concentrate 13.6 Wheat 10.8 Fish oil 7.5 Rapeseed oil 3.5 Camelina oil 3.5 Lactic bacteria 0.0 Lysine 0.9 DL-methionine 0.4 Monoammonium phosphate 1.2 Taurine 0.4 Vitaminsaand Mineralsb0.7 Proximate composition (%) Gross energy (MJ/kg) 18.92 Digestible energy, DE (MJ/kg) 17.26 Crude fat 18.0 Crude protein 43.8 Digestible protein, DP 38.8 DP/DE (mg/kJ or g/MJ) 22.5 Fiber 2.6 Nitrogen free extract 24.6 Starch 8.7 Non-starch polysaccharides 18.5 Diet B and C were formulated with the addition of probiotic (5 ×106CFU/g feed). aVitamin premix (IU or mg/kg diet): DL-αtocopherol acetate 60 IU; sodium menadione bisulfate 5 mg; retinyl acetate 15,000 IU; DL-cholecalciferol 3,000 IU; thiamine 15 mg; riboflavin 30 mg; pyridoxine 15 mg; vitamin B12 0.05 mg; nicotinic acid 175 mg; folic acid 500 mg; inositol 1,000 mg; biotin 2.5 mg; calcium pantothenate 50 mg. bMineral premix (g or mg/kg of diet) bi-calcium phosphate 500 g, calcium carbonate 215 g, sodium salt 40 g, potassium chloride 90 g, magnesium chloride 124 g, magnesium carbonate 124 g, iron sulfate 20 g, zinc sulfate 4 g, copper sulfate 3 g, potassium iodide 4 mg, cobalt sulfate 20 mg, manganese sulfate 3 g, sodium fluoride 1 g. and final sampling points (every 4 weeks), by using a FR-200 Fish Reader W (Trovan, Madrid, Spain) for data capture and preprocessing. The trial lasted 12 weeks (October 2019–December 2019). Fish were hand-fed once daily (12 a.m.), 5–6 days per week to visual satiety with either control or experimental diets for the entire duration of the trial. Feed intake and mortalities (<1%) were recorded daily and normal fish behavior was assessed routinely by camera monitoring. Sample Collection At the end of the feeding trial, four fish per replicate (12 fish/diet) were anesthetized with 0.1 g/L of tricaine-methasulfonate (MS222, Sigma-Aldrich) and then sacrificed by severing the spinal cord. The intestine (excluding the pyloric ceca) of each fish was dissected out, weighed, and measured aseptically to calculate the intestine weight index (IWI) and intestine length index (ILI). Then, anterior (AI) and posterior (PI) intestine tissue portions (∼0.4 cm) were put either into RNAlater, or in 10% neutral buffered formalin for subsequent molecular (AI) and histological (AI, PI) analyses. The remaining part of AI was opened and washed with sterile Hank’s balanced salt solution before collecting the autochthonous intestinal bacteria by scraping intestinal mucosa with the blunt end of a clean scalpel. Then, mucus samples were transferred to a sterile Eppendorf tube and stored in ice until subsequent (within 2 h) DNA extraction for microbiota analysis. To characterize feed-associated bacterial communities, two samples of 200 mg each from each feed were taken at the end of the trial and used for bacterial DNA extraction and sequencing. Histological Analysis Fixed samples of AI and PI were dehydrated in ethanol solutions with gradually increasing concentrations and then, embedded in paraffin. Sections of 5 µm were obtained with a microtome (Leica RM2245) and stained with hematoxylin and eosin (H&E), following standard histological protocols. The sections were examined under a stereomicroscope Eurotek Tecno NB50T (Orma Srl, Milan, Italy) and photographed with a digital camera Eurotek CMOS MDH5 (Orma Srl, Milan, Italy). Based on previous studies (Knudsen et al., 2007;Uran et al., 2008;Urán et al., 2009;Khojasteh, 2012), the semi-quantitative scoring system focused on five different gut morphological parameters (mucosal folds, connective tissue, lamina propria of simple folds, and supranuclear vacuoles). Histological alterations of each morphological parameter were classified using a score value ranging from one (normal condition) to five (severe alteration). The final values, obtained by the sum of score values for each parameter, were then used to classify the severity of the morphological damage by using a class-based scoring system: Class I (values ≤10)—normal tissue structure with