Camille Houdelet
email: camille.houdelet@univ-grenoble-alpes.fr

Michel Bocquet

Philippe Bulet
email: philippe.bulet@univ-grenoble-alpes.fr

MALDI Biotyping, an approach for deciphering and assessing the identity of the honeybee pathogen Nosema

Keywords: MALDI Biotyping, Peptide profiling, Microsporidia, Flow cytometry, Nosema, Apis mellifera Apis mellifera, Peptide profiling, MALDI Biotyping, Microsporidia, 39 Nosema, Flow cytometry

published or not. The documents may come

MALDI Biotyping, an approach for deciphering and assessing the identity of the honeybee pathogen Nosema

The microsporidia are obligate intracellular pathogenic fungi that parasitize a wide range of invertebrate and vertebrate hosts and have important impacts on health, food security and the economy. In this paper, we focus on Nosema ceranae and N. apis, which chronically infect the digestive tract of honeybees, altering their physiology and lifespan. We apply MALDI mass spectrometry for rapid molecular profiling of extracts of Nosema spores in order to identify the species and optionally the geographical origin and assess the viability status of Nosema microsporidia in conjunction with a flow cytometric approach. Pure solutions of spores were prepared for flow cytometry and MALDI profiling. A mechanical extraction of Nosema spores was conducted to obtain mass fingerprints of peptide/protein for samples of microsporidia from different geographical origins (MBO.NC01, MBO.NC02 and MBO.NA01), viable or heat killed. The present work demonstrates that MALDI-MS is a rapid, consumable cost-effective and simple method for identifying Nosema species. Moreover, a distinction in the peptide/protein profiles between two isolates with different geographical origins was observed. Mass fingerprints of viable and experimentally killed spores were also clearly distinguishable, regardless of Nosema species. Finally, using our computational models on the different Nosema species, we were able to classify five independent isolates of Nosema microsporidia. We demonstrated that MALDI Biotyping may represent a valuable tool for sanitary services and beekeepers to survey nosemosis in apiaries.

89

Regarding the pathogenic effect of nosemosis, their infectious capability or virulence 90 (increased mortality rates in infected individuals) depends of the number of spores 91 ingested by honeybees [START_REF] Ebert | The evolution and expression of parasite virulence[END_REF] and on temperature [START_REF] Higes | The differential 611 development of microsporidia infecting worker honey bee (Apis mellifera) at increasing 612 incubation temperature: Nosema apis infection is inhibited at high temperature[END_REF] . N. ceranae spores better resist high 92 temperatures (Mediterranean climate) than cold temperatures and complete their life 93 cycle faster at higher temperatures. With regards to the physiology of the bee, the 94 susceptibility or tolerance of bees to Nosema is age dependant: foragers are the most 95 intensively infected but have a better ability to survive if challenged than younger bees 96 [START_REF] Higes | How natural infection by Nosema ceranae 615 causes honeybee colony collapse: Natural Nosema ceranae infection[END_REF] . Nutritional status (i.e. pollen quality) and possibly genetic strain are also involved 97 [START_REF] Castelli | Impact of Nutritional Stress on Honeybee Gut 618 Microbiota, Immunity, and Nosema ceranae Infection[END_REF][START_REF] Tritschler | Protein 621 nutrition governs within-host race of honey bee pathogens[END_REF] .

98

The absence of official treatment against nosemosis in Europe 41 spurred 99 investigations into better understanding the detailed mechanisms of infection and their 100 variations, a key issue for better management of the disease, that requires development 101 of the most appropriate experimental design. To date, microsporidian spores have been 102 identified using morphological, immunological and molecular biological methods. In Two MALDI spectrometers are available on the market, MALDI Biotyper ® and 106 VITEK ® 42,43 . These MALDI-MS platforms perform by targeting the ribosomal proteins 107 as biomarkers for the identification of clinical bacterial, fungal and yeast isolates 108 (method referred to MALDI Biotyping [START_REF] Clark | Matrix-Assisted Laser Desorption Ionization-Time 634 of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical 635 Microbiology[END_REF][START_REF] Pranada | MALDI Biotyping for Microorganism Identification 637 in Clinical Microbiology[END_REF] . Proteins can be analyzed by mass The following method for purification of spores' solutions was inspired from the 145 recommendations of Fries and colleagues [START_REF] Fries | Standard methods for Nosema research[END_REF] . Briefly, abdomens of infected bees were 146 collected in 50 mL conical tubes (Falcon, Dominique Dutscher, France) containing 147 ultrapure water and strongly mixed manually using a spatula. The solution was filtered 148 with a 100 µm cell strainer (pluriSelect, Germany) and then centrifuged at 3,000 x g for 149 20 min at room temperature. After removing the supernatant, the residual pellet was 150 resuspended by adding 5 mL of ultrapure water. The spore suspension was filtered with 151 a 20 µm cell strainer (pluriSelect, Germany). The filtrate was centrifuged at 3,000 x g 152 for 1 hour and the residual pellet resuspended in 1 mL of Milli-Q water prior to 153 purification on a Percoll ® -gradient. The second step of purification was performed on a 154 gradient of Percoll ® from the bottom to the top: 100%, 75%, 50% and 25% of Percoll ® 155 prepared in 0.25 mol/L of sucrose (Fluka, Germany). The spore solution (300 µL) was 156 centrifuged twice at 8,000 x g for 20 min at 6°C. The spore pellets were collected prior 157 to a second step of centrifuging at 16,000 x g for 30 min at 6°C. The supernatant was 158 removed and the spore pellet was resuspended in 1 mL of Milli-Q water and centrifuged 159 at 16,000 x g at 6°C. This step was repeated five times. The spore pellet obtained was 160 resuspended in Milli-Q water and spore concentration was estimated using a Malassez 161 cell counting chamber (Assistent ® ). The purity of the spore solution was estimated by 162 dividing the total number of spores by the number of spores and debris. Counting was 163 performed in triplicate. All spore solutions were kept at room temperature until use. For these analyses, all solutions were prepared at 2.5 x 10 6 spores/mL in triplicate. To extract peptides and proteins from spores, three protocols were investigated using 175 the fresh spore solution MBO.NC02: (P1) ultrasonic, (P2) stainless steel balls and (P3) 176 zirconium oxide beads (Figure 1). For P1, the solution of spores was sonicated four 177 times for 45 sec (15 sec ON and 30 sec OFF) with an amplitude of 50% (130 Watt, 178 20kHz, VibraCell™, Sonics ® ). The extract was centrifuged at 15,800 x g for 4 min at 179 6°C before removing the supernatant and the pellet was vacuum-dried (Freezone Plus 180 2.5-liter freeze-dry-system; Labconco, Kansas City, MO, USA; Figure 1). For the two 181 other extraction protocols (P2 and P3), solutions were centrifuged at 15,800 x g for 4 182 min at 6°C before removing the supernatant and the pellets were vacuum-dried. The

253

To compare spore viability, data normality and homoscedasticity were evaluated.

254

When these two conditions were not respected, a non-parametric Mann-Whitney test 255 was applied; the confidence index was fixed at 0.95. [START_REF] Martín-Hernández | Microsporidia infection impacts the 549 host cell's cycle and reduces host cell apoptosis[END_REF] .

292

In literature, three protocols were referenced for spore's suspension purification: 293 centrifugation, triangulation and density gradient [START_REF] Chen | Morphological, molecular, and phylogenetic 652 characterization of Nosema ceranae, a microsporidian parasite isolated from the 653 European honey bee, Apis mellifera[END_REF][START_REF] Cole | Application of triangulation method to purification of Nosema spores from insect 656 tissues[END_REF] . With the two last protocols 294 authors obtained percent of purification up to 92 %. In this study, we propose an 295 optimized method derived from the Fries and colleagues (2013) [START_REF] Fries | Standard methods for Nosema research[END_REF] to fulfil the need of 296 highly purified spore solutions for molecular analyses by combining the methods of

Differential analysis by MALDI profiling of various spore extracts 306

The first part of our study was to establish the optimal parameters (in terms of We settled on extracts generated at a starting concentration of 2.5 x 10 6 fresh 311 spores/mL. Three protocols of peptide/protein extracts from spores were tested on the 312 MBO.NC02 isolate for its spore abundance: (P1) ultrasonic, (P2) stainless steel balls 313 and (P3) zirconium oxide beads (P1, P2 and P3, see Fig. 1) and the extracts were 314 analysed by MALDI-MS in triplicate (Figure 2). All measurements were done at the 315 same initial spore concentration and using the same MALDI-MS parameters and sample 316 dilution rate. Following data processing (5 successive steps, see details in Materials & 317 methods section), P3 resulted in the most complex mass spectra (Fig. 2C) compared to 318 P1 (Fig. 2A) and P2 (Fig. 2B) with 21, 17 and 5 significant detected peaks, respectively 319 (Table 1).

320

The identification by MALDI profiling is a well-recognized method in clinical 321 diagnostic microbiology but remains difficult for fungus, as the identification depends 322 on the phenotype of the fungus [START_REF] Croxatto | Applications of MALDI-TOF mass spectrometry in 658 clinical diagnostic microbiology[END_REF] . Different studies have used the MALDI-MS Table 1). This protocol was reproducible between replicates and an optical observation 342 after treatment did not show an alteration of spore structure. The mature stage of 343 Nosema presents a complex impermeable wall with a three layered-exospore, an 344 endospore and an internal plasma membrane [START_REF] Chen | Morphological, molecular, and phylogenetic 652 characterization of Nosema ceranae, a microsporidian parasite isolated from the 653 European honey bee, Apis mellifera[END_REF] , which explains difficulties encountered 345 in extracting proteins from the spores. 3A) and were kept for analysis.

