Fernanda Silva
email: fernandasildosan@hotmail.com

Santos

Raquel A F Neves
email: raquel.neves@unirio.br

Benoît Bernay
email: benoit.bernay@unicaen.fr

Natascha Krepsky
email: natascha@unirio.br

Valéria Laneuville Teixeira

Sébastien Artigaud
email: sebastien.artigaud@univ-brest.fr

Silva Dos Santos

The first use of LC-MS/MS proteomic approach in the brown mussel Perna perna after bacterial challenge: searching for key proteins on immune response

Keywords: Escherichia coli, Hepatopancreas, Immune-related proteins, LC-MS/MS, Perna perna, Protein profile, Salmonella enterica, Shellfish, Vibrio parahaemolyticus

Highlights

• A total of 3,805 proteins were found in the hepatopancreas of P. perna mussel • Vibrio parahaemolyticus seems to be virulent and suppresses P. perna immune response

• E. coli and S. enterica triggered a general immune response against bacteria

• Proteins altered in response to all challenge-bacteria were vital for immune defense

• This study points out critical proteins against bacteria, promising for biotechnology

Introduction

Coastal pollution is a concern worldwide and is often concentrated along densely-populated areas in low-and middle-income countries [START_REF] Landrigan | Human Health and Ocean Pollution[END_REF][START_REF] Tuholske | Mapping global inputs and impacts from of human sewage in coastal ecosystems[END_REF]. Continental runoff and sewage introduce excessive nutrients and pathogenic bacteria in the coastal waters, therefore altering the local microbial communities and, consequently, the ecological dynamics [START_REF] Buccheri | Investigating microbial indicators of anthropogenic marine pollution by 16S and 18S High-Throughput Sequencing (HTS) library analysis[END_REF][START_REF] Tuholske | Mapping global inputs and impacts from of human sewage in coastal ecosystems[END_REF][START_REF] Young | Vancomycin-resistant enterococci and bacterial community structure following a sewage spill into an aquatic environment[END_REF]. Additionally, anthropogenic pollution, not to mention climate change, can increase the abundance of introduced bacteria in the seawater and also alter the geographic ranges of indigenous marine pathogens [START_REF] Burge | Climate Change Influences on Marine Infectious Diseases: Implications for Management and Society[END_REF][START_REF] Cavicchioli | Scientists' warning to humanity: microorganisms and climate change[END_REF][START_REF] Chen | A pollution gradient contributes to the taxonomic, functional, and resistome diversity of microbial communities in marine sediments[END_REF].

Escherichia coli and Salmonella enterica belong to the Enterobacteriaceae family and reach the marine environment through anthropogenic sources, such as sewage [START_REF] Buccheri | Investigating microbial indicators of anthropogenic marine pollution by 16S and 18S High-Throughput Sequencing (HTS) library analysis[END_REF]. Escherichia coli naturally inhabit human and other warm-blooded animal guts [START_REF] Gomes | Diarrheagenic Escherichia coli[END_REF][START_REF] Jang | Environmental Escherichia coli: ecology and public health implications-a review[END_REF][START_REF] Yang | Current pathogenic Escherichia coli foodborne outbreak cases and therapy development[END_REF]. Since E. coli is discharged in the feces, its presence and abundance in the environment indicate the fecal contamination degree, as well as the presence of other intestinal pathogens [START_REF] Gomes | Diarrheagenic Escherichia coli[END_REF][START_REF] Jang | Environmental Escherichia coli: ecology and public health implications-a review[END_REF][START_REF] Yang | Current pathogenic Escherichia coli foodborne outbreak cases and therapy development[END_REF].

As part of the human microbiota, E. coli rarely induces disease in healthy individuals. However, some strains are known as diarrheagenic, because they cause diarrhea or extraintestinal diseases [START_REF] Gomes | Diarrheagenic Escherichia coli[END_REF][START_REF] Jang | Environmental Escherichia coli: ecology and public health implications-a review[END_REF][START_REF] Yang | Current pathogenic Escherichia coli foodborne outbreak cases and therapy development[END_REF]. Within diarrheagenic pathotypes, Enteropathogenic E. coli (EPEC) strains are described as those that cause diarrhea, render attaching and effacing lesions on the intestinal epithelium, and cannot produce Shiga toxins and heat-labile or heat-stable enterotoxins [START_REF] Gomes | Diarrheagenic Escherichia coli[END_REF][START_REF] Jang | Environmental Escherichia coli: ecology and public health implications-a review[END_REF][START_REF] Yang | Current pathogenic Escherichia coli foodborne outbreak cases and therapy development[END_REF].

Similarly, Salmonella spp. transmission can also be related to fecal contamination by the ingestion of contaminated water or food [START_REF] Balasubramanian | The global burden and epidemiology of invasive non-typhoidal Salmonella infections[END_REF][START_REF] Ferrari | Worldwide epidemiology of Salmonella serovars in animalbased foods: A meta-analysis[END_REF]. Depending on their specificity to human hosts, Salmonella enterica serovars are categorized as typhoid -TS or nontyphoid -NTS [START_REF] Balasubramanian | The global burden and epidemiology of invasive non-typhoidal Salmonella infections[END_REF][START_REF] Ferrari | Worldwide epidemiology of Salmonella serovars in animalbased foods: A meta-analysis[END_REF]. NTS serovars, such as S. typhimurium, infect a wide range of hosts and usually provoke a self-limiting diarrheal disease in humans [START_REF] Balasubramanian | The global burden and epidemiology of invasive non-typhoidal Salmonella infections[END_REF][START_REF] Ferrari | Worldwide epidemiology of Salmonella serovars in animalbased foods: A meta-analysis[END_REF].

In contrast to E. coli and S. enterica, Vibrio parahaemolyticus is indigenous to coastal and estuarine ecosystems being associated with several marine organisms -e.g., fish, mollusks, and crustaceans [START_REF] Bonnin-Jusserand | Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae, V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products[END_REF][START_REF] Ghenem | Vibrio Parahaemolyticus: A Review on Distribution, Pathogenesis, Virulence Determinants and Epidemiology[END_REF]. Vibrio parahaemolyticus together with V. cholerae and V. vulnificus comprise the three most important human pathogenic strains in the genus [START_REF] Bonnin-Jusserand | Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae, V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products[END_REF][START_REF] Ghenem | Vibrio Parahaemolyticus: A Review on Distribution, Pathogenesis, Virulence Determinants and Epidemiology[END_REF]. This first species is a relevant causal agent of human gastroenteritis associated with the consumption of marine foods [START_REF] Bonnin-Jusserand | Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae, V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products[END_REF][START_REF] Ghenem | Vibrio Parahaemolyticus: A Review on Distribution, Pathogenesis, Virulence Determinants and Epidemiology[END_REF]. Because of their filter-feeding habits, bivalve mollusks are directly in contact with bacteria suspended in the water column, either indigenous or introduced (Silva dos [START_REF] Silva Dos Santos | Immunology Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution[END_REF]. To thrive in a bacteria-rich environment, bivalves count with a robust innate immune system based on cellular and humoral responses [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. The main cellular effectors in the mussel hemolymph are known as hemocytes. These circulating cells act on phagocytosis, encapsulation, and infiltration to repair and defend damaged or infected tissues [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. Additionally, hemocytes can release reactive oxygen/nitrogen species (ROS/RNS), antimicrobial peptides (AMPs), and lysosomal enzymes as defense mechanisms [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF].

On the other response front, humoral factors in the hemolymph and tissues act synced with hemocytes [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. The first step to initiating an immune reaction against pathogens is the detection and recognition [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. Hence, evolutionarily conserved molecules -pattern recognition receptors (PRRs) -identify specific markers in the pathogens, known as pathogen-associated molecular patterns (PAMPs) [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. The main PRRs are toll-like receptors (TLRs), lectins (e.g., fibrinogen-related domain proteins -FReDs and C1q domain containing -C1qDC), thioester-containing proteins (TEPs), scavenger receptor cysteine-rich proteins (SRCRs), peptidoglycan recognition proteins (PGRPs) and gram-negative binding proteins (GNBPs) [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF].

After detection and recognition, signaling induces hemocytes to move to the reaction site and/or activate other humoral components [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. The main humoral effectors directly involved in pathogen killing are antimicrobial peptides (AMP). These molecules are categorized into defensins, big defensins, mytilins, myticins, and mytimacins [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. Other molecules that take part in humoral defense are bactericidal/permeability-increasing proteins (BPIs), proteases and lysozymes, heat shock proteins (HSPs), and protease inhibitors [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF].

As shown above, to compensate for the lack of an adaptative immune system, most marine invertebrates, including bivalves, resort to the production of a huge range of antimicrobial metabolites [START_REF] Nalini | Antibacterial macro molecules from marine organisms[END_REF]. Many of these include proteins or peptides and can be investigated by proteomic approaches [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Nalini | Antibacterial macro molecules from marine organisms[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. Through this, the protein profiles of organisms from different conditions can be compared to identify changes in protein expression, quantitative and qualitatively [START_REF] Campos | Proteomic research in bivalves: Towards the identification of molecular markers of aquatic pollution[END_REF][START_REF] Gonzalez | Omics in Aquatic Ecotoxicology: The Ultimate Response to Biological Questions?[END_REF]. Therefore, it is possible to understand organisms' responses at molecular levels as well as point out bioactive molecules of human interest (e.g., new compounds for the pharmaceutical industry) [START_REF] Nalini | Antibacterial macro molecules from marine organisms[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. Additionally, proteins can be used as molecular biomarkers related to the biological effects of stressors, including bacteria exposure. Then, it can be useful for environmental monitoring, not to mention the asses of health status and/or food safety in commercial species [START_REF] Campos | Proteomic research in bivalves: Towards the identification of molecular markers of aquatic pollution[END_REF][START_REF] Gonzalez | Omics in Aquatic Ecotoxicology: The Ultimate Response to Biological Questions?[END_REF].

The brown mussel Perna perna (Bivalvia, Mytilidae) is considered a valuable fishing resource for aquaculture, primarily in tropical and subtropical coastal regions [START_REF] Cunha | Wider sampling reveals a non-sister relationship for geographically contiguous lineages of a marine mussel[END_REF][START_REF] Lourenço | First record of the brown mussel (Perna perna) from the European Atlantic coast[END_REF]. This marine mussel is cultivated in countries such as Venezuela, South Africa, and primarily in Brazil [START_REF] Calvo-Ugarteburu | Rehabilitating mussel beds in Coffee Bay , South Africa : Towards fostering cooperative small-scale fisheries governance and enabling community upliftment[END_REF][START_REF] Narváez | Food availability and reproduction affects lipid and fatty acid composition of the brown mussel , Perna perna[END_REF]Silva dos Santos et al., 2022a), that responds to 99.96% of P. perna global production (FAO, 2016). Because it is a sessile and filterfeeder, the mussel can accumulate pollutants from the water column and then provide a picture of its bioavailability [START_REF] Birnstiel | Depuration reduces microplastic content in wild and farmed mussels[END_REF][START_REF] Pusceddu | Environmental risk assessment of triclosan and ibuprofen in marine sediments using individual and sub-individual endpoints *[END_REF]Silva dos Santos et al., 2022b). Therefore, P. perna has been proposed as a bioindicator species [START_REF] Birnstiel | Depuration reduces microplastic content in wild and farmed mussels[END_REF][START_REF] Pusceddu | Environmental risk assessment of triclosan and ibuprofen in marine sediments using individual and sub-individual endpoints *[END_REF]Silva dos Santos et al., 2022b). Likewise, the above-mentioned features make them possible vehicles for toxins, contaminants, and pathogenic bacteria for human beings [START_REF] Neves | Harmful algal blooms and shellfish in the marine environment: an overview of the main molluscan responses, toxin dynamics, and risks for human health[END_REF]Silva dos Santos et al., 2022a). Since it is a pivotal commercial species, bacteria also can cause diseases and massive mortality on mussel farms, leading as well to relevant economic losses [START_REF] Paillard | Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution[END_REF].