slight histological alterations; Class II (values 11–15)—moderate histological alterations; and Class III (values >15)—severe histological alterations of the organ. Gene Expression Analysis Total RNA from AI was extracted using a MagMax-96 total RNA isolation kit (Life Technologies, Carlsbad, CA, United States). The RNA yield was higher than 3.5 µg with absorbance measures (A260/280) of 1.9–2.1. cDNA was synthesized with the HighCapacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, United States), using random decamers and 500 ng of total RNA in a final volume of 100 µL. Reverse transcription (RT) reactions were incubated 10 min at 25◦C and 2 h at 37◦C. Negative control reactions were run without the enzyme. As reported previously (Estensoro et al., 2016), a customized PCR array layout was designed to simultaneously profile a panel of 44 selected genes, including markers of epithelial integrity (11), nutrient transport (4), mucins (3), cytokines (9), immunoglobulins (2), cell markers and chemokines (7), and pattern recognition receptors (8) (Table 2). qPCR reactions were performed using an iCycler IQ Real-Time Detection System (Bio-Rad, Hercules, CA, United States). Diluted RT Frontiers in Marine Science | www.frontiersin.org 4April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 5 Moroni et al. LAB-Fortified Feed for Fish TABLE 2 | PCR-array layout for intestine gene expression profiling. Function Gene Symbol GenBank Epithelial integrity Proliferating cell nuclear antigen pcna KF857335 Transcription factor HES-1-B hes1-b KF857344 Krueppel-like factor 4 klf4 KF857346 Claudin-12 cldn12 KF861992 Claudin-15 cldn15 KF861993 Cadherin-1 cdh1 KF861995 Cadherin-17 cdh17 KF861996 Tight junction protein ZO-1 tjp1 KF861994 Desmoplakin dsp KF861999 Gap junction Cx32.2 protein cx32.2 KF862000 Coxsackievirus and adenovirus receptor homolog cxadr KF861998 Nutrient transport Intestinal-type alkaline phosphatase alpi KF857309 Liver type fatty acid-binding protein fabp1 KF857311 Intestinal fatty acid-binding protein fabp2 KF857310 Ileal fatty acid-binding protein fabp6 KF857312 Mucus production Mucin 2 muc2 JQ277710 Mucin 13 muc13 JQ277713 Intestinal mucin i-muc JQ277712 Cytokines Tumor necrosis factor-alpha tnfαAJ413189 Interleukin 1 beta il1βAJ419178 Interleukin 6 il6 EU244588 Interleukin 7 il7 JX976618 Interleukin 8 il8 JX976619 Interleukin 10 il10 JX976621 Interleukin 12 subunit beta il12 JX976624 Interleukin 15 il15 JX976625 Interleukin 34 il34 JX976629 Immunoglobulins Immunoglobulin M igm JQ811851 Immunoglobulin T igt KX599201 Cell markers and chemokines CD4 cd4-1 AM489485 CD8 beta cd8b KX231275 C-C chemokine receptor type 3 ccr3 KF857317 C-C chemokine receptor type 9 ccr9 KF857318 C-C chemokine receptor type 11 ccr11 KF857319 C-C chemokine CK8/C-C motif chemokine 20 ck8/cl20 GU181393 Macrophage colony-stimulating factor 1 receptor 1 csf1r1 AM050293 Pattern recognition receptors (PRR) Galectin 1 lgals1 KF862003 Galectin 8 lgals8 KF862004 Toll-like receptor 2 tlr2 KF857323 Toll-like receptor 5 tlr5 KF857324 Toll-like receptor 9 tlr9 AY751797 C-type lectin domain family 10 member A clec10a KF857329 Macrophage mannose receptor 1 mrc1 KF857326 Fucolectin fcl KF857331 reactions (×6) were used for qPCR assays in a 25 µL volume in combination with a SYBR Green Master Mix (Bio-Rad, Hercules, CA, United States) and specific primers at a final concentration of 0.9 µM (Supplementary Table 1). The program used for PCR amplification included an initial denaturation step at 95◦C for 3 min, followed by 40 cycles of denaturation for 15 s at 95◦C and annealing/extension for 60 s at 60◦C. All the pipetting operations were executed by means of an EpMotion 5070 Liquid Handling Robot (Eppendorf, Hamburg, Germany) to improve data reproducibility. The efficiency of PCRs (>92%) was checked, and the specificity of reactions was verified by analyzing the melting curves (ramping rates of 0.5◦C/10 s over a temperature range of 55–95◦C), and linearity of serial