355

Regarding the Nosema species determination from live spores, according to Dim1, N. 356 apis (upper right, Fig. 3B) was clearly discriminated from N. ceranae (upper left, Fig. 357 3B). In addition, it seems that PCA data allowed to distinguish the two isolates of N. 358 ceranae collected from the non-European country and France (MBO.NC01 and 359 MBO.NC02, see Fig. 3B). Finally, PCA revealed a good separation between live (above 360 in Fig. 3B) and dead spores (below in Fig. 3B) and a tendency to discriminate the dead 361 spores of N. ceranae from the ones of N. apis (Fig. 3B, found on the rightmost side of 362 the clustered data points).

363

In order to explain the clustering, we analysed molecular ions and their contribution 364 to Dim1 and Dim2 (Figure 3C). Fifty-nine variables, corresponding to the 59 ion peaks 365 found in samples, were considered (see peak list and colour code in Table 2). Among 366 the 59 molecular ions, 11 were not discriminating (m1-2, m8, m12, m21, m26-27, m29-367 31 and m38, Fig. 3C, black labels in Table 2). Of the 48 discriminating peaks, 21 molecular ions (m3-4, m7, m10-11, m14, m16, m20, m23, m25, m32, m34, m36-37, 369 m42, m45-47, m49 and m58-59) were highly correlated to Dim1 and had a high 370 contribution to the discrimination of N. apis vs N. ceranae (green labels in Table 2).

371

Fifteen molecular ions (m5-6, m9, m13, m15, m17, m22, m24, m43 and m50-55, red 372 labels in Table 2) were negatively correlated to Dim1 and had a high contribution to the 373 discrimination of N. ceranae vs N. apis. Considering Dim2, the following 12 molecular 374 ions, m18-19, m28, m33, m35, m39-41, m44, m48 and m56-57, were negatively 375 correlated to Dim2 and had a limited contribution in the discrimination of fresh vs dead 376 spores regardless of Nosema species (Fig. 3C and blue labels in Table 2).

377

We visualised the average spectra (n=9) from technical replicates to first, highlight 378 modifications of the peptide/protein composition according to the Nosema species 379 (MBO.NC01 and .NC02 vs MBO.NA01, Figure 4A) and second, visualize a putative 380 difference between geographically distant Nosema isolates (MBO.NC01 vs 381 MBO.NC02, Fig. 4B). Between N. apis and N. ceranae isolates, the MALDI-MS 382 spectra showed a series of overall differences in MFPs regardless of the N. ceranae 383 isolate (see Fig. 4A1 and2). Several ions within the 2,800-5,500 mass ranges displayed 384 intensity variation according to the isolates considered (for enlarged mass area, see Fig. 385 4A3). Four major zones qualitatively discriminated the Nosema species, two in the mass 386 range between 2,800 -3,300 m/z and 4,200 -4,600, more complex for N. apis than N. 2). These molecular ions that are not 391 characterized in this study will be subjected to dedicated mass spectrometry approaches 392 in further studies. These results suggest that MALDI-MS represents a rapid and simple 393 method for assessing the identity of Nosema spore species.

394

As a next step, we analyzed two isolates of N. ceranae collected from two different 395 geographical origins, MBO.NC01 from a non-European apiary and NBO.NC02 from a 396 French apiary. Between the two N. ceranae isolates, the overall MALDI-MS spectra 397 showed minor differences (see Fig. 4B). Several ions within the 1,300 -3,600 mass 398 ranges displayed intensity variation according to the origin of the isolate (enlarged mass 399 area, Fig. 4B). Within this mass range, two qualitatively distinct areas discriminated the 400 origin of the Nosema isolates, one in the mass range between 1,300 -2,700 m/z with 401 five molecular ions (m/z 1,342, 1,455, 2,028, 2,473, and 2,646) only detectable in the 402 fresh extracts of the isolate MBO.NC02 (see Fig. 4B1). In contrast, the molecular ions 403 at m/z 1,834 and 3,519 were specific to the non-European isolate MBO.NC01. These 404 few differences explained the two distinct clusters within the PCA analyses (m1, m5, 405 m13, m21 and m27 for French isolate, and m8, m38 for non-European isolate; Fig. 4B1, 406 Fig. 6 and Table 2). These results suggest that, in addition to an approach for the 407 identification of Nosema species, MALDI-MS could be a promising method to classify 6B2). One molecular ion at 442 m/z 3062 was shared by the two geographically distinct extracts (see Fig. 6B3). Spectra 443 of dead spores were considerably less complex (m28, m39, m44 and m48 absent in dead 444 extracts) than fresh spore extracts (Fig. 3 and Fig. 6). With MALDI-MS we could 445 distinguish fresh and dead spores.

446

Measuring spore viability is important to evaluate the potential infectiousness of the 447 pathogen. The link between viability and infectiousness is still unclear because three 448 levels of information have to be considered: viability, extrusion rate of the polar tube 449 and germination rate [START_REF] Amigó | Longevity and effects of 679 temperature on the viability and polar-tube extrusion of spores of Glugea stephani, a 680 microsporidian parasite of commercial flatfish[END_REF][START_REF] Undeen | The Effects of Temperature on the Survival of 683 Edhazardia aedis (Microspora: Amblyosporidae), a Pathogen of Aedes aegypti[END_REF] . Extrusion refers to spores that extrude their polar tubes but 450 does not consider whether the sporoplasm of the spores will infect the host cell contrary 451 to the germination [START_REF] Amigó | Longevity and effects of 679 temperature on the viability and polar-tube extrusion of spores of Glugea stephani, a 680 microsporidian parasite of commercial flatfish[END_REF] . Even if viability cannot be equated to infectiousness, viability profiling may represent an essential step to better study the model and the mechanistic 453 of the infectious process. Two major studies worked on the longevity and viability of 454 spores in various conditions of conservation [START_REF] Peng | Quantifying spore viability of the honey 649 bee pathogen Nosema apis using flow cytometry[END_REF][START_REF] Collado | Flow cytometry analysis of Nosema 676 species to assess spore viability and longevity[END_REF] . Nowadays, to evaluate the ability for 455 spores to infect the host, scientists can infect bees with spore solutions and count the 456 number of spores after a few days of infection and measure the mortality [START_REF] Forsgren | Comparative virulence of Nosema ceranae and Nosema apis in 686 individual European honey bees[END_REF][START_REF] Huang | Infectivity and virulence of Nosema ceranae 689 and Nosema apis in commercially available North American honey bees[END_REF] . Even if 457 viability profiling does not reflect infectiousness [START_REF] Amigó | Longevity and effects of 679 temperature on the viability and polar-tube extrusion of spores of Glugea stephani, a 680 microsporidian parasite of commercial flatfish[END_REF][START_REF] Undeen | The Effects of Temperature on the Survival of 683 Edhazardia aedis (Microspora: Amblyosporidae), a Pathogen of Aedes aegypti[END_REF] , proteomic analyses could help to 458 identify factors of infectiousness. For example, the polar tube proteins are well known 459 for their involvement in invasion of the host cell [START_REF] Han | The role of microsporidian polar tube protein 4 (PTP4) in 692 host cell infection[END_REF][START_REF] Xu | The microsporidian polar tube: A highly specialised invasion organelle[END_REF] . However, masses of these 460 proteins are too high (up to 20 kDa) to be detected in our MALDI conditions.

461

Finally, between each extract (N. apis and the two N. ceranae, fresh and dead) analysed 462 with MALDI-MS, we detected one common molecular ion at mass 2,588 m/z (Fig. 4 and 463 Fig. 6), corresponding to the label peak m26 (see Table 2). We could not characterize 464 this ion, but it is a potential biomarker of the genus Nosema. and N. apis model we had generated. All isolates were identified as N. ceranae spores 470 with a high confidence (score up to 2.00, Figure 7).

44

 44 fighting Nosema infections in Apis mellifera.

103 2015 ,

 2015 FDA clearance was announced for more than 200 species of micro-organisms 104 using Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

  105

  109 spectrometry (MS); ionization of the proteins leads to measurement of the mass-to-110 charge (m/z) ratio, with the great benefit that the analysis is unbiased and a priori 111 knowledge of the proteins is thus not necessary. Molecular mass fingerprints (MFP) of 112 biological matrices by MALDI Biotyping is indeed a thriving approach, enabling the 113 rapid detection of peptide/protein components that can provide comparative 114 information. 115 In this work, we explore the potential of MALDI Biotyping for the identification of 116 Nosema species and its potential to evaluate Nosema spore viability as its represents a 117 challenge for monitoring the impact of Nosema on bee physiology. MALDI profiling 118 and also flow cytometry allow to rapidly discriminate viable and non-viable spores, 119 thanks to highly purified spore preparations. Finally, adapting MALDI Biotyping, a 120 technology used for the identification of bacterial strains, to the identification of 121 Nosema species, allowed us to obtain representative MFPs of the Nosema species (N. 122 apis versus N. ceranae) and also of the geographical origin. This demonstrated that 123 MALDI profiling represents an attractive and more robust alternative to optical 124 observation and is more cost effective, compared to PCR analyses, for identifying 125 Nosema species.

128

  For sample preparation and analyses, acetonitrile (ACN) and trifluoroacetic acid 129 (TFA), both of LC-MS grade quality or higher, were obtained from Carlo Erba 130 Reagents (Val de Reuil, France). Protein Calibration Standard I (ProtMix) was 131 purchased from Bruker Daltonics (Germany). For all experiments, ultrapure water 132 (MilliQ water) was used (Billerica, MA, USA). For a calibration solution APISCAL, 133 the A. mellifera peptides (apidaecin, abaecin and melittin) were synthesized by Smart 134 Bioscience and the peptide ETD was a donation from Dr. Philippe Bulet. The Percoll ® 135 and ammonium phosphate monobasic (NH 4 H 2 PO 4 ) were purchased from Sigma-Aldrich 136 (Missouri, USA). The two dyes, SYTO 16 and propidium iodide (PI) were obtained 137 from Invitrogen Corporation (Carlsbad, CA).