Although international environmental rules instruct shellfish farming in places with good water quality (FAO, 2017), the mussel is still harvested and cultivated in contaminated areas exposed to bacteria (Silva dos Santos et al., 2022a[START_REF] Silva Dos Santos | Immunology Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution[END_REF]. In this study, we aimed to assess the protein profile of the hepatopancreas of P. perna mussel challenged by bacteria to distinguish the molecular responses of introduced -E. coli and S. enterica -and marine indigenous bacteria -V. parahaemolyticus. The primary functions of the hepatopancreas are related to the metabolic balance, such as nutrient uptake and intracellular digestion, carbon and nitrogen metabolism, and detoxification [START_REF] Rőszer | The invertebrate midintestinal gland ("hepatopancreas") is an evolutionary forerunner in the integration of immunity and metabolism[END_REF]. Nevertheless, it also acts directly and indirectly on immunity [START_REF] Rőszer | The invertebrate midintestinal gland ("hepatopancreas") is an evolutionary forerunner in the integration of immunity and metabolism[END_REF]. Metabolic shifts occur induced by infection and impact the efficiency of the immune response [START_REF] Rőszer | The invertebrate midintestinal gland ("hepatopancreas") is an evolutionary forerunner in the integration of immunity and metabolism[END_REF]. Directly, the organ is involved in antigen processing and pathogen clearance, besides being a source of several innate immunity molecules [START_REF] Rőszer | The invertebrate midintestinal gland ("hepatopancreas") is an evolutionary forerunner in the integration of immunity and metabolism[END_REF]. As the bivalve hepatopancreas is a critical organ for innate immunity and metabolism, its protein profile furnishes a better understanding of the disturbance caused by bacteria and points to critical molecules in this process (Huang et al., 2021;[START_REF] Laith | Histopathological and immunological changes in green mussel, Perna viridis, challenged with Vibrio alginolyticus[END_REF][START_REF] Liu | Gender-specific metabolic responses in hepatopancreas of mussel Mytilus galloprovincialis challenged by Vibrio harveyi[END_REF][START_REF] Wu | Proteomic and metabolomic responses in hepatopancreas of Mytilus galloprovincialis challenged by Micrococcus luteus and Vibrio anguillarum[END_REF]. To the authors' knowledge, this is the first study using the LC-MS/MS proteomic approach in P. perna then allowing to elucidate factors related to mussel-bacteria interaction. This knowledge can support the development of strategies and tools to be applied to coastal marine resource management and contribute to the sustainability of coastal systems, not to mention the finding of molecules of human interest.

Material and methods

Mussels sampling

Adult individuals of P. perna mussel (shell length: 5.3-8.2 cm) were manually collected on September 2021, from the rocky shores at Vermelha Beach (VB), Rio de Janeiro, Brazil (22° 57′ 18.59″ S and 43° 9′ 52.91″ W). Mussels and seawater were transported to the laboratory facilities using 20 L containers, prior washed with a 2% solution of neutral Extran (Merck, MA02) and distilled water. As shown in Figure 1, the beach is located at the mouth of Guanabara Bay, facing the Atlantic Ocean. According to [START_REF] Krepsky | Dry Sand Quality: The Case Study of a Touristic Beach from Rio de Janeiro, Brazil[END_REF], VB composes the buffer zone of the Natural Monument of Sugar Loaf and Urca Hill's conservation unit and presents excellent water quality by microbiological standards.

Bacterial suspensions preparation

Escherichia coli 2348/EPEC and S. enterica subsp. enterica serovar Typhimurium str. SL1344 were grown at Tryptic Soy Broth (TSB; Himedia, Brazil) at 37 °C for 20 h, still in the bacteria exponential growth phase. Under the same conditions, V. parahaemolyticus ATCC 17802 was incubated in Alkaline Peptone Water 1% (1% peptone and 1% NaCl). Bacteria were washed twice by centrifuging at 3000 g and 15 °C for 10 min, and resuspended into PBS-NaCl (pH 7.4;[START_REF] Parisi | Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria[END_REF]. Bacteria suspension concentrations were obtained using the standard 0.5 (1.5 x 10 8 CFU mL -1 ) of the McFarland Scale and further diluted into sterile PBS-NaCl to 1.5 x 10 7 CFU mL -1 .

The above-mentioned strains were obtained from the Laboratory of Enteropathogens, Veterinary, Environmental and Food Microbiology/Department of Microbiology and Parasitology/Biomedical Institute/Fluminense Federal University -UFF; Laboratory of Pathogenic cocci and Microbiota/Department of Medical Microbiology/Professor Paulo de Goés Institute of Microbiology/Federal University of Rio de Janeiro -UFRJ; and Enterobacteria Laboratory/Oswaldo Cruz Institute/Oswaldo Cruz Foundation -FIOCRUZ, respectively.

Experimental design and challenge

At the laboratory, mussel shells (Figure 2) were cleaned to remove encrusting organisms and randomly distributed into 5 groups (n = 9) representing the following conditions: Non-injected control (NC); Injected control (IC); Escherichia coli (EC); Salmonella enterica (SE); and Vibrio parahaemolyticus (VP). Mussels from the bacteria-challenging groups were carefully injected with 100 μL of 1.5 x 10 7 CFU mL -1 of the corresponding bacterial suspension on their posterior adductor muscle (Figure 2), using a sterile syringe. Non-injected (NC) and injected (IC) control groups comprised, respectively, mussels not challenged and mussels injected with 100 μL sterile PBS-NaCl. After injections, mussels (n=9) were placed into separated glass aquaria, previously cleaned with neutral Extran solution (2% in distilled water), according to each condition. Aquariums were filled with 3 L of filtered seawater from VB (20 µm; salinity 30.9) and kept with constant aeration at 18 °C. Mussels were dissected 24h post-incubations using sterile stainless-steel scalpel and forceps, their hepatopancreas (Figure 2) were removed and individually conditioned in sterile Eppendorf tubes (2 mL), flash-frozen in liquid nitrogen, and kept in a freezer (-20°C) until lyophilization.

Total protein extraction and digestion

Lyophilized samples were added to microtubes containing 500 μL of extraction buffer (2M thiourea, 7M urea, 4% CHAPS, 50 mM tris-HCl; pH = 8.5), 5 μL of protease inhibitor and ceramic beads (mix of 1.4 mm and 2.8 mm). Samples were mechanically homogenized using FastPrep -24 TM 5G (MP Biomedicals) in the following mode: speed = 6.5 m/s; adapter = coolprep; time = 40 s. The program was run 3 times at the above conditions, with intervals of 30 s between each running and adding dry ice. Since the samples were not liquid enough, the above process was repeated by adding the same above amount of extraction buffer and protease inhibitor. Then, an equal volume of trichloroacetic acid/acetone (20/80) solution was added to the samples, which precipitated at 4 °C for 45 min. Samples were then centrifuged at 20.000 g and 4 °C for 50 min, and washed 5 times with acetone/tris-HCl 6.8 100mM (70/30) solution added by bromophenol blue pH indicator. Thereafter, samples were redissolved in the extraction buffer (500 μL) added to 1% of protease inhibitor, and diluted 100 times for protein quantification by the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Based on protein concentration, samples from each experimental condition were chosen (IC, EC, and SE: n = 5 and NC and VP: n = 4) to proceed to the next steps.

The volume necessary for 10 μg of total proteins was transferred to clean tubes and then samples were reduced with 6 μL dithiothreitol (final concentration 50 mM) for 20 min at room temperature. Then, 14 μL of acrylamide 30% were added to the samples (final concentration 20%). After shaking for 20 min, 5μL of tetramethylethylenediamine (TEMED) and 5 μL of ammonium peroxydisulfate 10% were added to each tube for gel polymerizing. Thereafter, samples were fixed with 100 μL of methanol, acetic acid, and H 2 O (50/40/10) solution for 10 min. About 500 μL of urea (6 M) were added to each tube and, after 10 min of shaking, 250 μL of acetonitrile were joined, vortexed and removed. Samples were rehydrated in 200 μL of ammonium bicarbonate (100 mM) and incubated for 10 min under shaking. An equal volume of acetonitrile (200 μL) was added, vortexed and removed. Once again, 200 μL of acetonitrile was added, and after complete sample dehydration, the liquid was removed and samples were left to air-dry. Five µg of each protein extract were prepared using a modified Gel-aided Sample Preparation protocol (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409837/). Samples were digested with trypsin/Lys-C overnight at 37 °C. For nano-LC fragmentation, protein or peptide samples were first desalted and concentrated onto a µC18 Omix (Agilent) before analysis.

LC-MS/MS analysis

The chromatography step was performed on a NanoElute (Bruker Daltonics) ultra-high-pressure nano flow chromatography system. Approximatively 200 ng of each peptide sample were concentrated onto a C18 pepmap 100 (5 mm x 300 µm i.d.) precolumn (Thermo Scientific) and separated at 50 °C onto a reversed phase Reprosil column (25cm x 75μm i.d.) packed with 1.6 μm C18 coated porous silica beads (Ionopticks). Mobile phases consisted of 0.1% formic acid, 99.9% water (v/v) (A) and 0.1 % formic acid in 99.9 % ACN (v/v) (B). The nanoflow rate was set at 250 nl/min, and the gradient profile was as follows: from 2 to 30 % B within 70 min, followed by an increase to 37 % B within 5 min and further to 85 % within 5 min and reequilibration.

MS experiments were carried out on an TIMS-TOF pro mass spectrometer (Bruker Daltonics) with a modified nano electrospray ion source (CaptiveSpray, Bruker Daltonics). A 1400 spray voltage with a capillary temperature of 180°C was typically employed for ionizing. MS spectra were acquired in the positive mode in the mass range from 100 to 1700 m/z and 0.60 to 1.60 1/k0 window. In the experiments described here, the mass spectrometer was operated in PASEF DIA mode with exclusion of single charged peptides. The DIA acquisition scheme consisted of 16 variable windows ranging from 400 to 1200 m/z.

Protein identification

Database searching and LFQ quantification (using XIC) was performed using DIA-NN (version 1.8; https://www.nature.com/articles/s41592-019-0638-x). An updated UniProt Mytilus coruscus database was used for library-free search/library generation. In the absence of genomic data for the species Perna perna or within the genus Perna, Mytilus coruscus was the closest species from Perna perna (same subfamily, Mytilinae) with the most comprehensive available proteome [START_REF] Li | The Whole-Genome Sequencing and Hybrid Assembly of Mytilus coruscus[END_REF]. The default parameter 0.0 was used for RT prediction and extraction mass accuracy, which means DIA-NN performed automatic mass and RT correction. Top six fragments (ranked by their library intensities) were used for peptide identification and quantification. The FDR was set to 1% at the peptide precursor level. The variable modifications allowed were as follows: Nterm-acetylation and Oxidation (M). In addition, C-Propionoamide was set as fix modification. "Trypsin/P" was selected. Data were filtering according to a FDR of 1%. Cross-run normalisation was performed using RT-dependent.

Data analysis

For the statistical analyses, label-free quantitative data were imported into the software Perseus 1.6.15.0 [START_REF] Tyanova | The Perseus computational platform for comprehensive analysis of (prote)omics data[END_REF]. Protein intensity values were log 2transformed and only identified proteins in a minimum of three samples of at least one of the tested conditions (i.e., non-injected control -NC; injected control -IC; Escherichia coli -EC; Salmonella enterica -SE; and Vibrio parahaemolitycus -VP) followed to the further analysis. Missed values were replaced from the normal distribution (width = 0.3 and downshift = 1.8).

Data from the different conditions were compared based on a one-way ANOVA (Benjamini-Hochberg FDR; significance ≥ 0.05) and a posteriori Tukey HSD (significance ≥ 0.05) tests. Protein functional annotation was carried out using the tools UniProt (https://www.uniprot.org/) and EggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale (http://eggnogmapper.embl.de/; [START_REF] Cantalapiedra | eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale[END_REF]. Then, the biological role of significantly different proteins fronting bacterial challenge was discussed taking into account the Clusters of Orthologous Group (COG) categories, biological process Gene Ontology (GO) terms, and literature review.

The UpSet graph for visualizing the intersecting sets of significantly different proteins among the tested conditions was created using the UpSetR package available at https://upset.app/ [START_REF] Conway | UpSetR: an R package for the visualization of intersecting sets and their properties[END_REF][START_REF] Lex | UpSet: Visualization of Intersecting Sets[END_REF].

Results and Discussion

A total of 3,805 proteins were found in the hepatopancreas of Perna perna mussels that underwent five different conditions (non-injected control -NC; injected control -IC; Escherichia coli -EC; Salmonella enterica -SE; and Vibrio parahaemolyticus -VP), of which 378 are uncharacterized (available as supplementary data). From the total, 597 were significantly different among conditions -upregulated or downregulated in at least one condition. Figure 3 shows the number of proteins significantly different for each intersecting group from pairwise comparisons. Eleven proteins showed significant differences between the two control groups (available as supplementary data), probably in response to the stress related to the injection, and for this reason were not discussed here. Mussels injected with V. parahaemolyticus presented a more marked response, with 343 proteins significantly different from all the other conditions that are discussed in an independent topic (3.2). Among the remaining significantly different proteins, only that different from both non-injected (NC) and injected (IC) controls are discussed herein (Topic 3.1).

Fig 3.

Upset plot for visualizing intersecting sets of significantly different proteins among the tested conditions. NC -non-injected control; IC -injected control; EC -Escherichia coli; SE -Salmonella enterica; and VP -Vibrio parahaemolyticus. SE_EC means a significant difference between these conditions and so on.

Response to bacterial challenge

Table 1 shows the 31 proteins that were upregulated or downregulated for one or more bacterial challenge groups (Escherichia coli -EC; Salmonella enterica -SE; and Vibrio parahaemolyticus -VP) compared with both controls (non-injected -NC and injected -IC). These proteins are discussed below according to each challenge group of P. perna.