dilutions of RT reactions (r2>0.98). Fluorescence Frontiers in Marine Science | www.frontiersin.org 5April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 6 Moroni et al. LAB-Fortified Feed for Fish data acquired during the extension phase were normalized by the delta-delta CT method (Livak and Schmittgen, 2001), using beta-actin as housekeeping gene due to its stability in different experimental conditions (average CT between experimental groups varied less than 0.2). Bacterial DNA Extraction The bacterial DNA was extracted from feeds (2 samples/feed) and from intestinal samples (7–10 fish/dietary group). Intestinal mucus samples (200 µl) were treated with 250 µg/ml of lysozyme (Sigma) for 15 min at 37◦C. Then, DNA was extracted using the High Pure PCR Template Preparation Kit (Roche) following the manufacturer’s instructions. DNA concentration, quality, and purity were measured using a NanoDrop 2000c (Thermo Fisher Scientific) and agarose gel electrophoresis (1% w/v in Tris-EDTA buffer). Samples were stored at −20◦C until sequencing. The same procedure was used to extract DNA from the control and experimental feeds (previously ground to a fine powder) to evaluate the concentration of the probiotic supplement. Illumina MiSeq Sequencing and Bioinformatic Analysis The V3-V4 region of the 16S rRNA gene (reference nucleotide interval 341–805 nt) was sequenced using the Illumina MiSeq system (2 ×300 paired-end run) at the Genomics Unit from the Madrid Science Park Foundation (FPCM, Spain). The details on the PCR and sequencing of amplicons have been described elsewhere (Piazzon et al., 2019). Raw sequence data were uploaded to the NCBI (National Center for Biotechnology Information) and Sequence Read Archive (SRA) under NCBI BIOPROJECT ID: PRJNA679278; NCBI BIOSAMPLE ID: SAMN16828235-61; and SRA ACCESSION: SRR13081673-99. Raw forward and reverse reads were quality filtered using FastQC2, and pre-processed using Prinseq (Rahlwes et al., 2019). Terminal N bases were trimmed at both ends and sequences with >5% of total N bases were discarded. Reads that were <150 bp long with a Phred quality score <28 in both of the sequence ends and with a Phred average quality score <26 were excluded. Then, forward and reverse reads were merged using fastq-join (Aronesty, 2013). Bacterial taxonomy was assigned using the Ribosomal Database Project (RDP) release 11 as a reference database (Cole et al., 2014). Reads were aligned with a custom-made pipeline using VSEARCH and BLAST (Altschul et al., 1990; Rognes et al., 2016). Alignment was performed establishing high stringency filters (≥90% sequence identity, ≥90% query coverage). Taxonomic assignment results were filtered and data were summarized in an Operational Taxonomic Units (OTUs) table. Sample depths were normalized by total sum scaling and then made proportional to the total sequencing depth, following previously described recommendations (McKnight et al., 2019). Species richness estimates and alpha diversity indexes were calculated using the R package Phyloseq (Mcmurdie and Holmes, 2013). Rarefaction curves were obtained by plotting 2http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ the number of observed taxonomic assignations in an OTU table against the number of sequences in each sample using the R package phyloseq. Inferred Metagenome and Pathway Analysis Piphillin was used to normalize the amplicon data by 16S rRNA gene copy number and to infer the metagenomics content (Iwai et al., 2016). This analysis was performed with the OTUs significantly driving the separation by probiotic in the PLS-DA analysis (described in the section “Statistics”). For the analysis, a sequence identity cut-off of 97% was implemented, and the inferred metagenomics functions were assigned using the Kyoto Encyclopedia of Genes and Genomes database (KEGG, Oct 2018 Release). Raw KEGG pathway output from Piphillin was analyzed with the R Bioconductor package DESeq2 using default parameters, after flooring