138 2 . 2

 22 Spore preparation 139 The isolates of N. ceranae spores (MBO.NC01 from a non-European apiary and 140 MBO.NC02 from a French apiary) and N. apis (MBO.NA01) spores were generous 141 gifts from the collections of Michel Bocquet and Robert Paxton, respectively. For each 142 isolate, live spores were obtained after multiplication in honeybees and dead spores 143 after heat shock (20 min at 121°C).

  144

164 2 . 3 For

 23 Identification of Nosema spores by polymerase chain reaction (PCR) PCR analysis, 200 µL of each purified N. ceranae spores' solution (total 2 x 10 5 166 spores) were analysed in triplicate by the public agency ANSES (Agence Nationale de 167 Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail) according to 168 their internal method developed by Martín-Hernández and colleagues 47 . The identity of 169 the N. apis isolate was confirmed by PCR as described by Chen and colleagues in 2008

173 2 . 4 . 1

 241 Peptide/Protein extraction 174

183

  extraction step was executed using either stainless steel (SSB16, Next Advance, USA) 184 or ceria stabilized zirconium oxide (ZrOB05, Next Advance, USA) beads for P2 and P3, 185 respectively. The purified spores were disrupted using a Mill-Mix 20 system (Domel, 186 Železniki, Slovenia). The spores were ground in 1.5 mL tubes (Eppendorf ® , Germany) 187 filled either with 1.6 mm stainless steel beads (no more than 3) or 0.5 mm zirconium 188 oxide beads (less than 10) for 30 sec at 30 Hz. The dried extracts were either used 189 immediately for MALDI analysis and then kept at -20°C.

190 2 . 4 . 2

 242 Sample preparation 191 Prior to MALDI-MS analyses, dried pellets were suspended in 25 µL of 0.1% TFA 192 and centrifuged at 15,800 x g for 2 min at 6°C. The cleared supernatant was transferred 193 to a clean low-binding microcentrifuge tube (Eppendorf ® , Germany). For MALDI 194 analysis, an equivalent of 100,000 spores or one µL of spore extract was spotted onto an 195 MTP 384 polished steel MALDI target (Bruker Daltonics) in triplicate and vacuum-196 dried. A fresh solution of matrix (10 mg/mL α-cyano-4-hydroxycinnamic acid in 70% 197 ACN, 2.5% TFA and 15mM NH 4 H 2 PO 4 ) was spotted onto the dried samples. The 198 instrument was calibrated using a mixture of two sets of peptides and proteins 199 (APISCAL, ProtMix) covering the dynamic range of interest. APISCAL is an in-house 200 calibration solution composed of apidaecin and abaecin, two antimicrobial peptides 201 from Apis mellifera (average m/z of 2,109 and 3,879, respectively), melittin, the major 202 venom component, (average m/z of 2,847); and ETD, a recombinant peptide, (average 203 m/z of 4,839). ProtMix (Protein Calibration Standard I, Bruker Daltonics) is a 204 manufacturer-available mixture of four peptides and proteins (Insulin, Ubiquitin, 205 Cytochrome C and Myoglobin at an average m/z of 5,734, 8,565, 12,360 and 16,952

  A sample of APISCAL and ProtMix were also heat treated 20 min at 207 121°C to serve as control for the heat killed spores analysed by MALDI Biotyping. The 208 sample spots were lightly vacuum-dried prior to analysis.

209 2 . 4 . 3

 243 Sample analysis 210 Mass spectra were acquired on a MALDI AutoFlex III Smartbeam instrument 211 (Bruker Daltonics) using the FlexControl 3.4 software (Bruker Daltonics) in positive 212 linear mode. The instrument was set up with the following parameters: 200 Hz laser at a 213 40% global attenuation offset, 20 kV source voltage 1, 18.7 kV source voltage 2, 9.25 214 kV lens voltage, 1.906 kV linear detector voltage, 120 ns of pulsed ion extraction delay 215 and 600 Da detector gating. MALDI-MS spectra were recorded at the mass range of 600 216 Da to 18 kDa by summing 2,000 laser shots. Data were previewed using the 217 FlexAnalysis 3.4 software. For each individual spectrum baseline correction was 218 performed with a TopHat algorithm, along with a smoothing according to the Savitzky-219 Golay algorithm (window size 5.0 m/z in one cycle). The peaks were automatically 220 picked using a signal-to-noise (S/N) ratio of 3.0, a picking height of 80 and an 221 application of baseline. Peak lists of each spectrum were extracted for data processing 222 and statistical analyses.

223 2 . 5

 25 Analyses of Nosema spores by flow cytometry 224For flow cytometry analysis, solutions of purified spores (10 6 spores) were used in 225 five and two replicates for N. ceranae and N. apis spores, respectively. Spore solutions 226 were dyed with 6.25 µM of SYTO 16 (Invitrogen Corporation, Carlsbad, CA) and 12 227 µM of propidium iodide (PI, Invitrogen Corporation, Carlsbad, CA), based on Peng and 228 colleagues' study 49 . Spore solutions (1 mL) were incubated on ice, in the dark for 30 229 min. After centrifugation at 20,800 x g for 5 min at 21°C, 680 µL of supernatant were 230 removed and the residual pellets were suspended in the remaining 320 µL. Flow 231 cytometry analysis was conducted on a GUAVA ® EasyCyte™ Plus Flow Cytometry 232 System (Millipore) with the Cytosoft software. SYTO 16 and PI were excited using a 233 488 nm laser. Spore solutions were analysed at a high flow rate (0.59 µL/s). A threshold 234 was established to exclude debris from spore events using Forward (FSC) and Side 235 Scatter (SSC); counting was based on 10,000 events. A logarithmic amplification 236 including FSC and SSC was applied. To establish settings, control solutions containing 237 50% dead spores were used. 238 Live spores were discriminated from dead spores using pulse height for PI and SYTO14 239 fluorescence, respectively. Compensations were made for N. ceranae (Green-%Yellow 240 54.9 and Yellow-%Green 27.4) and N. apis (Green-%Yellow 16.9 and Yellow-%Green 241 39.2). Parameters of acquisition were adjusted according to the species. Spore viability 242 was assessed by two technical replicates.

243 2 . 6

 26 Data processing and statistical analyses 244Data processing and statistical analyses were performed using R studio with R 3.4.2 245 software. The data processing of spectra was conducted in five steps. Peak lists of each sample were extracted (step 1) and masses lower than 1,000 Da were 247 suppressed (step 2). Peaks with an S/N of less than five were rejected (step 3). Peak lists 248 of each replicate from the same sample were compared and only common peaks were 249 conserved (step 4). Finally, potential double charged ions and modifications like 250 oxidation or dehydration were suppressed (step 5). Principal component analysis (PCA) 251 was performed on series of data including four variables for each individual spectrum: 252 molecular ions, resolution, peak area and intensity.

256 2 . 7 .

 27 Generation of MALDI mass spectra profiles257 A database of molecular profiles of the three isolates of N. spores (MBO.NC01, 258 MBO.NC02 and MBO.NA01) was generated using the MALDI Biotyper® procedure 259 (Bruker). For one average spectrum, each isolate was spotted eight times and each spot 260 was acquired three times. The quality of the 24 spectra generated was evaluated 261 following the Biotyper protocol and the main spectral profile was created using the 262 MBT Compass Explorer v4.1.70. Five isolates of Nosema spores (s1 to s5), were 263 collected from infected bees and analysed by MALDI Biotyping following the protocol 264 described above and were compared to referenced mass spectra from our local database.265For identification, a score was obtained depending of the confidence of the result. Three 266 ranges of identification exist: 0.00 -1.69, 1.70 -1.99 and 2.00 -3.00, corresponding to 267 no organism identification, low confidence or high confidence, respectively.

268 3 .

 3 Results and discussion269Nosema disease is quite complicated to diagnose without using laboratory equipment, as 270 the clinical observation of the intestinal tract retrieved after pulling the last abdominal 271 segment is not enough. A positive diagnosis is accomplished by visualizing the spores 272 under a microscope. However, the presence of spores is not enough, other elements are 273 necessary to affirm that we are in the presence of a Nosema disease. There are at least 274 three elements to consider regarding spores of Nosema, first detection and counting, 275 second identification of the species involved and third viability status, a factor often 276 associated with virulence. In this study, we developed MALDI Biotyping method to 277 serve these three elements and complemented this approach with flow cytometry 278 analysis. The approach of MALDI Biotyping was applied to identify (i) the Nosema 279 species, (ii) their geographical origins and (iii) their viability status. To validate our 280 experimental procedure, we analysed two species of Nosema (ceranae and apis) and 281 two isolates of N. ceranae from different geographical origins (one from France and one 282 from a non-European country). 283 2843.1 High degree of purification 285Nosema spores were obtained from different bee matrices (faecal residues, dead bees or ), but whatever the method for Nosema spp. identification, the spore 287 solution needs to be clarified and of the highest purity. For microscopic observation, a 288 simple centrifugation is sufficient to visualise spores while for molecular identification, 289 filtrated and centrifuged samples are requested for DNA extraction and PCR analysis 290 (ANSES recommendation based on protocols developed by Martín-Hernández and 291 colleagues)

  297 filtration and Percoll ® -gradient. The level of purity was estimated by counting the 298 number of spores using a Malassez cell counting chamber as recommended by ANSES, 299 following the recommendation of the World Organisation of Animal Health 300 (ANSES/SOP/ANA-I1.MOA.09 -Version 6). The purity of MBO.NC02 was 96.7% (sd 301 ± 0.2), the purity of MBO.NC01 was 99.2% (sd ± 0.3) and the purity of MBO.NA01 302 was 99.8 % (sd ± 0.04). Identities of the different spore solutions were confirmed by 303 PCR prior to our analyses. MBO.NC01 and MBO.NC02 were identified as Nosema 304 ceranae and MBO.NA01 as N. apis.

  305

  307 sensitivity, resolution, reproducibility and accuracy) of molecular ions between 1,000 308 and 18,000 m/z for recording the most representative MALDI peptide 309 signatures/molecular mass fingerprints (MFPs) of the different Nosema spore extracts.