Escherichia coli

Mussels challenged with E. coli presented only two proteins downregulated compared to controls, namely: a von Willebrand Factor A (vWA) domain-containing protein and a FERM ("four-point-one, ezrin, radixin, moesin") domain-containing protein. According to [START_REF] Whittaker | Distribution and Evolution of vonWillebrand/Integrin A Domains: Widely Dispersed Domainswith Roles in Cell Adhesion and Elsewhere[END_REF], the most ancient vWA domaincontaining proteins settle the intracellular environment of all eukaryotes and take part in transcription, DNA repair, ribosomal and membrane transport, and the proteasome. Probably, vWA domains mediate protein-protein interactions of multiprotein complexes involved in these functions [START_REF] Whittaker | Distribution and Evolution of vonWillebrand/Integrin A Domains: Widely Dispersed Domainswith Roles in Cell Adhesion and Elsewhere[END_REF]. Afterward in the evolution, vWA domains emerged in extracellular proteins of metazoan. Among these, the most representative in the extracellular matrix are the integrin β subunits, the major cell surface receptors, and also involved in cell adhesion [START_REF] Whittaker | Distribution and Evolution of vonWillebrand/Integrin A Domains: Widely Dispersed Domainswith Roles in Cell Adhesion and Elsewhere[END_REF]. Finally, nematodes and chordates separately diversified extracellular matrix proteins containing vWA domains, which compose the majority of known vWA domaincontaining proteins [START_REF] Whittaker | Distribution and Evolution of vonWillebrand/Integrin A Domains: Widely Dispersed Domainswith Roles in Cell Adhesion and Elsewhere[END_REF]. In general, the vWA domains are involved in protein-protein interactions, frequently using divalent cations such as Ca +2 [START_REF] Bowden | Extracellular vesicles and post-translational protein deimination signatures in mollusca-the blue mussel (Mytilus edulis), soft shell clam (mya arenaria), eastern oyster (crassostrea virginica) and atlantic jacknife clam (ensis leei)[END_REF][START_REF] Whittaker | Distribution and Evolution of vonWillebrand/Integrin A Domains: Widely Dispersed Domainswith Roles in Cell Adhesion and Elsewhere[END_REF]. Some mollusks' studies showed the vWA domain's important role in defense and immunity. [START_REF] Smith | RNA-Seq reveals a central role for lectin, C1q and von Willebrand Factor A domains in the defensive glue of a terrestrial slug[END_REF] found matrilin-like proteins rich in the vWA domain in the glue of the slug Arion subfuscus.

These proteins jointly with lectin and C1q domains seem to have a defensive role in the slug glue, one of the first barriers against pathogens. [START_REF] Bowden | Extracellular vesicles and post-translational protein deimination signatures in mollusca-the blue mussel (Mytilus edulis), soft shell clam (mya arenaria), eastern oyster (crassostrea virginica) and atlantic jacknife clam (ensis leei)[END_REF] showed posttranslational regulation of relevant pathways for immunity and metabolism via deimination of several proteins found in the hemolymph of the eastern oyster Crassostrea virginica, including vWA domain-containing protein.

The second protein downregulated in P. perna mussels injected with E. coli, compared to the controls, was a FERM domain-containing protein. A protein like that was also found in the transcriptome analysis of the marine gastropod Concholepas concholepas [START_REF] Cárdenas | Transcriptome analysis in Concholepas concholepas (Gastropoda, Muricidae): Mining and characterization of new genomic and molecular markers[END_REF]. The superfamily of proteins that hold a FERM domain is ubiquitous and performs the linkage between the plasm membrane and actin cytoskeleton, sometimes mediating the activation of their binders [START_REF] Frame | The FERM domain: Organizing the structure and function of FAK[END_REF][START_REF] Tanentzapf | An interaction between integrin and the talin FERM domain mediates integrin activation but not linkage to the cytoskeleton[END_REF][START_REF] Tepass | FERM proteins in animal morphogenesis[END_REF]. The FERM domain can interact with membrane lipids and several protein-binding partners, including transmembrane ion channels, adhesion molecules (e.g., integrins), and cytoplasmic proteins. Therefore, these proteins act in the structure, transport, and signal transduction of cells [START_REF] Frame | The FERM domain: Organizing the structure and function of FAK[END_REF][START_REF] Tanentzapf | An interaction between integrin and the talin FERM domain mediates integrin activation but not linkage to the cytoskeleton[END_REF][START_REF] Tepass | FERM proteins in animal morphogenesis[END_REF].

Figure 4 graphically sums up the immune-related role of these two down regulated proteins after E. coli challenging in a ciliated hepatopancreatic cell of P. perna mussel. The von Willebrand Factor A domain-containing protein (vWA) seems to behave as a humoral effector in mollusks, primarily in the extracellular matrix. The FERM domain-containing protein (FERM), in turn, is probably located close to the plasma membrane and involved in signal transduction, therefore acting at the initial phases after pathogen recognition to trigger some immune response. Immune-related role of the altered proteins in a ciliated hepatopancreatic cell of Perna perna mussel, 24h post-Escherichia coli injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). In mussels challenged with E. coli, a von Willebrand Factor A domain-containing protein (vWA) and a FERM domain-containing protein (FERM) were downregulated. FERM is probably located close to the plasma membrane and takes part in signal transduction. VWA seems to act as a humoral effector in mollusks, primarily in the extracellular matrix.

Salmonella enterica

Similar to mussels injected with E. coli, the FERM domain-containing protein was downregulated in mussels challenged by S. enterica. Likewise, the proteins splicing factor 3A subunit 1 (SF3A1), actin beta/gamma 1 (ACTB_G1), and galactokinase 2 (GALK2) were deregulated after S. enterica injection, whereas the following proteins were upregulated: leucine-rich repeat and guanylate kinase domain-containing protein (LRGUK), stress-activated protein kinase JNK, 60S ribosomal protein L18a (RPL18A), proteasome activator PA28γ (PA28γ or PSME3), Mitochondrial fission process protein 1 (MTFP1), Tubulin beta chain (β-Tubulin), axonemal dyneins (DNAH3 and DNAH9), EFHC1, Cilia-and flagella-associated protein 299, Glycerol-3-phosphate dehydrogenase (GPD2), Oxoglutarate dehydrogenase (OGDHL).

The protein LRGUK presents two essential domains for humoral immune response in bivalves -leucine-rich repeat (LRR) and guanylate kinase (GK). The first one characterizes a toll-like receptor (TLR), in other words, a membrane-bound pattern recognition receptor (PRR) [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. These receptors are crucial to detect countless classes of PAMPs (pathogen-associated molecular patterns) at microbial surfaces (e.g., LPS, components of the bacterial cell wall, flagellin, among others) and, therefore, trigger the immune response [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. According to [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF], TLRs consist of an evolutionarily conserved structure as follows: an intracellular tollinterleukin-1-receptor domain; a transmembrane region; and a variable extracellular region with an LRR domain. In the case of LRGUK found upregulated in the P. perna hepatopancreas, the intracellular domain seems to be a guanylate kinase (GK).

Membrane-associated guanylate kinases (MAGUKs) are a conserved protein family involved with tissue development, cell-cell communication, cell adhesion, and signal transduction [START_REF] Olsen | Functional Analysis of the Nucleotide Binding Domain of Membrane-associated Guanylate Kinases*[END_REF][START_REF] Zhu | Structure and function of the guanylate kinase-like domain of the MAGUK family scaffold proteins[END_REF]Zhu et al., , 2011)). Originally, GKs contain a GMP-binding site that catalyzes GMP to GDP conversion. However, several studies have shown that MAGUKS evolved, currently acting on protein-protein interaction [START_REF] Olsen | Functional Analysis of the Nucleotide Binding Domain of Membrane-associated Guanylate Kinases*[END_REF][START_REF] Zhu | Structure and function of the guanylate kinase-like domain of the MAGUK family scaffold proteins[END_REF]Zhu et al., , 2011)). Accordingly, the LRGUK protein found in this study seems to hold an LRR domain for pathogens recognition, and a GK domain performing signal transduction (e.g., interacting with microtubuleassociated protein; [START_REF] Reese | The guanylate kinase domain of the MAGUK PSD-95 binds dynamically to a conserved motif in MAP1a[END_REF]. Likewise, [START_REF] Huang | LRFN (leucine-rich repeat and fibronectin type-III domain-containing protein) recognizes bacteria and promotes hemocytic phagocytosis in the Pacific oyster Crassostrea gigas[END_REF] found a new transmembrane LRR (leucine-rich repeat and fibronectin type-III domain-containing protein) in the pacific oyster Crassostrea gigas. It was observed that the protein acts as a pattern recognition receptor for Vibrio spp. and promotes hemocyte phagocytosis. Additionally, it was found widely expressed in several tissues such as gills, adductor muscles, digestive glands, and hemocytes [START_REF] Huang | LRFN (leucine-rich repeat and fibronectin type-III domain-containing protein) recognizes bacteria and promotes hemocytic phagocytosis in the Pacific oyster Crassostrea gigas[END_REF].

Stress-activated protein kinase JNK participates in the mitogen-activated protein kinase (MAPK) cascade (Hatanaka et al., 2009;[START_REF] Irazoqui | Evolution of host innate defence: Insights from Caenorhabditis elegans and primitive invertebrates[END_REF][START_REF] Sun | Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis[END_REF]. MAPK cascade is a signaling pathway associated with innate immunity, evolutionarily conserved from invertebrates to mammals (Hatanaka et al., 2009;[START_REF] Irazoqui | Evolution of host innate defence: Insights from Caenorhabditis elegans and primitive invertebrates[END_REF][START_REF] Sun | Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis[END_REF]. Excluding variations, in invertebrates the pathway consists of three categories of protein kinases that are sequentially phosphorylated, activating the following ones (Hatanaka et al., 2009). In Drosophila and mammals, JNK or p38 protein kinases phosphorylate transcription factors at the end of the cascade, regulating the transcriptional activity of host response genes (Hatanaka et al., 2009). In the Yesso scallop Patinopecten yessoensis, it seems that only the JNK kinase participates in the immune response against Gram-negative and -positive bacteria [START_REF] Sun | Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis[END_REF]. In this way, after pathogen recognition by TLRs, the cascade is triggered and results in the transcription of host response genes that encode proteins related to innate immune response (e.g., antimicrobial peptides) (Hatanaka et al., 2009;[START_REF] Irazoqui | Evolution of host innate defence: Insights from Caenorhabditis elegans and primitive invertebrates[END_REF][START_REF] Sun | Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis[END_REF].

After recognition and signaling, the transcription of response genes and mRNA processing occurs in the nucleus. The protein splicing factor 3A (SF3A) complex accomplishes mRNA splicing via spliceosome and is composed of three proteins, as follows: SF3A1, SF3A2, and SF3A3 (De Arras and Alper, 2013). Hence, different mRNAs are constructed from the pre-mRNA splicing, depending on the cell demands. However, all three SF3A subunits (SF3A1, SF3A2, and SF3A3) are required for mRNA splicing [START_REF] De Arras | Limiting of the Innate Immune Response by SF3A-Dependent Control of MyD88 Alternative mRNA Splicing[END_REF]. In this study, the subunit SF3A1 was downregulated after S. enterica injection, suggesting a splicing brake by SF3A to build an alternative mRNA. Indeed, De Arras and Alper (2013) found the SF3A complex as a regulator of the innate immune response in mouse macrophages. If splicing by SF3A occurs or not, two alternative mRNA are created and drive to opposed immune responses [START_REF] De Arras | Limiting of the Innate Immune Response by SF3A-Dependent Control of MyD88 Alternative mRNA Splicing[END_REF]. The processed mRNA moves for translation by ribosomes at the cytoplasm. Herein, the 60S ribosomal protein L18a (RPL18A) was upregulated in response to S. enterica challenge. RPL18A is a structural constituent of ribosomes and its upregulation suggests an increase in protein translation [START_REF] Aoyama | The primary structure of rat ribosomal protein L18a[END_REF].

Proteasome activator PA28γ (PA28γ or PSME3) was also upregulated in P. perna in response to S. enterica challenge. Proteasomes are barrel-shaped protease complexes that perform the majority of the proteolytic processes in eukaryotic cells [START_REF] Fort | Evolution of Proteasome Regulators in Eukaryotes[END_REF][START_REF] Rechsteiner | Mobilizing the proteolytic machine: Cell biological roles of proteasome activators and inhibitors[END_REF]. Therefore, proteasomes are involved in countless basic cellular functions, such as protein quality control and cellular regulation [START_REF] Fort | Evolution of Proteasome Regulators in Eukaryotes[END_REF][START_REF] Rechsteiner | Mobilizing the proteolytic machine: Cell biological roles of proteasome activators and inhibitors[END_REF]. However, the binding of proteasome activators (PAs) is required to make proteasomes enzymatically active. The proteasome activator PA28 presents three homologs, namely: PA28α, PA28β, and PA28γ [START_REF] Fort | Evolution of Proteasome Regulators in Eukaryotes[END_REF][START_REF] Rechsteiner | Mobilizing the proteolytic machine: Cell biological roles of proteasome activators and inhibitors[END_REF]. The two first mentioned are restricted to jawed vertebrates and are related to important immune functions. On the other hand, PA28γ is found starting from the early invertebrates and is likely involved in transcription or apoptosis since it occurs in the nucleus [START_REF] Fort | Evolution of Proteasome Regulators in Eukaryotes[END_REF][START_REF] Rechsteiner | Mobilizing the proteolytic machine: Cell biological roles of proteasome activators and inhibitors[END_REF].