fractional counts to the nearest integer (Love et al., 2014;Bledsoe et al., 2016;Piazzon et al., 2019). Comparisons were also performed between different diets to evaluate possible pathway differences across diets. Statistics Data on growth and gene expression were analyzed by oneway ANOVA using SigmaPlot v14 (Systat Software Inc., San Jose, CA, United States). Normality of the data was verified by Shapiro-Wilk test, and Dunn’s post hoc test was used for multiple comparisons between groups. For analysis of qualitative histological data, we conducted the non-parametric Kruskall-Wallis test, followed by Dunn’s test for the multiple comparisons. GraphPad Prism8 (GraphPad Software, Inc., La Jolla, CA, United States) was used for both analyses. Microbiota species richness, alpha diversity indexes, and phylum abundance between experimental groups were determined by KruskalWallis test followed by Dunn’s post hoc test. Beta diversity was tested with permutational multivariate analysis of variance (PERMANOVA), using the non-parametric method adonis from the R package Vegan with 10,000 random permutations. To further study microbiota differences between dietary groups, supervised partial least-squares discriminant analysis (PLSDA) and hierarchical clustering of samples were sequentially applied using EZinfo v3.0 (Umetrics, Umea, Sweden) and hclust function (gplots R package), respectively. Hotelling’s T2 statistic was calculated by employing the multivariate software package, whereby points above the 95% confidence limit for T2were considered as outliers and discarded. Values of normalized counts of OTUs present in 3 or more samples were included in the analyses, and the significant contribution to the group separation was determined by the minimum variable importance in the projection (VIP) values (Wold et al., 2001;Li et al., 2012), which renders an accurate clustering using the average linkage method and Euclidean distance feasible. The quality of the PLS-DA model was evaluated by the parameters R2Y (cum) and Q2 (cum), which indicate the fit and prediction ability, respectively. To assess whether the supervised model was being overfitted, a validation test consisting Frontiers in Marine Science | www.frontiersin.org 6April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 7 Moroni et al. LAB-Fortified Feed for Fish on 600 random permutations was performed using SIMCAP+(v11.0, Umetrics). RESULTS Growth Performance Data on growth performance, feed intake, and feed conversion ratio (FCR) are reported in Table 3. All fish grew efficiently during the first 30 days of the trial (FCR =1.27–1.28), reaching an overall FCR of 1.55–1.60 at the end of trial. The decrease in the length of the day and temperature from October to December should be noted. No statistically significant differences were found between groups for the condition factor and specific growth rates (SGR), although the highest SGR tended to be achieved in fish fed diet C (high dose of probiotic). Indeed, the final body weight of these animals was higher than in the control group (diet A) (P<0.05) with intermediate values for fish fed diet B (low dose of probiotic). Thus, total weight gain varied from 97% in fish fed diet A to 106% in fish fed diet C. Histological and Biometric Scoring Histological analysis of gilthead sea bream intestine was performed according to the aforementioned morphological criteria. The intestinal scoring data are reported in Table 4. The AI (Figures 1A–C) and PI (Figures 1D–F) portions were not affected by probiotic administration. Although the mucosal folds of the PI were significantly different (P<0.05) between groups fed diets A and B, the total scores, calculated for each group, fall within an evaluation of Class I. In particular, the simple and complex folds appeared thin and regularly branched, lamina propria and connective tissue appeared normally proportioned and supranuclear vacuoles were numerous and well-distributed. Regarding the index of intestine