  310

323

  approaches to study microsporidian spores but only two were based on the MALDI 324 Biotyping workflow[START_REF] Kajiwara | Rapid detection of silkworm microsporidia by matrix-assisted 661 laser desorption/ionization time-of-flight mass spectrometry[END_REF][START_REF] Moura | Analysis of Four Human 664 Microsporidian Isolates by MALDI-TOF Mass Spectrometry[END_REF] . Moura and colleagues used a physical extraction with glass 325 beads while Kajiwara and Murakami employed a chemical extraction with formic acid 326 (70 %) and acetonitrile (100%). Other methods were focused on a target approach, proteins involved in spore maintenance or virulence (respectively 328 Polar Tube Proteins or Spore Wall Proteins). In the case of the silkworm pathogen 329 Nosema bombycis, the different studies show a complex protocol of extraction to 330 examine protein extracts by standard 2D gel electrophoresis[START_REF] Cai | Identification of a Nosema bombycis (Microsporidia) 667 spore wall protein corresponding to spore phagocytosis[END_REF][START_REF] Chen | Identification of a novel chitin-binding spore wall protein 670 (NbSWP12) with a BAR-2 domain from Nosema bombycis (microsporidia)[END_REF][START_REF] Wu | Proteomic analysis of spore wall proteins and identification of 673 two spore wall proteins from Nosema bombycis (Microsporidia)[END_REF] . For spore disruption 331 and extraction, Wu and colleagues used a Sodium Dodecyl Sulfate (SDS) extraction 332 buffer complemented with sucrose, β-mercaptoethanol, Tris and EDTA, whereas Cai333 and colleagues and Chen and colleagues, used another lysis buffer followed by the use 334 of acid-washed glass beads. After migration on the 2D-gel, proteins spots were excised 335 and prepared for MALDI-MS analyses. In our study, we developed and proposed a non-336 targeted approach to obtain spore extracts reliable for identification of species by 337 MALDI profiling. Different protocols were tested including three mechanical molecular 338 extractions using two models of beads (steel balls or zirconium beads) and an ultrasonic 339 treatment. The most complex spectrum in terms of number of molecular ions detected 340 was obtained from the extraction with zirconium beads (21 molecular ions, Fig 3.C and 341

346 3 . 3

 33 Statistical analyses for identification of spores by MALDI profiling 347In order to evaluate the possibility to use MALDI profiling as an alternative to PCR 348 to identify Nosema species (N. ceranae versus N. apis), geographical origin (European 349 vs non-European country) and viability status., we performed a series of statistical 350 analyses by principal component analysis (PCA, Figure3). 351 From the six different sets of samples (MBO.NC01.V, MBO.NC02.V, 352 MBO.NA01.V, MBO.NC01.NV, MBO.NC02.NV and MBO.NA01.NV) and the nine 353 technical replicates generated per samples, the first two dimensions reflected 65.2 % of 354 the variance (41.7% for Dim1 and 23.5% for Dim2, Fig.

  387 ceranae, and two others at m/z between 3,800 -4,200 and 4,600 -5,500 enriched in N. 388 ceranae compared to N. apis. The most part of these differences were relevant in the 389 statistical analyses (m14, m16, m36 and m46 for N. apis and m13, m17, m43 and m50-390 55 for N. ceranae; Fig. 4A3, Fig.3 and Table

408 3 . 3

 33 Fig.6A1). Within the two N. ceranae isolates, few distinct molecular ions were

465 3 . 4

 34 Blind tests on different isolates of Nosema spores 466Five spore isolates were freshly collected from infected bees. Each solution of spores 467 was purified following our protocol. For their identification, the spore extracts were 468 analyzed by MALDI Biotyping. The resulting spectra were compared to the N. ceranae

  469

472

  To conclude, for each individual spore isolate, we determined its mass 473 fingerprint and its viability using the combinatorial approach of MALDI-MS and flow 474 cytometry. Using this analytical strategy, we demonstrated the ability to discriminate N. 475 ceranae extracts from N. apis extracts and potentially N. ceranae from two distinct 476 geographical origins (France versus a non-European country). We also differentiated 477 live spores from dead spores. Using flow cytometry these two spore solutions were 478 segregated into two distinct clusters. For living spores, values averaged between 76 and 479 94%, while for dead spores less than 0.5% of living spores were detectable. Regarding 480 the MALDI profiling, the statistical analyses of this set of spore solutions are clearly 481 separated. Prognosis and/or Diagnosis have important implications for animal and 482 patient care, research, and policy. When they are, accurate and made in a timely and 483 cost-effective manner, the sanitary services and practitioners have the best opportunity 484 for a positive health outcome. In addition, public policy decisions are often influenced 485 by diagnostic information. The analytical strategy we are proposing has several 486 advantages over microscopic and PCR analyses for monitoring nosemosis in honeybee, 487 it is (i) cheaper in terms of consumables, (ii) less time consuming, (iii) requires less 488 spores (by a factor of two) for a robust identification, and (iv) allows estimation of a 489 viability status in contrast to PCR or direct microscopy. MALDI Biotyping may 490 represent a valuable tool for sanitary services and beekeepers to survey nosemosis in 491 apiaries.

  We would like to thank Omayra Martin-Rodriguez for help in 494 flow cytometry and Robert Paxton for donation of Nosema apis spores. We would like 495 to thank Karim Arafah and Sébastien Voisin from the BioPark for their training in mass 496 spectrometry and Cynthia McDonnell for English improvement of the manuscript. This

497

  work was supported by the EDCSV from University of Grenoble Alpes for the PhD 498 allocation of Camille Houdelet and from the Association Platform BioPark of Archamps 499 from its Research & Development budget. 500 Declaration of interest: The authors declare no conflict of interest including specific 501 financial interests and relationships and affiliations relevant to the subject of this 502 manuscript.

Figure 1 :

 1 Figure 1: Experimental workflow from biological material preparation to MALDI-

Figure 2 : 729 Figure 3 :Figure 4 :- 3 ,

 2729343 Figure 2: Flow cytometry analyses of Nosema spore isolates. The percentage of

Figure 5 :Figure 6 :

 56 Figure 5: Differential MALDI-MS spectra between fresh and dead spores of Nosema

769

  Differences between live (MBO.NA01.V (light blue), MBO.NC01.V (light green) and 770 MBO.NC02.V (light red)) and dead (MBO.NA01.NV (dark blue), MBO.NC01.NV (dark 771 green), MBO.NC02.NV (dark red)) spores are represented on the two dimensions Dim1 772 and Dim2. The ellipse represents the confidence of the results. For each sample nine 773 points are represented. The distribution of samples is based on the molecular ions and 774 their position on the correlation circle (C). Each arrow represents a variable and the colour 775 indicates the contribution of each variable in the principal component (dimension). 776 Representation of variation is explained by the two first dimensions (Dim1 and Dim2).

Figure 7 :

 7 Figure 7: Differential MALDI-MS spectra of five spore solutions and identification

Table 1 :

 1 Number of identified peaks in the peak lists of the three spore extracts

	503
	504

Table 2 :

 2 List of molecular ions (m/z: mass/charge) and their corresponding labels.

	703	Peaks positively and negatively correlated to the Dim1 of the PCA are in green and red,
	704	respectively. For Dim2, the peaks in blue are negatively correlated. The peaks in black
	705	are not discriminating.
		Labels Mass (m/z)	Labels Mass (m/z)
		m1	1342.69	m31	2920.42
		m2	1455.85	m32	2955.32
		m3	1476.71	m33	3062.63
		m4	1490.76	m34	3068.27
		m5	1495.52	m35	3077.79
		m6	1564.56	m36	3120.51
		m7	1664.07	m37	3168.88
		m8	1834.88	m38	3519.17
		m9	1887.27	m39	3583.21
		m10	1922.39	m40	3599.29
		m11	1964.36	m41	3630.81

http://mc.manuscriptcentral.com/rcm Rapid Communications in Mass Spectrometry


Miriam Reverter
email: mirireverter@gmail.com

Pierre Sasal
email: sasal@univ-perp.fr

Marcelino T Suzuki
email: suzuki@obs-banyuls.fr

Delphine Raviglione
email: delphine.raviglione@univ-perp.fr

Nicolas Inguimbert
email: nicolas.inguimbert@univ-perp.fr

Alan Pare
email: alan.pare@outlook.fr

Bernard Banaigs
email: banaigs@univ-perp.fr

Sébastien N Voisin
email: sebastien.voisin@biopark-archamps.org

Philippe Bulet
email: philippe.bulet@biopark-archamps.org

Nathalie Tapissier-Bontemps

Insights into the Natural Defenses of a Coral Reef Fish Against Gill Ectoparasites: Integrated Metabolome and Microbiome Approach

Keywords: fish mucus, host-parasite interactions, butterflyfish, metabolomics, microbiome, multiomics, Chaetodon, peptides

Understanding natural defense mechanisms against parasites can be a valuable tool for the development of innovative therapies. We have previously identified a butterflyfish species (Chaetodon lunulatus) that avoids gill monogenean parasites while living amongst closely related parasitized species. The metabolome and microbiome of several sympatric butterflyfish species from the island of Moorea (French Polynesia) were previously described. In this study, we used the previously generated datasets in an attempt to identify metabolites and bacteria potentially involved in parasite defense mechanisms. We investigated the interplay between the gill mucus metabolome and microbiome of the non-susceptible C. lunulatus versus sympatric butterflyfish species that were always found parasitized in the Central and Eastern Indo-Pacific. After observing significant differences between the metabolome and bacteria of susceptible versus non-susceptible fish, we obtained the discriminant metabolites and operational taxonomic units (OTUs) using a supervised analysis. Some of the most important discriminant metabolites were identified as peptides, and three new peptides derived from β-subunit hemoglobin from C. lunulatus (CLHbβ-1, CLHbβ-2, and CLHbβ-3) were purified, characterized and synthesized to confirm their structures. We also identified specific bacterial families and OTUs typical from low-oxygen habitats in C. lunulatus gill mucus. By using a correlation network between the two datasets, we found a Fusobacteriaceae strain exclusively present in C. lunulatus and highly correlated to the peptides. Finally, we discuss the possible involvement of these peptides and Fusobacteriaceae in monogenean avoidance by this fish species.