Another protein upregulated in P. perna mussels was MTFP1, which acts as a receptor to mediate mitochondrial fission in yeast and mammalian cells [START_REF] Sheridan | Mitochondrial fission/fusion dynamics and apoptosis[END_REF][START_REF] Van Der Bliek | Mechanisms of mitochondrial fission and fusion[END_REF]. In healthy cells, mitochondria continually fuse/divide and move through the cytoskeletal in response to cellular processes and to maintain the balance of the mitochondrial network [START_REF] Sheridan | Mitochondrial fission/fusion dynamics and apoptosis[END_REF][START_REF] Van Der Bliek | Mechanisms of mitochondrial fission and fusion[END_REF]. However, according to van der Bliek et al. ( 2013), fusion decreases and/or fission increases under cellular stress. Additionally, fission considerably rises during apoptosis [START_REF] Sheridan | Mitochondrial fission/fusion dynamics and apoptosis[END_REF][START_REF] Van Der Bliek | Mechanisms of mitochondrial fission and fusion[END_REF]. Therefore, MPFP1 upregulated in P. perna hepatopancreas suggests increased mitochondrial fission and, possibly, apoptosis. Indeed, S. enterica is a facultative intracellular pathogen that can survive and proliferate in the intracellular environment [START_REF] Jantsch | Cellular aspects of immunity to intracellular Salmonella enterica[END_REF][START_REF] Kehl | Eat in or Take out? Metabolism of Intracellular Salmonella enterica[END_REF]. Apoptosis may be part of mussel cells' defense against S. enterica, to remove damaged and infected cells.

Several proteins related to cytoskeleton structure and cell motility significantly responded to Salmonella injection. ACTB_G1 was downregulated, whereas β-tubulin, axonemal dyneins (DNAH3 and DNAH9), EFHC1, and cilia-and flagella-associated protein 299 were upregulated in P. perna hepatopancreas compared with controls. Actin monomers (ACTB_G1) polymerize into actin filaments that, in turn, are arranged into bundles or networks at the cell edge [START_REF] Fletcher | Cell mechanics and the cytoskeleton[END_REF][START_REF] Ramaekers | The cytoskeleton and disease[END_REF]. Actin filaments are usually associated with the membrane proteins and are continually polymerized and depolymerized in response to local signaling [START_REF] Fletcher | Cell mechanics and the cytoskeleton[END_REF][START_REF] Ramaekers | The cytoskeleton and disease[END_REF]. As a result, actin drives cell movement and shape (e.g., pseudopods and phagocytosis) and cell-cell communication [START_REF] Fletcher | Cell mechanics and the cytoskeleton[END_REF][START_REF] Ramaekers | The cytoskeleton and disease[END_REF]. β-and α-tubulin heterodimers compose microtubules that handle the anchoring and transport of vesicles and organelles [START_REF] Fletcher | Cell mechanics and the cytoskeleton[END_REF][START_REF] Ramaekers | The cytoskeleton and disease[END_REF].

Microtubules, together with axonemal dyneins, also constitute cilia and flagella [START_REF] Mirvis | Cilium structure, assembly, and disassembly regulated by the cytoskeleton[END_REF][START_REF] Smith | Primary Cilia, Ciliogenesis and the Actin Cytoskeleton: A Little Less Resorption, A Little More Actin Please[END_REF]. Dyneins are microtubule-associated motors that hydrolyze ATP to mediate cilia and flagella movement [START_REF] Mirvis | Cilium structure, assembly, and disassembly regulated by the cytoskeleton[END_REF][START_REF] Smith | Primary Cilia, Ciliogenesis and the Actin Cytoskeleton: A Little Less Resorption, A Little More Actin Please[END_REF]. EFHC1 is a microtubule-associated protein in humans, conserved in cells that hold cilia and/or flagella. The protein holds an EF-hand motif at the C-terminus that binds Ca +2 [START_REF] Ikeda | The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella[END_REF][START_REF] Murai | Characterization of the C-terminal half of human juvenile myoclonic epilepsy protein EFHC1: Dimer formation blocks Ca2+ and Mg2+ binding to its functional EF-hand[END_REF][START_REF] Rossetto | Defhc1.1, a homologue of the juvenile myoclonic gene EFHC1, modulates architecture and basal activity of the neuromuscular junction in Drosophila[END_REF]. Cilia-and flagella-associated proteins act on the assembly of protein complexes, such as axoneme [START_REF] Feng | Hemocyte Clusters Defined by scRNA-Seq in Bombyx mori: In Silico Analysis of Predicted Marker Genes and Implications for Potential Functional Roles[END_REF]. Indeed, hepatopancreatic tubules of bivalves comprise two types of cells, as follows: cylindrical-shaped acidophilic digestive cells and pyramid-shaped basophilic cells [START_REF] Tunali | A structural study of functional cells in hepatopancreas of Mytilus galloprovincialis Lamarck, 1819[END_REF][START_REF] Yang | Histopathology, antioxidant responses, transcriptome and gene expression analysis in triangle sail mussel Hyriopsis cumingii after bacterial infection[END_REF]. In most bivalve species, basophilic cells have cilia on their apical surfaces and are specialized in protein secretion [START_REF] Tunali | A structural study of functional cells in hepatopancreas of Mytilus galloprovincialis Lamarck, 1819[END_REF][START_REF] Yang | Histopathology, antioxidant responses, transcriptome and gene expression analysis in triangle sail mussel Hyriopsis cumingii after bacterial infection[END_REF]. According to [START_REF] Mirvis | Cilium structure, assembly, and disassembly regulated by the cytoskeleton[END_REF] and [START_REF] Smith | Primary Cilia, Ciliogenesis and the Actin Cytoskeleton: A Little Less Resorption, A Little More Actin Please[END_REF], actin can promote cilia disassembly. Therefore, the above protein pattern with actin downregulated and all the other ciliary proteins upregulated suggests an increased cilia activity, probably related to the secretory role of these cells.

The enzymes GALK2, GPD2, and OGDHL act on energy production and metabolism [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. GALK2 converts D-galactose into α-D-galactose-1-phosphate, the first reaction to form glucose-6-phosphate from galactose [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. Afterward, glucose-6-phosphate can proceed to glycolysis or glucogenesis pathways [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. GPD2 catalyzes the oxidation of glycerol-3phosphate to dihydroxyacetone phosphate, an intermediate in the glycolytic pathway [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. It is worth highlighting that glycerol is a lipid constituent, converted into glycerol-3-phosphate by the enzyme glycerol kinase, to be metabolized in glycolysis [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. Moreover, GPD2 participates indirectly in the respiratory chain [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. Last but not least, OGDHL converts 2oxoglutarate into S-succinyl dihydrolipoamide in the tricarboxylic acid (TCA) cycle [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. The enzyme is a major determinant of the metabolic flux in the TCA cycle since 2-oxoglutarate is a key intermediate in carbon metabolism in that cycle and also acts as the major carbon skeleton for nitrogen-assimilatory reactions [START_REF] Huergo | The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite[END_REF][START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. Therefore, the enzyme fluctuates depending on carbon and nitrogen availability and energy demand [START_REF] Huergo | The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite[END_REF][START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF].

The previously mentioned scenario suggests high energy consumption and metabolism regulation as a result of the S. enterica challenge. Indeed, cellular and molecular immune responses demand a high energy cost (Brokordt et al., 2019). According to [START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF], energy balance is crucial for aquatic invertebrates to cope with stress. During moderate stress, the energy demand to cover protection and damage repair increases [START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF]. GALK2 downregulated and GPD2 upregulated in P. perna hepatopancreas suggest a change from carbohydrate to lipid metabolism, together with OGDHL upregulated to maintain the raised flux in the TCA cycle and the energy production. Similarly, a significant decline in glycogen reserves was observed in the hepatopancreas of the scallop Chlamys farreri, 3 h after Vibrio anguillarum challenge [START_REF] Wang | Immune response and energy metabolism of Chlamys farreri under Vibrio anguillarum challenge and high temperature exposure[END_REF]. These results corroborate and show the preference of bivalves to use carbohydrate reserves as a fast energy source for immune response. Under normal circumstances and moderate stress, aerobic metabolism is enough to overlay energy demand and is mostly fueled by lipids and carbohydrates [START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF]. Proteins are metabolized only during extreme energy deficiency [START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF]).

An overview of altered proteins -downregulated or upregulated -in the hepatopancreas of P. perna mussel in response to the S. enterica injection is shown in Figure 5. Before all else, the leucine-rich repeat (LRR) and guanylate kinase (GK) domain-containing protein (LRGUK) acts on pathogens recognition -through the LRR domain which characterizes a membrane-bound toll-like receptor (TLR) -and signal transduction -via the GK domain. As shown by the mussels injected with E. coli, the FERM domain-containing protein (FERM) possibly takes part in signal transduction close to the plasma membrane. At the end of the mitogen-activated protein kinase (MAPK) cascade, the stress-activated protein kinase JNK (JNK) phosphorylates transcription factors that, in turn, regulate the transcription of response genes. The proteasome activator PA28γ (PA28γ) located in the nucleus is also presumably involved in transcription or apoptosis. After transcription, the splicing factor 3A subunit 1 (SF3A1) takes part in mRNA processing, which is translated at the cytoplasm by ribosomes constituted by the 60S ribosomal protein L18a (RPL18A). Mitochondrial fission process protein 1 (MTFP1) mediates mitochondrial fission, a process that rises under cellular stress and during apoptosis. The proteins actin beta/gamma 1 (ACTB_G1), Tubulin beta chain (β-Tubulin), axonemal dyneins (DNAH3 and DNAH9), EFHC1, Cilia-and flagella-associated protein 299 (CF-299) constitute cytoskeleton and cilia structure, which are probably related to the protein secretion function of the ciliated basophilic cells of hepatopancreas. Last but not least, the enzymes GALK2, GPD2, and OGDHL (not shown in Figure 5) act on energy production and metabolism, suggesting a high energy consumption and metabolism regulation to deal with S. enterica challenge. Immune-related role of the altered proteins -downregulated or upregulated -in a ciliated hepatopancreatic cell of Perna perna mussel, 24 h post-Salmonella enterica injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). First of all, the leucine-rich repeat (LRR) and guanylate kinase (GK) domain-containing protein (LRGUK) acts on pathogens recognition and signal transduction, through the LRR and GK domains, respectively. As in the mussels injected with E. coli, the FERM domain-containing protein (FERM) probably also takes part in signal transduction close to the plasma membrane. Afterward, the stressactivated protein kinase JNK (JNK) participates at the end of the mitogen-activated protein kinase (MAPK) cascade, phosphorylating transcription factors that regulate the transcription of response genes. The proteasome activator PA28γ (PA28γ) located in the nucleus is also presumably involved in transcription or apoptosis. After transcription, the splicing factor 3A subunit 1 (SF3A1) takes part in mRNA processing, which is translated at the cytoplasm by ribosomes constituted by the 60S ribosomal protein L18a (RPL18A). Mitochondrial fission process protein 1 (MTFP1) mediates mitochondrial fission, which rises under cellular stress and during apoptosis. Last but not least, the proteins actin beta/gamma 1 (ACTB_G1), Tubulin beta chain (β-Tubulin), axonemal dyneins (DNAH3 and DNAH9), EFHC1, Cilia-and flagellaassociated protein 299 (CF-299) constitute cytoskeleton and cilia structure, which are probably related to the protein secretion function of the ciliated basophilic cells of hepatopancreas.

Vibrio parahaemolyticus

Regarding mussels' proteomic response to V. parahaemolyticus, the proteins leucine-rich repeat and guanylate kinase domain-containing protein (LRGUK), splicing factor 3A subunit 1 (SF3A1), 60S ribosomal protein L18a (RPL18A), β-Tubulin and EFHC1 presented the same profile than mussels challenged with S. enterica. That is, the protein SF3A1 was downregulated and all the others were upregulated compared with controls. Hence, there were similar response patterns primarily concerning recognition, signaling, transcription, and translation, besides cytoskeleton and cilia-associated proteins. Additionally, for mussels injected with V. parahaemolyticus, the proteins rhomboid protease (RHBDL1_2_3), RNA 3-phosphate cyclase-like 1 (RCL1), and Histone H2A were downregulated, whereas the following proteins were upregulated: hepatocyte nuclear factor-4α (HNF4α or NR2A1), poly-U-binding factor 60 kDa (PUF60), THOC1, nuclear GTP-binding protein (NUG1), ABC transmembrane type-1 domain-containing protein, motor dyneins (DNAH), ARMC3, malate dehydrogenase (MDH2) and mitochondrial-processing peptidase subunit beta (PMPCB).