length (ILI) (Table 4), diet B showed a significantly lower ILI than the control group (diet A) (P<0.05), but no differences were observed between the other groups. No differences in the intestine weight index (IWI) were observed between groups. Gene Expression Profiling All genes included in the PCR-array were found at detectable levels with the highest expression level for markers of nutrient transport (alpi, fabp1, and fabp2), epithelial integrity (cx32.2), mucus production (muc2, muc13) and pattern recognition receptors (fcl) (Supplementary Table 2). Regarding the probiotic effect, statistically significant changes were found in the expression patterns of 5 out of 44 genes (P<0.05) (Figure 2). In particular, expression of interleukin 10 (il10), interleukin (il12), and toll-like receptor 2 (tlr2) was upregulated in fish fed diet C (high probiotic dose) with intermediate values (not statistically different from the control group) in fish fed diet B (low probiotic dose). In contrast, the highest values of toll-like receptor 5 (tlr5) and galectin-8 (lgals8) were seen in fish fed diet B, whereas intermediate values were found in fish fed diet C. The probiotic treatment altered other markers (desmoplakin, dsp; interleukin 34, il34; C-C chemokine receptor 3, ccr3; and macrophage mannose receptor 1, mrc1) to a lesser extent, with an overall enhancement of gene expression that was especially evident in fish fed diet C (P<0.1). TABLE 3 | Growth performance of gilthead sea bream (Sparus aurata). Diet Mean body weight (g) WG1(%) SGR2(%) Feed intake CF3FCR4 Initial Final (g dry feed/fish) Period T0-T1, 24/09/2019–24/10/2019 A 82.67 ±0.86 130.53 ±1.35 57.9 ±0.4 1.52 ±0.01 61.82 ±0.59ab 2.89 ±0.02 A 1.27 ±0.01 B 83.45 ±0.74 130.01 ±1.20 55.8 ±0.8 1.48 ±0.02 59.58 ±0.73a2.84 ±0.02 B 1.28 ±0.01 C 83.28 ±0.83 132.08 ±1.32 58.6 ±0.8 1.54 ±0.02 60.61 ±0.75b2.86 ±0.01 C 1.27 ±0.01 Period T1-T2, 25/10/2019–15/11/2019 A 130.53 ±1.35 149.43 ±1.56 14.5 ±0.6 0.66 ±0.03 38.78 ±2.17 2.76 ±0.02 A 1.80 ±0.02 B 130.01 ±1.20 150.08 ±1.40 15.4 ±0.4 0.68 ±0.01 36.99 ±1.19 2.74 ±0.01 B 1.84 ±0.04 C 132.08 ±1.32 152.99 ±1.66 15.8 ±0.8 0.70 ±0.02 33.93 ±2.06 2.73 ±0.01 C 1.86 ±0.06 Period T2-T3, 15/11/2019–18/12/2019 A 149.43 ±1.56 163.04 ±2.02a9.1 ±0.6 0.28 ±0.02 35.71 ±0.69 2.78 ±0.02 A 2.40 ±0.05 B 150.08 ±1.40 166.30 ±1.90ab 10.8 ±0.3 0.31 ±0.03 33.74 ±1.37 2.73 ±0.03 B 2.31 ±0.03 C 152.99 ±1.66 171.24 ±2.07b11.9 ±1.8 0.36 ±0.02 32.69 ±1.46 2.81 ±0.01 C 2.02 ±0.25 Overall, 24/09/2019–18/12/2019 A 82.67 ±0.86 163.04 ±2.02a97.2 ±1.4 0.80 ±0.01 135.85 ±3.29 2.78 ±0.02 A 1.57 ±0.05 B 83.45 ±0.74 166.30 ±1.90ab 99.3 ±0.7 0.81 ±0.01 129.62 ±2.71 2.73 ±0.03 B 1.60 ±0.03 C 83.28 ±0.83 171.24 ±2.07b105.6 ±2.5 0.85 ±0.02 126.44 ±6.86 2.81 ±0.01 C 1.55 ±0.02 Data are reported as mean ±SEM, different superscript letters indicate significant differences (P <0.05) between diet groups in the same sub-column. 1Weight gain, WG =(100 ×body weigh increase)/initial body weight. 2Specific growth rate, SGR =100 ×(ln final body weight–ln initial body weight)/days. 3Condition factor, CF =100 ×(body weight/standard length). 4Feed conversion ratio, FCR =dry feed intake/wet weight gain [total feed supplied (g DM, dry matter)/WG (g)]. Frontiers in Marine Science | www.frontiersin.org 7April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 8 Moroni et al. LAB-Fortified Feed for Fish TABLE 4 | Histological scoring (for anterior and posterior intestine) and biometric measurement [intestinal length index (ILI) and intestinal weight index (IWI)] of gilthead sea bream (Sparus aurata) juveniles fed the control (A) and experimental (B and C) diets. Diet Mucosal folds Connective tissue Lamina propria of simple folds Supranuclear vacuoles Total score ILI1(cm) IWI2(g) Anterior intestine Biometric measurement A 1.1 ±0.1 1.7 ±0.06 1.7 ±0.06 1.5 ±0.04 6.1 ±0.2 97.21 ±7.62a2.43 ±0.06 B 1.0 ±0.04 1.5 ±0.2 1.5 ±0.1 2.2 ±0.5 6.3 ±0.9 75.73 ±6.74b2.38 ±0.12 C 1.1 ±0.04 1.7 ±0.1 1.5 ±0.2 2.0 ±0.4 6.2 ±0.7 86.43 ±8.02ab 2.40 ±0.17 Posterior intestine A 1.2 ±0.2a1.6 ±0.09 1.9 ±0.2 2.2 ±0.3 6.9 ±0.8 B 1.8 ±0.3b2.0 ±0.2 1.7 ±0.1 2.1 ±0.2 7.6 ±0.8 C 1.3 ±0.07ab 1.8 ±0.04 1.7 ±0.08 2.0 ±0.07 6.8 ±0.07 Data are reported as mean ±SEM of 12 fish per diet. Different superscript letters indicate significant differences (Dunn’s pot-hoc test, P <0.05) between dietary groups in the same sub-column. 