Introduction

Parasites are crucial for a well-balanced ecosystem, contributing to its organization by modifying competitive and trophic interactions and driving natural selection [START_REF] Hudson | Is a healthy ecosystem one that is rich in parasites?[END_REF]. However, disruption of ecosystem equilibrium due to activities that degrade the environment and compromise the hosts (e.g., pollution, climate change, culture intensification) or through the introduction of new species that can introduce new pathogens (enemy release hypothesis), can lead to parasite outbreaks and can result in high host mortalities [START_REF] Fox | Climate-driven tipping-points could lead to sudden, high-intensity parasite outbreaks[END_REF][START_REF] Cable | Global change, parasite transmission and disease control: Lessons from ecology[END_REF]. Increasing the connectivity of systems (e.g., global animal trade) also heightens the risks of emergence and transmission of infectious agents [START_REF] Perry | Current drivers and future directions of global livestock disease dynamics[END_REF][START_REF] Sana | Origin and invasion of the emerging infectious pathogen Sphaerothecum destruens[END_REF]. Understanding the factors underpinning host-parasite interactions is thus critical to prevent the emergence of infectious diseases that can result in biodiversity decrease and severe economic losses [START_REF] Sana | Origin and invasion of the emerging infectious pathogen Sphaerothecum destruens[END_REF][START_REF] Cunningham | Disease invasion: Impacts on biodiversity and human health[END_REF]. In this context, a better understanding of natural defense mechanisms can contribute to the development of alternative sustainable therapies both in human and animal medicine [START_REF] Rakers | Antimicrobial Peptides (AMPs) from Fish Epidermis: Perspectives for Investigative Dermatology[END_REF][START_REF] Hahn | Experimental Models to Study the Role of Microbes in Host-Parasite Interactions[END_REF].

Host-parasite interactions have been traditionally studied as an interplay between host defense and parasite evasion mechanisms [START_REF] Quigley | The mode of host-parasite interaction shapes coevolutionary dynamics and the fate of host cooperation[END_REF]. However, recent research suggests that host and parasite associated-microorganisms play major roles in host and parasite fitness, highlighting the need to study host-parasite interactions in the light of the microbiota [START_REF] Hahn | Experimental Models to Study the Role of Microbes in Host-Parasite Interactions[END_REF][START_REF] Dheilly | Biological warfare: Microorganisms drivers of host-parasite interactions[END_REF]. Despite the poor understanding of the role of native host microbiota against eukaryotic parasites is poorly understood, some studies suggest they can deploy multiple strategies to control parasitic infections. For example, Cirimotich et al. found that an Enterobacter bacterium from mosquito inhibited the development of malaria parasites by the production of reactive oxygen species (ROS), rendering mosquitoes resistant to infection [START_REF] Cirimotich | Natural Microbe-Mediated Refractoriness to Plasmodium Infection in Anopheles Gambiae[END_REF]. Other studies in reptiles and teleostean fish showed that associated bacteria could be a source of antibacterial and antifungal metabolites, but few studies have explored their activities against eukaryote parasites [START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF][START_REF] Stevens | Bacteria associated with lionfish (Pterois volitans/miles complex) exhibit antibacterial activity against known fish pathogens[END_REF]. The involvement of microbiota in the modulation of the host innate immune system is well recognized in many animals, including fish, and is considered to influence pathogen control strategies [START_REF] Gómez | A review on the interactions between gut microbiota and innate immunity of fish: Table 1[END_REF]. For example, in a recent study, Sepahi and collaborators [START_REF] Sepahi | Symbiont-derived sphingolipids modulate mucosal homeostasis and B cells in teleost fish[END_REF] found that a bacterium from external mucosal surfaces of rainbow trout was able to regulate the symbiont communities and the production of immunoglobulins, T-cells, and B-cells through the production of sphingolipids.

Mucosal surfaces are the main zones of contact between animals and the environment and thus play a key role in both entry and defense against pathogens [START_REF] Reverter | Biological and Ecological Roles of External Fish Mucus: A Review[END_REF]. Teleost mucus is mainly composed of mucin glycoproteins, which are continuously produced and shed to limit pathogen contact with epithelial cells and promote their clearance [START_REF] Hansson | Role of mucus layers in gut infection and inflammation[END_REF]. Mucosal surfaces also contain a wide-array of immune-related molecules (e.g., lysozyme, immunoglobulins, lectins, and antimicrobial peptides) and diverse associated microbial communities that orchestrate to control pathogens [START_REF] Gómez | A review on the interactions between gut microbiota and innate immunity of fish: Table 1[END_REF][START_REF] Sepahi | Symbiont-derived sphingolipids modulate mucosal homeostasis and B cells in teleost fish[END_REF][START_REF] Reverter | Biological and Ecological Roles of External Fish Mucus: A Review[END_REF][START_REF] Gómez | The mucosal immune system of fish: The evolution of tolerating commensals while fighting pathogens[END_REF]. Despite the recent increase in fish mucus research, its role in parasite infection and control remains poorly understood. An increasing body of research suggests some fish mucus cues are used by parasites to detect their hosts [START_REF] Kallert | No shot in the dark: Myxozoans chemically detect fresh fish[END_REF][START_REF] Brooker | Role of kairomones in host location of the pennellid copepod parasite, Lernaeocera branchialis (L. 1767)[END_REF], and mucosal macromolecules such as IgM and the glycoprotein WAP 65-2 have been found to attract and induce attachment of several monogenean parasites in tiger pufferfish (Takifugu rubripes) [START_REF] Ohashi | Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)[END_REF][START_REF] Igarashi | Mucosal IgM Antibody with d-Mannose Affinity in Fugu Takifugu rubripes Is Utilized by a Monogenean Parasite Heterobothrium okamotoi for Host Recognition[END_REF]. Fish mucus is known to display antimicrobial, antifungal, and antiparasitic activities [START_REF] Hellio | Antibacterial, antifungal and cytotoxic activities of extracts from fish epidermis and epidermal mucus[END_REF][START_REF] Wang | Analysis of enzyme activity, antibacterial activity, antiparasitic activity and physico-chemical stability of skin mucus derived from Amphiprion clarkii[END_REF], but to date, few antiparasitic molecules have been identified. Antimicrobial peptides (AMPs), which are normally small cationic amphipathic peptides with less than 50 amino acids, are commonly found in fish mucus and fish epithelium [START_REF] Masso-Silva | Antimicrobial Peptides from Fish[END_REF]. Some peptides isolated from gill epithelium (hemoglobin-derived peptide Hbβ-P1 and piscidin-2 are active against several parasites, but whether these AMPs are also found in fish mucus or whether other AMPs isolated from fish mucus display antiparasitic activities is not clear [START_REF] Colorni | Activity of the antimicrobial polypeptide piscidin 2 against fish ectoparasites[END_REF][START_REF] Ullal | Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)[END_REF][START_REF] Ullal | Antiparasitic activity of the antimicrobial peptide HbbetaP-1, a member of the betahaemoglobin peptide family[END_REF].

Butterflyfishes (Chaetodontidae), a diverse and emblematic family of coral reef fishes, are naturally parasitized by monogeneans belonging to the family Dactylogyridae [START_REF] Plaisance | Description and redescription of Haliotrema species (Monogenoidea: Poloyonchoinea: Dactylogyridae) parasitizing butterfly fishes (Teleostei: Chaetodontidae) in the Indo-West Pacific Ocean[END_REF]. Dactylogyrids are direct cycle flatworms that attach to fish gills by two pairs of hooks [START_REF] Kearn | Evolutionary expansion of the Monogenea[END_REF]. Despite the fact that, all parasites are considered to impose a cost on their hosts, it is extremely difficult to determine the physiological impacts of parasites in the natural environment [START_REF] Frantz | Parasitic versus nutritional regulation of natural fish populations[END_REF]. As such, the effects of dactylogyrids on butterflyfish communities have not been determined, but other Dactylogyrus species are recognized as major pathogens in cultured fish [START_REF] Dash | Effect of Dactylogyrus catlaius (Jain 1961) infection in Labeo rohita (Hamilton 1822): Innate immune responses and expression profile of some immune-related genes[END_REF][START_REF] Li | Grass carp which survive Dactylogyrus ctenopharyngodonid infection also gain partial immunity against Ichthyophthirius multifili[END_REF]. In a recent study, we found that only one butterflyfish species (Chaetodon lunulatus) out of 34 species studied through the Indo-Pacific (including the present study site, Moorea in French Polynesia), was never parasitized by gill monogeneans [START_REF] Reverter | Gill monogenean communities (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF]. Butterflyfish phylogeny, ecology, and behavior have been studied extensively [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF][START_REF] Bellwood | Evolutionary history of the butterflyfishes (f: Chaetodontidae) and the rise of coral feeding fishes[END_REF], and C. lunulatus does not present any striking difference in lifestyle compared to other sympatric and phylogenetically close butterflyfishes such as C. ornatissmus and C. reticulatus, which are always found highly parasitized [START_REF] Reverter | Gill monogenean communities (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF]. In this study, we aimed to investigate the mechanisms behind the species-specific resistance of C. lunulatus to gill monogeneans. We focused on the study of the gill mucus as the main zone of contact between fish and parasites, and we used an integrative approach to identify potential metabolites and operational taxonomic units (OTUs) that might be involved in either parasite attraction or deterrence. This study is a continuation of our previous research on the characterization of different butterflyfish species gill mucus metabolome and microbiome [START_REF] Reverter | Fish mucus metabolome reveals fish life-history traits[END_REF][START_REF] Reverter | Characterisation of the gill mucosal bacterial communities of four butterflyfish species: A reservoir of bacterial diversity in coral reef ecosystems[END_REF], focused on the integration of metabolome and microbiome data in an attempt to elucidate parasite attraction or deterrence mechanisms. The use of this approach allowed us to characterize three previously unknown β-subunit hemoglobin-derived peptides from C. lunulatus gill mucus and explore the correlations between C. lunulatus overexpressed features and the identified bacteria.