A rhomboid protease (RHBDL1_2_3) was downregulated in P. perna hepatopancreas in response to Vibrio challenge. Rhomboid proteases compose a subfamily of enzymes that catalyze intramembrane proteolysis [START_REF] Urban | The rhomboid protease family: A decade of progress on function and mechanism[END_REF]. These serine endopeptidases are found in all domains of life and, in animals, act by initiating cell signaling [START_REF] Urban | The rhomboid protease family: A decade of progress on function and mechanism[END_REF]. For example, in D. melanogaster and C. elegans, rhomboid proteases mediate the activation of epidermal growth factor receptor (EGFR) signaling to trigger several pathways related to development processes [START_REF] Dutt | EGF Signal Propagation during C. elegans Vulval Development Mediated by ROM-1 Rhomboid[END_REF][START_REF] Van Buskirk | Epidermal growth factor signaling induces behavioral quiescence in Caenorhabditis elegans[END_REF][START_REF] Wasserman | A family of rhomboid-like genes: Drosophila rhomboid-1 and roughoid/rhomboid-3 cooperate to activate EGF receptor signaling[END_REF][START_REF] Yogev | Drosophila EGFR signalling is modulated by differential compartmentalization of Rhomboid intramembrane proteases[END_REF]. One of the main pathways activated by EGFR is the MAPK cascade [START_REF] Guichard | brother of rhomboid, a rhomboid-Related Gene Expressed during Early Drosophila Oogenesis, Promotes EGF-R/MAPK Signaling[END_REF][START_REF] Moghal | The epidermal growth factor system in Caenorhabditis elegans[END_REF], discussed in the prior section (3.1.2). MAPK pathway regulates the cell's transcriptional activity, including that related to immune responses (Hatanaka et al., 2009). Therefore, rhomboid proteases may have some role in signal transduction involved in defense against bacteria in P. perna, probably after recognition and signaling by leucine-rich repeat and guanylate kinase domain-containing protein (LRGUK) (discussed in the prior section 3.1.2).

Another protein upregulated in Vibrio-challenged mussels was the hepatocyte nuclear factor-4α (HNF4α or NR2A1). NR2A1 is part of a superfamily of transcription factors called nuclear receptors (NRs) [START_REF] Miglioli | Nuclear receptors and development of marine invertebrates[END_REF][START_REF] Sladek | What are nuclear receptor ligands?[END_REF]. Through two highly conserved domains -DNA binding (DBD) and ligand binding (LBD) domains -NRs regulate transcription in response to small lipophilic ligands [START_REF] Miglioli | Nuclear receptors and development of marine invertebrates[END_REF][START_REF] Sladek | What are nuclear receptor ligands?[END_REF]. Hence, NR2A1 controls several hepatic functions at the transcriptional level [START_REF] Jiang | Immunohistochemical detection of hepatocyte nuclear factor-4α in vertebrates[END_REF]. In mammals, NR2A1 regulates several genes related to hepatocyte differentiation and the control of lipids, lipoproteins, and glucose metabolism, among other functions [START_REF] Jiang | Immunohistochemical detection of hepatocyte nuclear factor-4α in vertebrates[END_REF]. In Drosophila, [START_REF] Storelli | Drosophila HNF4 Directs a Switch in Lipid Metabolism that Supports the Transition to Adulthood[END_REF] showed NR2A1 as a crucial regulator of lipid metabolism in early adulthood. HNF4α drives the production of very long-chain fatty acids (VLCFA) and VLCFAderived hydrocarbons from lipid stores [START_REF] Storelli | Drosophila HNF4 Directs a Switch in Lipid Metabolism that Supports the Transition to Adulthood[END_REF]. However, these lipids are not fated for energy production but are secreted and transported to the body surfaces to act as waterproofing agents and pheromones [START_REF] Storelli | Drosophila HNF4 Directs a Switch in Lipid Metabolism that Supports the Transition to Adulthood[END_REF]. Through this, HNF4 also maintains glucose homeostasis [START_REF] Storelli | Drosophila HNF4 Directs a Switch in Lipid Metabolism that Supports the Transition to Adulthood[END_REF]. A similar process also occurs in mice, whereby HNF4a from hepatocytes acts on the expression of fatty acids elongases that contributes to the waterproof epidermis [START_REF] Storelli | Drosophila HNF4 Directs a Switch in Lipid Metabolism that Supports the Transition to Adulthood[END_REF]. Therefore, NR2A1 upregulated in P. perna hepatopancreas indicates the regulation of hepatic genes likely related to lipid metabolism, not necessarily for energy production but for some process to deal with the bacterial challenge.

After transcription, mRNA is processed to form messenger ribonucleoprotein complexes (mRNPs) to be translated in the cytoplasm [START_REF] Kew | Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60[END_REF][START_REF] Li | PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis[END_REF][START_REF] Ren | The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis[END_REF]. Splicing is an essential step in mRNA processing, whereby protein-coding sequences known as exons are attached together after removing noncoding sequences (introns) from the primary transcripts [START_REF] Kew | Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60[END_REF][START_REF] Li | PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis[END_REF][START_REF] Ren | The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis[END_REF]. Hence, different combinations of exons are possible to form alternative mRNAs, which allows expanding the range of proteins encoded from the same primary sequence [START_REF] Kew | Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60[END_REF][START_REF] Li | PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis[END_REF][START_REF] Ren | The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis[END_REF]. Splicing is catalyzed by a family of specialized proteins called splicing factors, including poly-U-binding factor 60 kDa (PUF60). PUF60 seems to have a direct role in the modulation of innate immunity [START_REF] Kew | Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60[END_REF][START_REF] Li | PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis[END_REF][START_REF] Ren | The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis[END_REF]. When the sea cucumber Stichopus monotuberculatus was challenged with lipopolysaccharides (LPS) -a constituent of the extern membrane of Gram-negative bacteria assumed as a PAMP -PUF60 mRNA was upregulated [START_REF] Ren | The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis[END_REF]. Additionally, PUF60 overexpressed may induce apoptosis in the sea cucumber [START_REF] Ren | The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis[END_REF]. In the Japanese flounder Paralichthys olivaceus, PUF60 gene expression was upregulated after bacterial infection and, in contrast, was downregulated after viral infection [START_REF] Li | PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis[END_REF]. Additionally, after PUF60 knockdown, bacterial dissemination in the tissues of the flounder rose whereas viral replication was blocked [START_REF] Li | PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis[END_REF]. In the opposite situation, PUF60 overexpression inhibited bacterial dissemination but promoted viral replication [START_REF] Li | PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis[END_REF]. Similar to the sea cucumber, PUF60 was found to be involved in autophagy and apoptosis processes in the Japanese flounder [START_REF] Li | PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis[END_REF][START_REF] Ren | The first echinoderm poly-U-binding factor 60 kDa (PUF60) from sea cucumber (Stichopus monotuberculatus): Molecular characterization, inducible expression and involvement of apoptosis[END_REF]. In C. elegans, bacterial infection induces a PUF60-dependent splicing remodeling [START_REF] Kew | Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60[END_REF]. [START_REF] Kew | Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60[END_REF] showed that PUF60 leads to sensitivity to bacterial infection, but increases lifespan likely by limiting the inflammatory process induced by infection. Therefore, PUF60 upregulated in P. perna challenged by Vibrio corroborates with the above-mentioned results and suggests its role in immunity modulation in response to bacterial infection.

Still about mRNA processing, the protein THOC1 is part of the multi-subunit THO complex that, in turn, composes the transcription and export (TREX) complex [START_REF] Jimeno | The THO complex as a key mRNP biogenesis factor in development and cell differentiation[END_REF][START_REF] Reed | Coupling transcription, splicing and mRNA export[END_REF]. TREX complex is found in all eukaryotes and also takes part in mRNA processing to be transported from the nucleus to the cytoplasm as messenger ribonucleoprotein complexes (mRNPs) [START_REF] Jimeno | The THO complex as a key mRNP biogenesis factor in development and cell differentiation[END_REF][START_REF] Reed | Coupling transcription, splicing and mRNA export[END_REF]. Particularly, the THO complex couple transcription and export, as it has a functional role related to RNA polymerase II (RNAPII) transcription elongation and mediates mRNA export [START_REF] Castellano-Pozo | The Caenorhabditis elegans THO Complex Is Required for the Mitotic Cell Cycle and Development[END_REF][START_REF] Jimeno | The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability[END_REF][START_REF] Rehwinkel | Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster[END_REF]. Additionally, it was shown that the THO complex has a key role in the cell cycle and differentiation [START_REF] Castellano-Pozo | The Caenorhabditis elegans THO Complex Is Required for the Mitotic Cell Cycle and Development[END_REF]. Therefore, THOC1 (homolog Hpr1 in Drosophila, C. elegans, and yeast) upregulated in P. perna, together with other proteins, suggests an increase in protein transcription and translation activity in the cell in response to a signal. Indeed, the Vibrio-challenged group had the highest response, with approximately 500 proteins upregulated or downregulated. Thus, it seems that THOC1 and, therefore, THO complex, have some role in the transcription and translation of proteins related to the immune response in the mussel P. perna.

Two proteins related to ribosome biogenesis were altered in response to Vibrio challenge -RCL1 downregulated and NUG1 upregulated. The primary transcripts of rRNAs for the small and large ribosomal subunits are co-transcribed as a single transcript to be cleavage afterward [START_REF] Horn | Rcl1 protein, a novel nuclease for 18 S ribosomal RNA production[END_REF][START_REF] Zhu | Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and upregulates a cohort of ribosome biogenesis genes in zebrafish[END_REF]. RCL1 is an evolutionarily conserved endonuclease that mediates this cleave [START_REF] Horn | Rcl1 protein, a novel nuclease for 18 S ribosomal RNA production[END_REF][START_REF] Zhu | Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and upregulates a cohort of ribosome biogenesis genes in zebrafish[END_REF]. In yeasts, RCL1 cleaves the pre-rRNA at the A2 site [START_REF] Horn | Rcl1 protein, a novel nuclease for 18 S ribosomal RNA production[END_REF], whereas in zebrafish cleaves at the A1 site [START_REF] Zhu | Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and upregulates a cohort of ribosome biogenesis genes in zebrafish[END_REF]. Therefore, depletion in RCL1 impairs ribosome maturation [START_REF] Horn | Rcl1 protein, a novel nuclease for 18 S ribosomal RNA production[END_REF][START_REF] Zhu | Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and upregulates a cohort of ribosome biogenesis genes in zebrafish[END_REF], and at the phenotypical level in zebrafish, rcl1-/-mutants exhibited alterations in the digestive organogenesis, such as a diminutive liver [START_REF] Zhu | Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and upregulates a cohort of ribosome biogenesis genes in zebrafish[END_REF]. According to [START_REF] Zhu | Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and upregulates a cohort of ribosome biogenesis genes in zebrafish[END_REF], RCL1 deficiency possibly triggers an ordinary mechanism that upregulates the expression of other genes related to ribosome biogenesis. Indeed, concomitant to RCL1 downregulation, GTPase NUG1 (nucleostemin in humans) was upregulated.

NUG1 is essential for 60S ribosome (large subunit) assembly and nuclear export [START_REF] Bassler | The Nug1 GTPase reveals an N-terminal RNAbinding domain that is essential for association with 60 S pre-ribosomal particles[END_REF][START_REF] Manikas | The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation[END_REF]. The enzyme consists of a GTPase middle domain flanked by the N-and C-terminal domains [START_REF] Bassler | The Nug1 GTPase reveals an N-terminal RNAbinding domain that is essential for association with 60 S pre-ribosomal particles[END_REF][START_REF] Manikas | The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation[END_REF]. According to [START_REF] Bassler | The Nug1 GTPase reveals an N-terminal RNAbinding domain that is essential for association with 60 S pre-ribosomal particles[END_REF], the N-terminal domain is crucial for nucleolar targeting and association with pre-60S particles since it displays RNA binding activity. The highly evolutionary-conserved C-domain is also essential for ribosome biogenesis, although its function is not well known [START_REF] Bassler | The Nug1 GTPase reveals an N-terminal RNAbinding domain that is essential for association with 60 S pre-ribosomal particles[END_REF][START_REF] Manikas | The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation[END_REF]. The central GTPase domain is stimulated by potassium ions and, as opposed to the other domains, plays a non-essential regulative role in pre-60 S subunit biogenesis [START_REF] Manikas | The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation[END_REF]. [START_REF] Manikas | The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation[END_REF] showed that NUG1 and the RNA helicase DBP10 bind close to each other in the pre-ribosome, where posteriorly the peptidyl-transferase center (PTC) is localized in the mature ribosome. Therefore, it suggests these enzymes are involved in the formation of the PTC in ribosomes. Defects or depletion of NUG1 results in ribosome 60S biogenesis issues [START_REF] Manikas | The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation[END_REF]. In C. elegans, NUG1 has a role in cell growth and proliferation by enabling ribosome biogenesis [START_REF] Kudron | C. elegans Nucleostemin Is Required for Larval Growth and Germline Stem Cell Division[END_REF]. In Drosophila, NUG1 depletion impairs midgut precursor cells and cell growth likely as a result of blocking ribosome biogenesis [START_REF] Rosby | Knockdown of the Drosophila GTPase nucleostemin 1 impairs large ribosomal subunit biogenesis, cell growth, and midgut precursor cell maintenance[END_REF]. Therefore, it seems that NUG1 upregulated in P. perna hepatopancreas likely compensates for the RCL1 depletion to maintain ribosome biogenesis fronting disturbances caused by the Vibrio injection in mussels.