1Intestinal length index, ILI =100 ×(intestine length/standard length). 2Intestinal weigth index, IWI =100 ×(intestine weight/fish weight). FIGURE 1 | Light microscope images obtained from anterior (A–C) and posterior (D–F) intestine of gilthead sea bream juveniles (Sparus aurata) fed with diets A, B, and C, stained with hematoxylin and eosin (H&E). Scale bar =500 µm. Characterization of Feed-Associated Bacterial Communities At the end of the trial, the normalized counts of L. lactis subsp. lactis resulted 8–11 in diet A (<0.0001% total bacterial counts); 30,204 in diet B (2.5% total counts); and 61,828 (5.4% total counts) in diet C (Figure 3). By excluding Cyanobacteria/Chloroplast (>90% total counts), Firmicutes and Proteobacteria proved to be the most highly represented bacterial phyla in the three feeds, whereas the rest of the bacterial population consisted of Bacteriodetes and Fusobacteria phyla (Supplementary Figure 1A). However, the percentage of Firmicutes varied considerably between feeds, with higher values in feed B (4.2%) and C (7.8%) than in the control feed, in which Firmicutes represented only 2% of the total counts. Thus, by recalculating the relative bacterial abundances after excluding Cyanobacteria/Chloroplast, the percentage of Firmicutes rose from 34% in the control diet A to 70% in diet B and 79% in diet C (Supplementary Figure 1B). Then, by specifically analyzing the relative abundance of the probiotic L. lactis subsp. lactis in comparison to the most representative genera within the phylum Firmicutes, the percentage of L. lactis subsp. lactis was close to 0% in the control diet, whereas in B and C diets, it was significantly higher, reaching values of 64 and 71%, respectively (Supplementary Figure 1C). Alpha Diversity and Gut Microbiota Composition Illumina sequencing of AI-adherent bacteria yielded 3,677,860 high-quality and merged reads, with an average value of 136,217 reads per sample (Supplementary Table 3). When annotated, the Frontiers in Marine Science | www.frontiersin.org 8April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 9 Moroni et al. LAB-Fortified Feed for Fish FIGURE 2 | Fold change of differentially expressed genes (Dunn’s post hoc test; **P<0.05; *P<0.1) in the anterior intestine of fish fed experimental diets (diets B and C) relative to the control diet (A). Data are the mean + SEM of 9–12 fish per diet. White columns (fish fed diet B). Black columns fish fed diet C. FIGURE 3 | Relative abundance of Firmicutes phylum and Lactococcus lactis subsp. lactis in control (Diet A) and experimental diets (Diets B and C). reads were assigned to 1,313 OTUs at 97% identity threshold. Rarefaction analysis showed curves that approximated saturation (horizontal asymptote); thus, a good coverage of the bacterial community was achieved and the number of sequences for analysis was considered appropriate (Supplementary Figure 2). Indeed, up to 85% of the OTUs were classified at the level of species and more than 90% at the level of genus (94.1%), family (96%), order (97%), class (97.2%), and phylum (99%). As shown in Table 5, the richness estimator (ACE) indicated a higher OTU richness in fish fed diet B than in fish fed diet A or diet C. At the same time, alpha diversity estimators (Shannon and Simpson) disclosed a reduced evenness in fish fed diet C, which Frontiers in Marine Science | www.frontiersin.org 9April 2021 | Volume 8 | Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 16 Moroni et al. LAB-Fortified Feed for Fish bream (Sparus aurata L.) specimens. 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