Results

Metabolomic Analysis of Chaetodon Gill Mucus

The metabolomics dataset used in this article was previously obtained and described in Reverter et al. [START_REF] Reverter | Fish mucus metabolome reveals fish life-history traits[END_REF]. Metabolomics analyses were performed on both polar (fraction H2O/MeOH) and apolar (fraction MeOH/CH2Cl2) fractions. Principal coordinate analysis (PCoA) of the apolar fraction did not show a significant difference (ANOSIM, P = 0.20) between the susceptible and non-susceptible fish (C. lunulatus) to gill monogeneans (Supplementary Figure S1). Therefore, we did not perform further analysis and all the following results, including variable of importance projection (VIP) selection, identification, and correlation with bacterial OTUs were based on the analysis of the polar fraction.

A PCoA of the polar fraction showed significant differences between the gill mucus metabolome composition of susceptible and non-susceptible fish (ANOSIM, P < 0.001, Figure 1). The PLS-DA model accurately predicted differences between the metabolic profiles of susceptible and nonsusceptible fish (NMC = 0.043, p-value = 0.001). After "cleaning" of the matrix (removal of isotopes and adducts), we found 69 features with a VIP score higher than 1, all significantly over-expressed (Kruskal-Wallis, P < 0.001) in the non-susceptible fish (Figure 2a). Eight out of the 69 VIPs were identified as peptides due to their characteristic multicharged ions and typical fragmentation patterns observed from the high-resolution mass spectrometry data, whilst the rest of the VIP were unknowns (Figure 2b and Figure 3).

Peptide Characterisation and Synthesis

Since LC-ESI-HRMS/MS profiles of the most important VIPs were characteristic of peptides, we optimized the extraction procedure using an acidic extraction to selectively precipitate large proteins while enhancing the solubility of peptides. After solid-phase extraction (SPE) pre-purification of the acidic extract from C. lunulatus mucus, analysis by ultra-high performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) of this sample compared to the polar extract (used for the metabolomics pipeline), showed enrichment in two major peptides with m/z 657.1599 [M + 5H] 5+ (CLHbβ-1) and 644.8738 [M + 2H] 2+ (CLHbβ-2) (Figure 3, Supplementary Figures S2 andS3). Another VIP peptide (CLHbβ-3), not detectable on the Total Ion Chromatogram (TIC) presented in Figure 3, was detected using selected ion extraction mode at m/z 766.0947 [M + 3H] 3+ (Supplementary Figure S4).

Acidic extractions of two heavily parasitized species (C. reticulatus and C. ornatissimus) [START_REF] Reverter | Gill monogenean communities (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF] were done as control and did not show any of the VIPs previously observed in the non-susceptible species, even using the selected ion extraction mode. Additionally, the comparison of the TIC obtained from the peptide-enriched fractions extracted from susceptible and non-susceptible fish species highlighted the presence of two additional peptides with m/z 679.7758 [M + 5H] 5+ (CLHbβ-4) and 702.3929 [M + 5H] 5+ (CLHbβ-5) in the mucus samples of all analyzed species (Figure 4, Supplementary Figures S5 andS6). The mass differences between CLHbβ-1 and CLHbβ-4 (113 Da) and CLHbβ-1 versus CLHbβ-5 (226 Da) correspond to one and two additional leucine residues (Figure 4, Table 1).

The SPE pre-purified sample was analyzed for peptide de novo sequencing for confirmation and further characterization of these five peptides. MALDI-MS analysis of the 60% acetonitrile (MeCN) eluted fraction showed the presence of several potential peptides, including five peaks whose MH + m/z values would match the expected average MH + m/z values for the five peptides discussed above (Supplementary Figure S7). After nanoLC-MS/MS analysis, de novo sequencing of the spectra of CLHbβ-1, CLHbβ-2 and CLHbβ-3 revealed they were composed of 30, 10, and 22 amino acid residues, respectively (Supplementary Figure S8, Table 1). CLHbβ-4 and CLHbβ-5 were shown to have the same sequence as CLHbβ-1, but with one and two extra leucine on the C-terminal end, respectively. Search for amino acid sequence homology against bioactive peptide databases BIOPEP-UWM (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) showed no significant sequence identity to any sequence in these databases. Homology search with the Swissprot-UniprotKB database using Mascot and Sequest highlighted peptides derived from the hemoglobin subunit beta-B of the Japanese amberjack Seriola quinqueradiata with high scores (see Supplementary Table S1). Further comparison with a beta subunit hemoglobin sequence of a closely related species Chaetodon austriacus [START_REF] Dibattista | Draft genome of an iconic Red Sea reef fish, the blacktail butterflyfish (Chaetodon austriacus): Current status and its characteristics[END_REF] confirmed that the five VIP peptides showed 100% identity with beta subunit hemoglobin (Hbβ N-terminus (Figure 5). The five peptides identified (CLHbβ-1 to 5) were synthesized according to Fmoc/tBu solid-phase method using preloaded Leu or Arg Wang resin in order to confirm the previously obtained structures. After elongation was completed, the peptides were cleaved from the resin using TFA, affording targeted compounds with yields ranging from 42% to 92% and a purity of at least 90% for each of the synthetic peptides as judged by mass spectrometry analysis. Peptides CLHbβ-2 to CLHbβ-5 tended to form gels; therefore, they were used without further purification. The accuracy of their sequence was confirmed by comparing the chromatographic (identical retention times) and spectrometric data (i.e., exact mass and fragmentation pattern) between the synthetic and the natural forms (Supplementary Figures S4 to S8).

Bacterial Community of Chaetodon Gill Mucus

The bacterial communities of one non-susceptible butterflyfish (C. lunulatus) and three highly susceptible butterflyfish species (C. ornatissimus, C. reticulatus, and C. vagabundus) have been previously described [START_REF] Reverter | Characterisation of the gill mucosal bacterial communities of four butterflyfish species: A reservoir of bacterial diversity in coral reef ecosystems[END_REF]. Here, we analyzed the same data in the context of parasite presence and absence. PCoA showed significant differences between the gill mucosal bacterial communities of parasitized and non-parasitized fishes (ANOSIM, P < 0.001, Figure 6). The PLS-DA model accurately predicted bacterial differences between susceptible and non-susceptible fish (NMC = 0.016, p-value = 0.015). We identified 133 OTUs with VIP scores higher than 1, which belonged to 19 bacterial orders (Figure 7). The mean relative abundance of these orders in susceptible and non-susceptible fish was obtained by grouping the 133 retained OTUs by bacterial order. The Actinomycetales and Pseudomonadales were the only bacterial orders that presented a significantly higher mean relative abundance in the susceptible fish (4.7 ± 4.8%, and 0.4 ± 0.5% P < 0.005). Twelve bacterial orders presented significantly higher mean relative abundances in non-susceptible fish (Figure 7): Fusobacteriales (3.5 ± 2.9%), Bacteroidales (3.6 ± 2.4%), Oceanospirillales (2.8 ± 4.8%), Alteromonadales (0.4 ± 0.7%), Spirochaetales (0.33 ± 0.30), Rhodobacterales (0.6 ± 0.8%), Desulfovibrionales (0.6 ± 0.6%), Vibrionales (33.7 ± 27.7%), Verrucomicrobiales (5.8 ± 4.8%), Clostridiales (3.9 ± 3.4%), Rickettsiales (0.13 ± 0.2%) and Erysipelotrichales (0.12 ± 0.10%).

Correlation Between VIP Metabolites and VIP OTUs

A correlation analysis between metabolites and bacterial OTUs that were significantly overexpressed in non-susceptible fish, with a VIP score higher than 1 and present in all C. lunulatus individuals, was performed. All peptides were significantly correlated to at least one OTU. We identified seven OTUs that were correlated (ρ ≥ 0.5, P < 0.005) to at least one of the VIP metabolites (three Vibrionaceae, one Verrucomicrobiaceae, one Ruminococcaceae, one Fusobacteriaceae and one Bacteroidales that could not be identified to family level). The Fusobacteriaceae and the Bacteroidales were the OTUs correlated to the highest number of metabolites (Figure 8A). The Fusobacteriaceae was also the OTU correlated to the highest number of peptides (4 out of 7) and the only OTU 8B). Other OTUs correlated to peptides included the Bacteroidales (correlated to CLHbβ-2 and CLHbβ-3), the Ruminococcaceae, the Verrucomicrobiaceae, and one Vibrionaceae (Figure 8B).

Discussion

The emergence of infectious diseases in animal production systems is a major problem causing severe economic losses (estimated US 9.5$ billion per year) through decreased production of farmed species or increased production costs [START_REF] Shinn | Economic impacts of aquatic parasites on global finfish production[END_REF]. Treatment of ectoparasite infections in aquaculture normally involves bathing fish in chemical substances (e.g., praziquantel, formaldehyde) that have nefarious environmental consequences, are hazardous for the animals and sometimes display reduced efficacy due to the emergence of resistance [START_REF] Picón-Camacho | An assessment of the use of drug and nondrug interventions in the treatment of Ichthyophthirius multifiliis Fouquet, 1876, a protozoan parasite of freshwater fish[END_REF][START_REF] Forwood | Efficacy of current and alternative bath treatments for Lepidotrema bidyana infecting silver perch, Bidyanus bidyanus[END_REF]. Therefore, the development of more environmentally-friendly therapies and sustainable/integrated alternatives is urgently needed [START_REF] Reverter | Aquaculture at the crossroads of global warming and antimicrobial resistance[END_REF]. Within this context, a better understanding of the natural defense mechanisms deployed by fish to avoid parasites can be extremely useful for the development of innovative therapies. In this study, we have investigated the natural interplay between the gill mucus metabolome and microbiome of Chaetodon lunulatus, as potential mechanisms involved in parasite avoidance. C. lunulatus is the only butterflyfish species that has never been found parasitized by gill monogeneans, despite living among parasitized butterflyfishes with similar ecology, behavior, and phylogenetic position [START_REF] Reverter | Gill monogenean communities (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF]. For this reason, it provides a good model to study natural parasite evasion mechanisms.