It is widely known that histones are structural components of chromatin, also playing crucial roles in DNA processes [START_REF] Nikapitiya | The role of histones in the immune responses of aquatic invertebrates[END_REF]. However, histones or histone-derived peptides have been also reported to have antimicrobial activity, since their N-terminus correspond to antimicrobial peptides (AMPs) [START_REF] Nikapitiya | The role of histones in the immune responses of aquatic invertebrates[END_REF][START_REF] Vizioli | Antimicrobial peptides from animals: focus on invertebrates[END_REF]. Therefore, in addition to their first known function, histones participate in innate immune responses through multiple mechanisms of action both in invertebrates and vertebrates [START_REF] Nikapitiya | The role of histones in the immune responses of aquatic invertebrates[END_REF][START_REF] Vizioli | Antimicrobial peptides from animals: focus on invertebrates[END_REF]. Free histones can be found out of the nucleus (e.g., cytoplasm, membranes, and extracellular fluids) to be easier to interact with pathogens [START_REF] Nikapitiya | The role of histones in the immune responses of aquatic invertebrates[END_REF]. Histones bound to lipid droplets may be released into the extracellular environment or, in an alternative process, form extracellular traps (ETs) after hemocyte cell death to catch microorganisms [START_REF] Nikapitiya | The role of histones in the immune responses of aquatic invertebrates[END_REF]. As histones also have LPS-binding activity, they may be present on the hemocyte surface functioning as PRRs [START_REF] Nikapitiya | The role of histones in the immune responses of aquatic invertebrates[END_REF]. Additionally, histones and their fragments, combined with other molecules, can be released in the hemolymph to act as free AMPs or PRRs [START_REF] Nikapitiya | The role of histones in the immune responses of aquatic invertebrates[END_REF]. Indeed, histone H2A has been shown as a precursor of antimicrobial peptides in several mollusks [START_REF] De Zoysa | Abhisin: A potential antimicrobial peptide derived from histone H2A of disk abalone (Haliotis discus discus)[END_REF][START_REF] Li | Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri[END_REF][START_REF] Sathyan | Identification and Molecular Characterization of Molluskin, a Histone-H2A-Derived Antimicrobial Peptide from Molluscs[END_REF]. Therefore, histone H2A downregulated in P. perna hepatopancreas in response to Vibrio suggests that these proteins have a role in some sort of antimicrobial mechanism in the mussel defense.

An ABC transmembrane type-1 domain-containing protein was upregulated in P. perna hepatopancreas after Vibrio injection in mussels. The ATP-binding cassette (ABC) comprises a superfamily of transmembrane transport proteins highly conserved from bacteria to humans [START_REF] Ogasawara | ABC proteins in evolution[END_REF][START_REF] Sheps | The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes[END_REF]. These proteins share the same basic structure -two ATP-binding and two transmembrane domainsand present high similarity in the sequence of their ATP-binding domain [START_REF] Ogasawara | ABC proteins in evolution[END_REF][START_REF] Sheps | The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes[END_REF]. Since they are able to interact with a huge range of substrates, ABC transporters present diverse functions [START_REF] Ogasawara | ABC proteins in evolution[END_REF][START_REF] Sheps | The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes[END_REF]. Among the four types of ABC proteins, type 1 seems to be one of the most ancient in evolution [START_REF] Ogasawara | ABC proteins in evolution[END_REF]. ABC transmembrane type-1 proteins are specialized in importing specific nutrients [START_REF] Ogasawara | ABC proteins in evolution[END_REF] and their upregulation in P. perna hepatopancreas suggests some role of cellular importing fronting bacterial challenge.

Two motor dyneins heavy-chain were also upregulated in mussels injected with V. parahaemolyticus. As opposed to axonemal dyneins that are involved in the cilia and flagella beating, cytoplasmatic dyneins are one of the three families of cytoskeletal motor proteins [START_REF] Kardon | Regulators of the cytoplasmic dynein motor[END_REF][START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF]. Regarding structure, the cytoplasmic dynein heavy chain consists primarily of a C-terminal motor domain and an N-terminal tail domain. The motor domain comprises six AAA domains arranged in a ring that can bind ATP and force movement along cytoskeletal filaments [START_REF] Kardon | Regulators of the cytoplasmic dynein motor[END_REF][START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF]. The tail domain, in turn, mediates cargo interactions directly or by recruiting accessory proteins [START_REF] Kardon | Regulators of the cytoplasmic dynein motor[END_REF][START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF]. Therefore, dyneins are able to move along the microtubules for minus enddirected transport, that is, driving to the microtubule organizing center close to the nucleus [START_REF] Kardon | Regulators of the cytoplasmic dynein motor[END_REF][START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF]. Dynein cargoes include organelles, vesicles, transcription factors, cytoskeletal filaments, mRNPs, among others [START_REF] Kardon | Regulators of the cytoplasmic dynein motor[END_REF][START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF]. Hence, cytoplasmic dyneins are involved in the vast majority of cellular processes, including transmitting signals between cells or different parts of the cell for immune defense [START_REF] Kardon | Regulators of the cytoplasmic dynein motor[END_REF][START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF].

The armadillo repeat-containing protein 3 (ARMC3) was upregulated in Vibriochallenged P. perna. Armadillo repeat-containing proteins (ARMCs) compose a family with a domain consisting of tandem repeats of approximately 42 amino acids, a structure that overall allows protein-protein binding [START_REF] Gul | Metazoan evolution of the armadillo repeat superfamily[END_REF]Huang et al., 2021;[START_REF] Tewari | Armadillo-repeat protein functions: Questions for little creatures[END_REF]. ARMCs can also contain additional domains in the N-or Cterminus and, therefore, present additional functions related to these domains [START_REF] Gul | Metazoan evolution of the armadillo repeat superfamily[END_REF]Huang et al., 2021;[START_REF] Tewari | Armadillo-repeat protein functions: Questions for little creatures[END_REF]. This is the case of ARMC3, which in metazoans also presents an EDR1 additional domain [START_REF] Gul | Metazoan evolution of the armadillo repeat superfamily[END_REF]. ARMC3 is found from early metazoans to human beings, although the identity between ARMC3 in nonmammals vertebrates and Homo sapiens is low (49%) (Huang et al., 2021). Hence, it can present different functions among these groups. As far as known, ARMC3 takes part in cilio/flagellogenesis in animals [START_REF] Lonergan | Identification of Novel Lung Genes in Bronchial Epithelium by Serial Analysis of Gene Expression[END_REF][START_REF] Pausch | A frameshift mutation in ARMC3 is associated with a tail stump sperm defect in Swedish Red (Bos taurus) cattle[END_REF]. Possibly, ARMC3 upregulation in P. perna suggests a rise in cilia activity demand on basophilic cells of the hepatopancreas, probably related to the secretory role of these cells. Additionally, it corroborates with the cilia-related proteins β-Tubulin and EFHC1, also upregulated in Vibrio-challenged mussels (discussed in the prior topic 3.1.2).

As discussed in the prior section (3.1.2), all the alterations associated with defense spend a lot of energy (Brokordt et al., 2019). As expected, an enzyme related to energy production and the metabolism was upregulated in Vibrio-challenged mussels compared to controls. The mitochondrial enzyme malate dehydrogenase isoform 2 (MDH2) catalyzes the reversible conversion of malate to oxaloacetate in the citric acid cycle [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. Additionally, MDH2 takes part in the malate-aspartate shuttle, which acts in molecule traffic between cytosol and mitochondria to integrate different metabolic pathways [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. Besides participating in the citric acid cycle, oxaloacetate is an intermediate for gluconeogenesis in the cytosol [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. Further, several amino acids are deaminated in the mitochondria to be converted into oxaloacetate and then, enter the gluconeogenesis pathway [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. However, to be transported by the shuttle from mitochondria to cytosol, oxaloacetate has to be reduced to malate by MDH2 and then, oxidated into oxaloacetate again [START_REF] Nelson | Lehninger Principles of Biochemistry[END_REF]. Therefore, MDH2 upregulated suggests an increase in energy demand and, possibly, the activation of the gluconeogenesis pathway.

Mitochondrial-processing peptidase subunit beta (PMPCB) together with the subunit alpha (PMPCA) form mitochondrial processing proteases (MPPs), which act n protein targeting from the cytosol to mitochondria [START_REF] Gakh | Mitochondrial processing peptidases[END_REF]. Hence, proteins destined for the mitochondrial matrix are targeted as precursor polypeptides carrying Nterminal prolongations [START_REF] Gakh | Mitochondrial processing peptidases[END_REF]. Once these presequences reach their final location within the mitochondrion, these prolongations are cleaved off by MPPs [START_REF] Gakh | Mitochondrial processing peptidases[END_REF]. For this, PMPCA recognizes and binds to the substrate whereas PMPCB is the catalytic subunit [START_REF] Gakh | Mitochondrial processing peptidases[END_REF]. Therefore, PMPCB upregulated in Vibriochallenged mussels suggests an increase in protein supply for mitochondrion. Indeed, together with MDH2 upregulation, it indicates the rise in mitochondrion activity and, perhaps, an increase in protein supply to be forwarded to the gluconeogenesis pathway.

To recap the above discussion, Figure 6 shows a frame of the immune-related roles of the altered proteins -downregulated or upregulated -in P. perna mussel after the V. parahaemolyticus challenge. As in the mussels injected with S. enterica, the leucine-rich repeat (LRR) and guanylate kinase (GK) domain-containing protein (LRGUK) was upregulated. In this protein, the LRR domain typifies a toll-like receptor (TLR) for pathogens recognition and the GK domain seems to act on signal transduction. Thereafter, a rhomboid protease (RHBDL1_2_3) seems to participate in signal transduction, triggering some pathway likely related to transcriptional response against pathogens. The transcription factor hepatocyte nuclear factor-4α (NR2A1) regulates the transcription of genes related to hepatic functions and lipid metabolism. The protein THOC1 also acts on transcription and mediates mRNA export to the cytoplasm. However, before mRNA export, the proteins splicing factor 3A subunit 1 (SF3A1) and poly-U-binding factor 60 kDa (PUF60) take part in mRNA processing. In cooperation, the proteins RNA 3-phosphate cyclase-like 1 (RCL1) and nuclear GTPbinding protein (NUG1) participate in ribosome biogenesis. Thereafter, mRNA can be translated at the cytoplasm by ribosomes structurally constituted by the 60S ribosomal protein L18a (RPL18A). The mitochondrial-processing peptidase subunit beta (PMPCB) takes part in protein supply and targeting from the cytosol to mitochondria, indicating increased activity of this organelle. Corroborating with PMPCB function, the mitochondrial enzyme malate dehydrogenase isoform 2 (MDH2) (not shown in Figure 6) takes part in the citric acid cycle and acts in molecule traffic between cytosol and mitochondria. ABC transmembrane type-1 domain-containing protein (ABC) is specialized in importing specific nutrients, presumably critical for fronting bacteria. Histone H2A, besides its known nuclear function, may be found outside of the nucleus acting as a humoral effector via multiple mechanisms of action, because of its antimicrobial activity. Finally, the proteins β-Tubulin, EFHC1, and ARMC3 constitute cilia structure, which is probably related to the protein secretion function of the ciliated basophilic cells of the hepatopancreas. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). As in the mussels injected with S. enterica, the leucine-rich repeat (LRR) and guanylate kinase (GK) domaincontaining protein (LRGUK) presents an LRR domain, which typifies a toll-like receptor (TLR) for pathogens recognition, and the GK domain, that seems to perform signal transduction. Thereafter, a rhomboid protease (RHBDL1_2_3) seems to participate in signal transduction, triggering some pathway likely related to transcriptional response against pathogens. Then, the transcription factor hepatocyte nuclear factor-4α (NR2A1) regulates the transcription of genes related to hepatic functions and lipid metabolism. THOC1 also acts on transcription and mediates mRNA export to the cytoplasm. Nevertheless, before mRNA export, the proteins splicing factor 3A subunit 1 (SF3A1) and poly-U-binding factor 60 kDa (PUF60) take part in mRNA processing. In concert, the proteins RNA 3-phosphate cyclase-like 1 (RCL1) and nuclear GTP-binding protein (NUG1) are required for ribosome biogenesis. Then, mRNA can be translated at the cytoplasm by ribosomes constituted by the 60S ribosomal protein L18a (RPL18A). The mitochondrial-processing peptidase subunit beta (PMPCB) takes part in protein supply and targeting from the cytosol to mitochondria, indicating increased activity of this organelle. ABC transmembrane type-1 domain-containing protein (ABC) is specialized in importing specific nutrients, presumably important for fronting bacteria. Histone H2A, besides its known nuclear function, may be found out of the nucleus acting as a humoral effector via multiple mechanisms of action, because of its antimicrobial activity. Finally, the proteins β-Tubulin, EFHC1, and ARMC3 constitute cilia structure, which is probably related to the protein secretion function of the ciliated basophilic cells of the hepatopancreas.

Table 1. List of proteins significantly different from both non-injected (NC) and injected (IC) controls, identified in the hepatopancreas of Perna perna mussels after bacterial challenge -Escherichia coli (EC), Salmonella enterica (SE), and Vibrio parahaemolyticus (VP).

Specific response to Vibrio parahaemolyticus

Mussels challenged with Vibrio parahaemolyticus (VP) presented 343 proteins significantly different from all the other conditions (NC -non-injected control; ICinjected control; EC -Escherichia coli; and SE -Salmonella enterica), within a total of 597 proteins that significantly differed from at least one condition. Among these 343 proteins, 327 were downregulated whereas only 16 were upregulated (Figure 7.a). These altered proteins in specific response to Vibrio were classified into functional categories based on the Database of Clusters of Orthologous Genes (COGs) and the most frequent (%) COGs among them are shown in Figure 7.b, as follows: T -Signal transduction mechanisms (13%); O -Posttranslational modification, protein turnover, chaperones (12%); S -Function unknown (10%); U -Intracellular trafficking, secretion, and vesicular transport (9%); A -RNA processing and modification (6%); Z -Cytoskeleton (6%); K -Transcription (6%); J -Translation, ribosomal structure, and biogenesis (5%); and remaining categories (33%). The 16 upregulated proteins did not fit in any COG functional category, therefore significantly different proteins in specific response to Vibrio of all the above COG categories were downregulated. Figure 8 compares the percentage (%) of proteins by each one of the eight most frequent COGs, between proteins significantly different (downregulated) only for mussels challenged by V. parahaemolyticus (VP) and the total of proteins found in P. perna mussels (all the other condition-groups). In the specific response to Vibrio, that is, proteins significantly different from all the other conditions, there was a lower percentage of proteins fitted in the categories cytoskeleton (Z) and function unknown (S) compared with the total of proteins found (Figure 8). On the other hand, the percentage of proteins included in the categories transcription (K), RNA processing and modification (A), and posttranslational modification, protein turnover, and chaperones (O) was smoothly higher than in the total of proteins found in P. perna (Figure 8). Although the functional categories translation, ribosomal structure and biogenesis (J), signal transduction mechanisms (T), and intracellular trafficking, secretion, and vesicular transport (U) comprise one of the main portions of significantly different proteins in specific response to Vibrio, the percentage of proteins fitted into these categories did not change quantitatively compared with the total of proteins found (Figure 8). ) of proteins between all groups (total proteins found in Perna perna mussels) and proteins altered in specific response to Vibrio parahaemolyticus (VP; significantly different from all the other groups), according to the eight most frequent COG functional categories.

Unlike most marine bacteria, the genus Vibrio comprises multiple species harmful to shellfish [START_REF] Gosling | Marine Bivalve Molluscs[END_REF]. Indeed, vibrioses constitute most reports of bacterial diseases of bivalves, often leading to mass mortality [START_REF] Gosling | Marine Bivalve Molluscs[END_REF]. Among the approximately 100 species in the genus, the following Vibrio species are often associated with diseases in fish and shellfish: V. parahaemolyticus, V. vulnificus, V. furnissii, V. campbellii, V. harveyi, V. alginolyticus, and V. anguillarum [START_REF] Liu | Comprehensive analysis of the phylogeny and extracellular proteases in genus Vibrio strain[END_REF]. These pathogenic strains produce several virulence factors (e.g., enterotoxins, cytotoxins, proteases, among others), which are crucial to their invasive mechanisms and subsequent infection [START_REF] Liu | Comprehensive analysis of the phylogeny and extracellular proteases in genus Vibrio strain[END_REF]. In fact, the proteomic response of P. perna specifically to V. parahaemolyticus was more substantial (343 altered proteins) than that to the marine introduced bacteria -E. coli and S. enterica.

Since bivalves throve in a rich-Vibrio environment throughout their evolutionary history, they likely assembled countless pattern recognition receptors (PRRs) to identify conserved pathogen-associated molecular patterns (PAMPs) of Vibrio species [START_REF] Gosling | Marine Bivalve Molluscs[END_REF][START_REF] Liu | Comprehensive analysis of the phylogeny and extracellular proteases in genus Vibrio strain[END_REF]. The first phase for generating a defense response is recognition and signal transduction [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. However, it can be impaired as the pathogen virulence increases [START_REF] Liu | Comprehensive analysis of the phylogeny and extracellular proteases in genus Vibrio strain[END_REF][START_REF] Yu | Comparative transcriptomic analysis of surf clams (Paphia undulate) infected with two strains of Vibrio spp. reveals the identity of key immune genes involved in host defense[END_REF]. Indeed, the majority of significantly different proteins of P. perna in specific response to Vibrio fitted in the COG T -signal transduction mechanisms. Nevertheless, Vibrio seems to suppress P. perna immune response since among the 343 altered proteins in specific response to V. parahaemolyticus, 326 proteins were downregulated (Figure 7). These proteins were directly or indirectly related to defense, including proteins comprised in the COG T. Accordingly, this result suggests that Vibrio is virulent for P. perna mussels and likely suppresses key-proteins related to immune response.

Vibrio parahaemolyticus was also found to be highly virulent for surf clams Paphia undulate [START_REF] Yu | Comparative transcriptomic analysis of surf clams (Paphia undulate) infected with two strains of Vibrio spp. reveals the identity of key immune genes involved in host defense[END_REF]. The rate of survival of the clams abruptly declined at 24 h (87.2%) and 48 h (65.3%) post-infection with V. parahaemolyticus [START_REF] Yu | Comparative transcriptomic analysis of surf clams (Paphia undulate) infected with two strains of Vibrio spp. reveals the identity of key immune genes involved in host defense[END_REF]. Additionally, the clams presented an extensive range of downregulated genes (1433), which were associated with cellular and molecular mechanisms for pathogen recognition, and immunity triggering [START_REF] Yu | Comparative transcriptomic analysis of surf clams (Paphia undulate) infected with two strains of Vibrio spp. reveals the identity of key immune genes involved in host defense[END_REF]. The razor clam Sinonovacula constricta also presented a strong response to V. parahaemolyticus infection [START_REF] Zhao | Comparative transcriptome analysis of Sinonovacula constricta in gills and hepatopancreas in response to Vibrio parahaemolyticus infection[END_REF]. After injection, 1,781 significant differentially expressed transcripts were obtained in the gills and 490 in the hepatopancreas [START_REF] Zhao | Comparative transcriptome analysis of Sinonovacula constricta in gills and hepatopancreas in response to Vibrio parahaemolyticus infection[END_REF]. Several transcripts in both tissues were specifically related to immune response, such as pathogen recognition receptors (PRRS) and immune signaling and cell communication [START_REF] Zhao | Comparative transcriptome analysis of Sinonovacula constricta in gills and hepatopancreas in response to Vibrio parahaemolyticus infection[END_REF]. Corroborating these results, proteomic studies with other Vibrio species (e.g., V. anguillarum and V. tapetis) injected in the mussel Mytilus galloprovincialis and the Manila clam Ruditapes philippinarum have also altered several proteins related to recognition and signaling, among others immune roles (Ji et al., 2013;[START_REF] Smits | A proteomic study of resistance to Brown Ring disease in the Manila clam, Ruditapes philippinarum[END_REF][START_REF] Wu | Proteomic and metabolomic responses in hepatopancreas of Mytilus galloprovincialis challenged by Micrococcus luteus and Vibrio anguillarum[END_REF].

After recognition and signaling, the response at the subcellular level involves the transcription of response genes and RNA processing, including splicing to obtain alternative mRNAs [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF]Hatanaka et al., 2009;[START_REF] Irazoqui | Evolution of host innate defence: Insights from Caenorhabditis elegans and primitive invertebrates[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. Then, the translation and posttranslational modifications of recent-constructed proteins occur to make them functional and targeted [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF]Hatanaka et al., 2009;[START_REF] Irazoqui | Evolution of host innate defence: Insights from Caenorhabditis elegans and primitive invertebrates[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. These recent-formed molecules, in turn, perform different roles at several levels to trigger both cellular and humoral immune responses, including a huge range of recognition factors and humoral effectors [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF][START_REF] Sousa | Review: Antibacterial components of the Bivalve's immune system and the potential of freshwater bivalves as a source of new antibacterial compounds[END_REF]. The main functional categories of proteins significantly different (downregulated) in specific response to Vibrio corresponded with the above processes and reinforce Vibrio virulence to P. perna, namely: O -Posttranslational modification, protein turnover, chaperones (12%); A -RNA processing and modification (6%); K -Transcription (6%); J -Translation, ribosomal structure and biogenesis (5%) (Figure

7.b).

Last but not least, the functional categories cytoskeleton and intracellular traffic, secretion, and vesicular transport had an increased percentage of proteins downregulated in specific response of P. perna to Vibrio (Figure 7.b). The cytoskeleton is the basis of all cellular movements and works together with transporter proteins, to carry organelles, molecules, and vesicles [START_REF] Kardon | Regulators of the cytoplasmic dynein motor[END_REF][START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF]. Consequently, the cytoskeleton is closely linked to secretion [START_REF] Kardon | Regulators of the cytoplasmic dynein motor[END_REF][START_REF] Roberts | Functions and mechanics of dynein motor proteins[END_REF]. As discussed in the prior topics (3.1.1, 3.1.2, and 3.1.3), the main role of ciliated basophilic cells of hepatopancreas is protein secretion [START_REF] Tunali | A structural study of functional cells in hepatopancreas of Mytilus galloprovincialis Lamarck, 1819[END_REF][START_REF] Yang | Histopathology, antioxidant responses, transcriptome and gene expression analysis in triangle sail mussel Hyriopsis cumingii after bacterial infection[END_REF]. In front of a bacterial challenge, basophilic cells likely secret proteins and other molecules related to mussel defense, such as humoral effectors observed in other bivalves infected with Vibrio [START_REF] Ji | Responses of Mytilus galloprovincialis to bacterial challenges by metabolomics and proteomics[END_REF][START_REF] Smits | A proteomic study of resistance to Brown Ring disease in the Manila clam, Ruditapes philippinarum[END_REF]Wu et al., 2013;[START_REF] Yang | Cloning of a big defensin gene and its response to Vibrio parahaemolyticus challenge in the noble scallop Chlamys nobilis (Bivalve: Pectinidae)[END_REF]. Therefore, the high percentage of downregulated proteins in the hepatopancreatic cells of P. perna, which are involved in transcription, RNA processing, translation and protein production, cytoskeleton, and secretion, again support the hypothesis that V. parahaemolyticus is virulent to the mussel and impairs several crucial processes to triggering defense response.

Conclusion

This is the first study using a shotgun proteomic approach in the mussel Perna perna after bacterial challenge. Therefore, for the first time, it is possible to notice a global profile of P. perna hepatopancreas proteins (a total of 3,805), focusing on critical proteins for the mussel-bacteria relationship. As expected, mussels injected with the marine indigenous bacterium V. parahaemolyticus presented a more prominent response -343 proteins significantly different from all the other conditions, from a total of 597 significantly different -than those challenged with marine introduced bacteria -E. coli and S. enterica. Additionally, as the large majority of proteins in specific response to V. parahaemolyticus were downregulated (327), this bacterium seems to be virulent to the mussel P. perna since it suppresses mussel's immune response. In general, that is, for the three challenge bacteria, significantly different proteins -downregulated or upregulated -were found to perform critical roles in immune response at all levels in the mussel hepatopancreas, as follows: recognition and signal transduction; transcription; RNA processing; translation and protein processing; secretion; and humoral effectors against bacteria. Hence, this study was the first step to better understanding P. perna immune responses at molecular levels and pointing out critical and strategic proteins against bacteria. Going beyond, these proteins can be helpful in biotechnology as molecular biomarkers related to bacteria exposure. Then, it can be applied in environmental monitoring and to assess the health status and/or food safety of commercial species (such as the brown mussel P. perna). For example, the early detection of Vibrio species in mussel farming and its treatment. Additionally, among the huge range of antimicrobial compounds in bivalves, it can be found bioactive molecules of human interest. However, further studies are needed to go deeper into this fertile field and explore promising molecules to be applied in several areas, including coastal marine resource management such as bivalve farms. Immune-related role of the altered proteins in a ciliated hepatopancreatic cell of Perna perna mussel, 24h post-Escherichia coli injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). In mussels challenged with E. coli, a von Willebrand Factor A domain-containing protein (vWA) and a FERM domain-containing protein (FERM) were downregulated. FERM is probably located close to the plasma membrane and takes part in signal transduction. VWA seems to act as a humoral effector in mollusks, primarily in the extracellular matrix. Immune-related role of the altered proteins -downregulated or upregulated -in a ciliated hepatopancreatic cell of Perna perna mussel, 24 h post-Salmonella enterica injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). First of all, the leucine-rich repeat (LRR) and guanylate kinase (GK) domain-containing protein (LRGUK) acts on pathogens recognition and signal transduction, through the LRR and GK domains, respectively. As in the mussels injected with E. coli, the FERM domain-containing protein (FERM) probably also takes part in signal transduction close to the plasma membrane. Afterward, the stressactivated protein kinase JNK (JNK) participates at the end of the mitogen-activated protein kinase (MAPK) cascade, phosphorylating transcription factors that regulate the transcription of response genes. The proteasome activator PA28γ (PA28γ) located in the nucleus is also presumably involved in transcription or apoptosis. After transcription, the splicing factor 3A subunit 1 (SF3A1) takes part in mRNA processing, which is translated at the cytoplasm by ribosomes constituted by the 60S ribosomal protein L18a (RPL18A). Mitochondrial fission process protein 1 (MTFP1) mediates mitochondrial fission, which rises under cellular stress and during apoptosis. Last but not least, the proteins actin beta/gamma 1 (ACTB_G1), Tubulin beta chain (β-Tubulin), axonemal dyneins (DNAH3 and DNAH9), EFHC1, Cilia-and flagellaassociated protein 299 (CF-299) constitute cytoskeleton and cilia structure, which are probably related to the protein secretion function of the ciliated basophilic cells of hepatopancreas. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). As in the mussels injected with S. enterica, the leucine-rich repeat (LRR) and guanylate kinase (GK) domaincontaining protein (LRGUK) presents an LRR domain, which typifies a toll-like receptor (TLR) for pathogens recognition, and the GK domain, that seems to perform signal transduction. Thereafter, a rhomboid protease (RHBDL1_2_3) seems to participate in signal transduction, triggering some pathway likely related to transcriptional response against pathogens. Then, the transcription factor hepatocyte nuclear factor-4α (NR2A1) regulates the transcription of genes related to hepatic functions and lipid metabolism. THOC1 also acts on transcription and mediates mRNA export to the cytoplasm. Nevertheless, before mRNA export, the proteins splicing factor 3A subunit 1 (SF3A1) and poly-U-binding factor 60 kDa (PUF60) take part in mRNA processing. In concert, the proteins RNA 3-phosphate cyclase-like 1 (RCL1) and nuclear GTP-binding protein (NUG1) are required for ribosome biogenesis. Then, mRNA can be translated at the cytoplasm by ribosomes constituted by the 60S ribosomal protein L18a (RPL18A). The mitochondrial-processing peptidase subunit beta (PMPCB) takes part in protein supply and targeting from the cytosol to mitochondria, indicating increased activity of this organelle. ABC transmembrane type-1 domain-containing protein (ABC) is specialized in importing specific nutrients, presumably important for fronting bacteria. Histone H2A, besides its known nuclear function, may be found out of the nucleus acting as a humoral effector via multiple mechanisms of action, because of its antimicrobial activity. Finally, the proteins β-Tubulin, EFHC1, and ARMC3 constitute cilia structure, which is probably related to the protein secretion function of the ciliated basophilic cells of the hepatopancreas.