The untargeted metabolomics study showed that parasitized and non-parasitized fish presented significantly different polar metabolome fingerprints. In a previous study, we found that butterflyfish species taxonomy was not a significant driver of metabolome variability of the polar fraction [START_REF] Reverter | Fish mucus metabolome reveals fish life-history traits[END_REF], suggesting other C. lunulatus specific traits behind these differences. The coupling of the untargeted metabolomics study with high-resolution mass spectrometry analysis allowed us to identify several metabolites, of which eight peptides (mass range between 1.2-4.5 kDa), significantly overexpressed in C. lunulatus gill mucus. We characterized and chemically synthesized three of these peptides (CLHbβ-1, CLHbβ-2, CLHbβ-3) found uniquely in C. lunulatus, which presented 100% homology to N-terminal region of β-subunit hemoglobin (Hb), suggesting that these peptides might be cleaved off from the N-terminal of Hb by specific proteases after arginine residues (i.e., trypsin-like). The other identified peptides were present in smaller quantities and could not be characterized. We also identified two additional peptides (CLHbβ-4 and CLHbβ-5) present in both parasitized and nonparasitized fish, probably resulting from the cleavage of the same protein (Hb) after leucine residues by other types of proteases (i.e., carboxypeptidase-like). Several studies have previously reported fish AMPs cleaved from functional proteins such as histones (e.g., parasin-I and hipposin) [START_REF] Birkemo | Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)[END_REF][START_REF] Park | Parasin I, an antimicrobial peptide derived from histone H2A in the catfish, Parasilurus asotus[END_REF], L- amino acid oxidases [START_REF] Salles | Identification and characterization of proteases from skin mucus of tambacu, a Neotropical hybrid fish[END_REF], ribosomal proteins [START_REF] Fernandes | A novel antimicrobial function for a ribosomal peptide from rainbow trout skin[END_REF], and Hb [START_REF] Ullal | Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)[END_REF]. Hb is an iron-containing oxygentransport metalloprotein with a quaternary structure composed of two α-globin and two β-globin chains [START_REF] Anthea | Human Biology and Health[END_REF]. Several Hb-derived peptides with antimicrobial and antiparasitic activities have been isolated from different fish species (channel catfish Ictalurus punctatus, Japanese eel Anguilla japonica, and skipjack tuna Katsuwonus pelamis) and tissues (gill epithelium, liver) [START_REF] Ullal | Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)[END_REF][START_REF] Zhang | Isolation and characterization of a novel antibacterial peptide derived from hemoglobin alpha in the liver of Japanese eel, Anguilla japonica[END_REF][START_REF] Seo | Antimicrobial function of SHβAP, a novel hemoglobin β chain-related antimicrobial peptide, isolated from the liver of skipjack tuna, Katsuwonus pelamis[END_REF]. Interestingly, CLHbβ-1 presented some homology to Hbβ-P3, a β-chain hemoglobin peptide isolated from gill epithelium of channel catfish resulting from the same cleavage of the protein, which displays antibacterial activity [START_REF] Ullal | Antiparasitic activity of the antimicrobial peptide HbbetaP-1, a member of the betahaemoglobin peptide family[END_REF].

Host commensal microbiota influences host physiology, playing an active role in regulating host immune homeostasis, which is crucial to avoid the proliferation of pathogens [START_REF] Wu | The role of gut microbiota in immune homeostasis and autoimmunity[END_REF][START_REF] Boutin | Probiotic treatment by indigenous bacteria decreases mortality without disturbing the natural microbiota of Salvelinus fontinalis[END_REF][START_REF] Boutin | Inter Individual Variations of the Fish Skin Microbiota: Host Genetics Basis of Mutualism?[END_REF]. In a previous study, we characterized the core microbiota of three highly parasitized butterflyfishes and the nonparasitized C. lunulatus [START_REF] Reverter | Characterisation of the gill mucosal bacterial communities of four butterflyfish species: A reservoir of bacterial diversity in coral reef ecosystems[END_REF]. Here, we found that some bacteria typically associated with low-oxygen habitats (Fusobacteriaceae, Clostridiales), were significantly more abundant in C. lunulatus compared to other Chaetodon species. Increased expression of Hb under low-oxygen levels has been observed in organisms such as fish, plants, or humans [START_REF] Gaulke | Acclimation to a low oxygen environment alters the hematology of largemouth bass (Micropterus salmoides)[END_REF][START_REF] Heinicke | Long-term exposure to intermittent hypoxia results in increased hemoglobin mass, reduced plasma volume, and elevated erythropoietin plasma levels in man[END_REF][START_REF] Hunt | Increased level of hemoglobin 1 enhances survival of hypoxic stress and promotes early growth in Arabidopsis thaliana[END_REF]. These results suggest that C. lunulatus gill mucus might have lower-oxygen contents and possibly higher levels of hemoglobin than in other butterflyfish species, but this is a non-trivial analysis that will have to be developed in further studies.

Associated bacteria can also play major roles in pathogen control through the production of bioactive metabolites [START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF][START_REF] Sepahi | Symbiont-derived sphingolipids modulate mucosal homeostasis and B cells in teleost fish[END_REF][START_REF] Sanchez | Examining the Fish Microbiome: Vertebrate-Derived Bacteria as an Environmental Niche for the Discovery of Unique Marine Natural Products[END_REF]. We have explored the correlations between metabolites and OTUs to investigate whether some bacteria are specifically related to some metabolites in C. lunulatus. We found that all peptides were highly correlated to at least one OTU. CLHbβ-1, CLHbβ-2, and CLHbβ-3 were highly correlated to a Fusobacteriaceae strain. Fusobacteria are frequently found inhabiting mucosal tissues and are known to produce a short-chain fatty acid, butyrate, which is the end-product of fermentation of carbohydrates, including those found in mucins [START_REF] Bennett | Fusobacteria: New taxonomy and related diseases[END_REF]. Studies show that butyrate provides benefits to mammal hosts, enhancing mucus production and acting as anti-carcinogen and anti-inflammatory [START_REF] Engelhardt | Role of short-chain fatty acids in the hind gut[END_REF][START_REF] Andoh | Physiological and Anti-Inflammatory Roles of Dietary Fiber and Butyrate in Intestinal Functions[END_REF], but its effects in fish are yet unknown. If Fusobacteria enhances mucus production in fish, this could increase gill mucus thickness, increasing oxygen diffusion distance, and eventually promoting higher levels of hypoxia as previously described in clownfish [START_REF] Hess | Exposure of clownfish larvae to suspended sediment levels found on the Great Barrier Reef: Impacts on gill structure and microbiome[END_REF]. Loweroxygen levels could then promote higher hemoglobin levels, which could be cleaved by Fusobacteria or other microbial proteases (maybe for nutrition purposes), leading to the VIP peptides. Several studies have shown that extracellular microbe proteases can specifically cleave Hb and produce Hbderived peptides with antimicrobial activities [START_REF] Jiang | Respiratory protein-generated reactive oxygen species as an antimicrobial strategy[END_REF][START_REF] Du | Rapid reprogramming of haemoglobin structure-function exposes multiple dualantimicrobial potencies[END_REF][START_REF] Lee | A Perspective on the Role of Extracellular Hemoglobin on the Innate Immune System[END_REF]. Furthermore, some studies on Fusobacterium nucleatum, a human pathogenic Fusobacteria, suggest peptides and amino acids are the main energy source of Fusobacteria [START_REF] Bakken | Utilization of amino acids and peptides by Fusobacterium nucleatum[END_REF][START_REF] Rogers | The breakdown and utilization of peptides by strains of Fusobacterium nucleatum[END_REF] and several extracellular serine proteases have been isolated from F. nucleatum [START_REF] Brokstad | Purification and characterization of a 65-kilodalton diisopropylfluorophosphate-binding protein in the outer membrane of Fusobacterium nucleatum Fev1[END_REF][START_REF] Doron | Identification and Characterization of Fusolisin, the Fusobacterium nucleatum Autotransporter Serine Protease[END_REF]. As CLHbβ-1, CLHbβ-2 and CLHbβ-3 are cleaved after an arginine residue, these peptides are probably produced by a trypsin-like protease, possibly from bacterial origin.

Proteases in skin mucus are involved in the natural resistance of fish to an infection either by acting directly on pathogens or by modifying the mucus consistency to allow pathogen removal [START_REF] Kallert | No shot in the dark: Myxozoans chemically detect fresh fish[END_REF][START_REF] Esteban | Ángeles An Overview of the Immunological Defenses in Fish Skin[END_REF]. Proteases can also activate and enhance the production of other innate immune components present in fish mucus [START_REF] Esteban | Ángeles An Overview of the Immunological Defenses in Fish Skin[END_REF]. We can suggest two hypotheses by which C. lunulatus specific Fusobacteriaceae could participate in monogenean control by contributing to the production of bioactive Hb-derived peptides or producing proteases acting directly on the parasite. However, findings discussed here come from different animal models and bacterial strains, and therefore more research is required to elucidate the role of Fusobacteria in C. lunulatus gill mucus and its putative involvement in the production of hemoglobin peptides. Equally, more studies are required to elucidate the origin (bacterial or fish) of the proteases cleaving Hb, as well as determine the putative antiparasitic activity of CLHbβ-1, CLHbβ-2, and CLHbβ-3. Since the Fusobacteriaceae OTU in C. lunulatus gills has no close cultivated counterpart, future metagenomic analysis of the fish mucus, and multi-metagenome assembly (e.g., [START_REF] Albertsen | Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes[END_REF]) could help to access the peptidases associated this OTU and test these hypotheses.