Fig 1 .

 1 Fig 1. Vermelha Beach (VB), Rio de Janeiro, Brazil: mussels and seawater sampling location.

Fig 2 .

 2 Fig 2. The external and internal anatomical features of the mussel Perna perna.

Fig

  Fig 4.Immune-related role of the altered proteins in a ciliated hepatopancreatic cell of Perna perna mussel, 24h post-Escherichia coli injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). In mussels challenged with E. coli, a von Willebrand Factor A domain-containing protein (vWA) and a FERM domain-containing protein (FERM) were downregulated. FERM is probably located close to the plasma membrane and takes part in signal transduction. VWA seems to act as a humoral effector in mollusks, primarily in the extracellular matrix.

Fig 5 .

 5 Fig 5.Immune-related role of the altered proteins -downregulated or upregulated -in a ciliated hepatopancreatic cell of Perna perna mussel, 24 h post-Salmonella enterica injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). First of all, the leucine-rich repeat (LRR) and guanylate kinase (GK) domain-containing protein (LRGUK) acts on pathogens recognition and signal transduction, through the LRR and GK domains, respectively. As in the mussels injected with E. coli, the FERM domain-containing protein (FERM) probably also takes part in signal transduction close to the plasma membrane. Afterward, the stressactivated protein kinase JNK (JNK) participates at the end of the mitogen-activated protein

Fig 6 .

 6 Fig 6.Immune-related role of the altered proteins -downregulated or upregulated -in a ciliated hepatopancreatic cell of Perna perna mussel, 24 h post-Vibrio parahaemolyticus injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). As in the mussels injected with S. enterica, the leucine-rich repeat (LRR) and guanylate kinase (GK) domaincontaining protein (LRGUK) presents an LRR domain, which typifies a toll-like receptor (TLR) for pathogens recognition, and the GK domain, that seems to perform signal transduction. Thereafter, a rhomboid protease (RHBDL1_2_3) seems to participate in signal transduction, triggering some pathway likely related to transcriptional response against pathogens. Then, the transcription factor hepatocyte nuclear factor-4α (NR2A1) regulates the transcription of genes

  production and metabolism; IR -Immune response

Fig 7

 7 Fig 7. a) Number of proteins downregulated (n= 327) and upregulated (n= 16) in Perna perna mussel among the 343 proteins significantly different between Vibrio parahaemolyticus (VP) and all the others conditions (NC -non-injected control; IC -injected control; EC -Escherichia coli; and SE -Salmonella enterica). b) Frequency (%) of functional categories of the proteins altered after VP challenge, which are significantly different from all other conditions, based on the Database of Clusters of Orthologous Genes -COGs. T -Signal transduction mechanisms; O -Posttranslational modification, protein turnover, chaperones; S -Function unknown; U -Intracellular trafficking, secretion, and vesicular transport; A -RNA processing and modification; Z -Cytoskeleton; K -Transcription; J -Translation, ribosomal structure, and biogenesis. As the 16 upregulated proteins did not fit in any COG functional category, significantly different proteins in specific response to VP in all functional categories shown in the graph were downregulated.

Fig 8 .

 8 Fig 8.Comparison of the percentage (%) of proteins between all groups (total proteins found in Perna perna mussels) and proteins altered in specific response to Vibrio parahaemolyticus (VP; significantly different from all the other groups), according to the eight most frequent COG functional categories.

Fig 1 .

 1 Fig 1. Vermelha Beach (VB), Rio de Janeiro, Brazil: mussels and seawater sampling location.

Fig 2 .

 2 Fig 2. The external and internal anatomical features of the mussel Perna perna.

Fig 3 .

 3 Fig 3. Upset plot for visualizing intersecting sets of significantly different proteins among the tested conditions. NC -non-injected control; IC -injected control; EC -Escherichia coli; SE -Salmonella enterica; and VP -Vibrio parahaemolyticus. SE_EC means a significant difference between these conditions and so on.

Fig

  Fig 4.Immune-related role of the altered proteins in a ciliated hepatopancreatic cell of Perna perna mussel, 24h post-Escherichia coli injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). In mussels challenged with E. coli, a von Willebrand Factor A domain-containing protein (vWA) and a FERM domain-containing protein (FERM) were downregulated. FERM is probably located close to the plasma membrane and takes part in signal transduction. VWA seems to act as a humoral effector in mollusks, primarily in the extracellular matrix.

Fig 5 .

 5 Fig 5.Immune-related role of the altered proteins -downregulated or upregulated -in a ciliated hepatopancreatic cell of Perna perna mussel, 24 h post-Salmonella enterica injection. Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). First of all, the leucine-rich repeat (LRR) and guanylate kinase (GK) domain-containing protein (LRGUK) acts on pathogens recognition and signal transduction, through the LRR and GK domains, respectively. As in the mussels injected with E. coli, the FERM domain-containing protein (FERM) probably also takes part in signal transduction close to the plasma membrane. Afterward, the stressactivated protein kinase JNK (JNK) participates at the end of the mitogen-activated protein kinase (MAPK) cascade, phosphorylating transcription factors that regulate the transcription of response genes. The proteasome activator PA28γ (PA28γ) located in the nucleus is also presumably involved in transcription or apoptosis. After transcription, the splicing factor 3A subunit 1 (SF3A1) takes part in mRNA processing, which is translated at the cytoplasm by ribosomes constituted by the 60S ribosomal protein L18a (RPL18A). Mitochondrial fission process protein 1 (MTFP1) mediates mitochondrial fission, which rises under cellular stress and during apoptosis. Last but not least, the proteins actin beta/gamma 1 (ACTB_G1), Tubulin beta chain (β-Tubulin), axonemal dyneins (DNAH3 and DNAH9), EFHC1, Cilia-and flagellaassociated protein 299 (CF-299) constitute cytoskeleton and cilia structure, which are probably related to the protein secretion function of the ciliated basophilic cells of hepatopancreas.

Fig 6 .

 6 Fig 6. Immune-related role of the altered proteins -downregulated or upregulated -in a ciliated hepatopancreatic cell of Perna perna mussel, 24 h post-Vibrio parahaemolyticus injection.Herein, the primary steps for initiating an immune reaction against pathogens, according to the cell compartment, are displayed as: recognition and signaling (cell periphery); signal transduction (cytoplasm); and transcription and mRNA processing (nucleus). As in the mussels injected with S. enterica, the leucine-rich repeat (LRR) and guanylate kinase (GK) domaincontaining protein (LRGUK) presents an LRR domain, which typifies a toll-like receptor (TLR) for pathogens recognition, and the GK domain, that seems to perform signal transduction. Thereafter, a rhomboid protease (RHBDL1_2_3) seems to participate in signal transduction, triggering some pathway likely related to transcriptional response against pathogens. Then, the transcription factor hepatocyte nuclear factor-4α (NR2A1) regulates the transcription of genes related to hepatic functions and lipid metabolism. THOC1 also acts on transcription and mediates mRNA export to the cytoplasm. Nevertheless, before mRNA export, the proteins splicing factor 3A subunit 1 (SF3A1) and poly-U-binding factor 60 kDa (PUF60) take part in mRNA processing. In concert, the proteins RNA 3-phosphate cyclase-like 1 (RCL1) and nuclear GTP-binding protein (NUG1) are required for ribosome biogenesis. Then, mRNA can be translated at the cytoplasm by ribosomes constituted by the 60S ribosomal protein L18a (RPL18A). The mitochondrial-processing peptidase subunit beta (PMPCB) takes part in protein supply and targeting from the cytosol to mitochondria, indicating increased activity of this organelle. ABC transmembrane type-1 domain-containing protein (ABC) is specialized in importing specific nutrients, presumably important for fronting bacteria. Histone H2A, besides its known nuclear function, may be found out of the nucleus acting as a humoral effector via multiple mechanisms of action, because of its antimicrobial activity. Finally, the proteins β-Tubulin, EFHC1, and ARMC3 constitute cilia structure, which is probably related to the protein secretion function of the ciliated basophilic cells of the hepatopancreas.

Fig 7

 7 Fig 7. a) Number of proteins downregulated (n= 327) and upregulated (n= 16) in Perna perna mussel among the 343 proteins significantly different between Vibrio parahaemolyticus (VP) and all the others conditions (NC -non-injected control; IC -injected control; EC -Escherichia coli; and SE -Salmonella enterica). b) Frequency (%) of functional categories of the proteins altered after VP challenge, which are significantly different from all other conditions, based on the Database of Clusters of Orthologous Genes -COGs. T -Signal transduction mechanisms; O -Posttranslational modification, protein turnover, chaperones; S -Function unknown; U -Intracellular trafficking, secretion, and vesicular transport; A -RNA processing and modification; Z -Cytoskeleton; K -Transcription; J -Translation, ribosomal structure, and biogenesis. As the 16 upregulated proteins did not fit in any COG functional category, significantly different proteins in specific response to VP in all functional categories shown in the graph were downregulated.

Fig 8 .

 8 Fig 8. Comparison of the percentage (%) of proteins between all groups (total proteins found in Perna perna mussels) and proteins altered in specific response to Vibrio parahaemolyticus (VP; significantly different from all the other groups), according to the eight most frequent COG functional categories.

Acknowledgements

The authors are grateful to Dr. Rosana Barreto Rocha Ferreira (UFRJ), Dr. Júlia Peixoto de Albuquerque (UFF) and Dr. Renata Garcia Costa (Fiocruz), for providing the bacteria strains used in this work. The authors thank Mr. Ivanildo Chagas dos Santos and Débora Silva dos Santos, the first author's father and sister respectively, and Professor Luciano N. Santos for assisting with mussels sampling. The authors also thank Dr. Leandro Machado Rocha, MSc. Jorge Andrés Duarte Duarte and the technician Camila Soares Silvestre Toledo from the faculty of pharmacy (UFF) for helping with sample lyophilization. This work was supported by National Council for Scientific and Technological Development -CNPq [Fernanda Silva dos Santos -Sandwich doctoral fellowship 203434/2020-0] and by ISblue project, Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program "Investissements d'Avenir" embedded in France 2030. Additionally, this study was partially financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001 -[Fernanda Silva dos Santos -doctoral fellowship 88882.457000/2019.1], and financially supported by the Universidade Federal do Estado do Rio de Janeiro (UNIRIO) -[INOVA UNIRIO IN-01/2019 attributed to Natascha Krepsky].This study is also part of the project "PELD/ Guanabara: Pesquisas Ecológicas de Longa Duração" financially supported by Brazilian National Council for Scientific and Technological Development (CNPq; 558083/2009-9 and 441373/2016-0) and Foundation Carlos Chagas Filho Research Support of the State of Rio de Janeiro (FAPERJ; 136375 and E-26/110.144/2013). Raquel A. F. Neves is grateful to the research grant attributed by FAPERJ (E-26/201.283.2021).