In summary, we identified and characterized three novel hemoglobin-derived peptides (CLHbβ-1, CLHbβ-2, and CLHbβ-3) exclusively found in C. lunulatus gill mucus using a metabolomics-microbial metabarcoding approach. We also identified specific bacterial families and OTUs typical from low-oxygen habitats in C. lunulatus gill mucus and found a Fusobacteriaceae strain exclusively present in C. lunulatus and also highly correlated to the three identified peptides. We discuss here the possible involvement of these peptides and Fusobacteriaceae to the absence of monogenean parasites in this species. Further research is in progress in order to evaluate in vitro peptide activities against butterflyfish monogeneans at different stages of their life cycle and to characterize the expression of trypsin-like proteases in the skin mucus of different fish species of this genus. In order to further study our hypothesis, the maturation process required to generate the Hbderived peptides needs to be elucidated.

Materials and Methods

Chemicals

MilliQ water (Merck Millipore, Billerica, MA, USA) was used; LC-MS-grade formic acid (FA), trifluoroacetic acid (TFA) and acetonitrile (MeCN) were from Carlo-Erba Reagents (Val de Reuil, France). All Fmoc-protected amino acids, DIC (N,N-diisopropylcarbodiimide), Oxyma (Ethyl cyanoglyoxylate-2-oxime), Wang resin preloaded with arginine (loading 0.29 mmol/g) or leucine (loading 0.33 mmol/g) were purchased from Iris Biotech (Marktredwitz, Germany). DCM (dichloromethane), DMF (N,N-dimethylformamide), cHex (Cyclohexane), TFA, TIS (Triisopropylsilane), FA, MeCN and piperidine for peptide synthesis were obtained from Sigma-Aldrich (Saint Louis, MI, USA), TIPS (triisopropyl silane, Sigma-Aldrich).

Animal Ethics

Animal handling and sampling protocols were pre-approved by animal experimentation experts from our institute, following the European Union directive 2010/63UE.

Fish Mucus Sampling

Fish mucus samples used for this study had been previously obtained, processed, and analyzed in previous works [START_REF] Reverter | Fish mucus metabolome reveals fish life-history traits[END_REF][START_REF] Reverter | Characterisation of the gill mucosal bacterial communities of four butterflyfish species: A reservoir of bacterial diversity in coral reef ecosystems[END_REF]. Briefly, the fish were spear-fished in Moorea (French Polynesia), and the mucus was collected from scrapping the gill filaments (Supplementary material 1a).

Study of the Gill Mucus Metabolome

Untargeted Metabolomics

The metabolome dataset used for this study was previously obtained and described in Reverter and collaborators [START_REF] Reverter | Fish mucus metabolome reveals fish life-history traits[END_REF]. Briefly, the metabolome of eight butterflyfish species, Chaetodon auriga, C. lunulatus, C. lunula, C. ornatissimus, C. quadrimaculatus, C. reticulatus, C. vagabundus, and C. ulietensis was explored using a non-targeted LC-MS metabolomics pipeline. Freeze-dried mucus was extracted using a two-step biphasic extraction, which yielded polar and apolar extracts, respectively. Both extracts were analyzed using an LC-ESI-MS system, and the metabolomic data was processed using the R package XCMS, providing a matrix containing the retention time, m/z values, and integrated peak areas of the identified features (Supplementary material 1b).

Optimized Peptide Extraction and Peptides Purification

An acidic extraction was performed following the method described in Vidal-Dupiol et al. [START_REF] Vidal-Dupiol | Innate Immune Responses of a Scleractinian Coral to Vibriosis*[END_REF] to obtain a peptide-enriched extract. Briefly, 10 mg of freeze-dried mucus were suspended in 1 mL of 2 M glacial acetic acid. The suspension was maintained in a 4 °C water bath during sonication (Vibra-cellTM 75185, 50% power, three pulses of 30 s) and centrifuged at 10,000× g for 20 min at 4 °C, to remove the cellular debris. The obtained supernatant was immediately collected and prepurified by solid-phase extraction using a Strata C18 cartridge (500 mg/6 mL; Phenomenex, CA, USA) washed using acidified water (0.05% TFA), and three successive elutions were performed with 10%, 60%, and 80% acetonitrile (MeCN) in acidified water. The fractions obtained were freeze-dried and reconstituted with 1 mL of H2O/MeCN (1:1 v/v). C. lunulatus and C. ornatissimus mucus samples were extracted in triplicates.

Peptide Sequence Analysis

MALDI-MS analysis of the SPE-prepurified peptides was done on a Bruker AutoFlex™ III (Bruker Daltonics, Germany). Samples were diluted 10-fold in acidified water (0.1% trifluoroacetic acid), and 0.5 μL of a given sample was mixed with 0.5 μL of 4-HCCA (Sigma-Aldrich, France) on a MALDI MTP 384 polished ground steel plate (Bruker Daltonics). Following co-crystallization, MALDI MS spectra were recorded in positive linear mode using FlexControl 4.0 software (Bruker Daltonics). The following instrument settings were used: 1.5 kV of electric potential difference, dynamic range of detection of 600 to 18,000 Da, global attenuator offset of 46% with 200 Hz laser frequency, and 2000 accumulated laser shots per sample. A suppression mass gate up to m/z 600 to prevent detector saturation by clusters of the 4-HCCA matrix. An external calibration of the mass spectrometer was performed using a standard mixture of peptides and proteins (Peptide Standard Calibration II and Protein Standard Calibration I, Bruker Daltonics) covering the dynamic range of analysis.

NanoLC-MS/MS analyses were carried out using an Ultimate 3000 nano-HPLC coupled with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, MA, USA). The peptides were loaded onto a C18 PepMap100 precolumn (5 μm, 300 μm × 5 mm) at 10 μL.min -1 , and separated onto an Acclaim C18 PepMap100 column (75 μm × 250 mm, 3 μm 100 A) at 300 nL min -1 , both maintained at 35 °C (both columns from Thermo Fisher Scientific). Peptide samples were eluted by a gradient of H2O/MeCN (A/B), both containing 0.1% formic acid, from 2% to 50% B in 31 min. For MS analysis, the Q-Exactive mass spectrometer was used in positive mode and data-dependent acquisition. The voltage applied to the nano-tips was adjusted to produce 0.3 μA and the entrance capillary was maintained at 320 °C. The Q-Exactive Orbitrap acquired a full-range scan from 380 to 2000 m/z at 70,000 resolution, automatic gain control (AGC) target 3.10 6 , maximum ion trap time (IT) 200 ms, and then fragmented the top ten-peptide ions in each cycle (range 200-2000 m/z, 17,500 resolution, AGC target 2.10 5 , maximum IT 100 ms, intensity threshold 4.10 4 , excluding charge-unassigned ions, normalised collision energy at 27). Parent ions were then excluded from MS/MS for the next 10 s. The software Chromeleon Xpress and Xcalibur 2.2 (Thermo Fisher Scientific) were used to control the HPLC and the mass spectrometer, respectively. Exact masses were calculated with the Xtract algorithm from Xcalibur software.

Characterization of peptide sequences was performed by Proteome Discoverer 1.4 (Thermo Fisher Scientific). Homology searches of the amino acid sequence were performed on the Swissprot-UniprotKB database (November 2016, https://www.uniprot.org/) using Mascot and Sequest.

Solid-Phase Synthesis and Peptide Analysis

Peptides were synthesized by solid-phase peptide synthesis (SPPS) using Fmoc/O-tbutyl chemistry on an automated microwave peptide synthesizer (CEM liberty one, Orsay, France). SPPS was performed at a 0.10 mmol scale with DIC/Oxyma as a coupling reagent and 20% piperidine in DMF for Fmoc deprotection. The elongation was carried out automatically using a 5-fold excess of protected amino acids and coupling reagent. The mixture was irradiated in a microwave cavity at 70 °C for 20 min. Fmoc deprotection was achieved using 20% piperidine in DMF (7 mL for 30 s at 33 °C and 7 mL for 3 min at 70 °C). After completion of the synthesis, the peptidyl-resin was washed with 2 × 10 mL of DCM. Cleavage of the peptide and removal of the protecting groups were performed by treating the peptidyl-resin with 10 mL of TFA/DCM/H2O/TIPS 4.7:4.7:4:2 (v/v) for 60 min. After the resins' filtration, the filtrate was concentrated and co-evaporated with cyclohexane. The peptide was precipitated with diethyl ether and recovered by centrifugation. Following purification, peptide purity was established by HPLC-ELSD analysis, and identity was confirmed by comparison of either retention times and spectral data of natural peptides using UHPLC-HRMS/MS. These analyses were performed on a UHPLC system (Vanquish, Thermo Scientific) interfaced with an Orbitrap mass spectrometer (Q-Exactive Plus Hybrid, Thermo Scientific) using a Luna Omega Polar C18 column (2.1 mm × 100 mm, 1.6 μm particle size, 100 A porosity, Phenomenex). The elution rate was set to 0.3 mL.min -1 at a constant temperature of 30 °C. The elution was performed using a gradient of H2O/MeCN (A/B), both containing 0.1% formic acid, from 2% to 50% B in 11 min. Mass spectrometer parameters were set as follows: spray voltage 3500 V, capillary temperature 320 °C, sheath gas N2 pressure 35 (arbitrary unit), auxiliary gas pressure 10 (arbitrary unit). Mass spectrometric data were collected in continuum full-range scan mode from m/z 300-1000 in positive mode with a resolution of 70,000.

CLHbβ-1: LLIVYPWTQR Crude peptide (152 mg, 100% yield) was purified by semi-preparative HPLC using a linear gradient from 10% to 100% B in A over 50 min with a flow rate of 5 mL/min to yield CLHbβ-1 (55 mg, 42% yield, > 98% purity

Study of the Gill Mucus Associated Bacterial Communities

The bacterial community dataset was previously acquired and described in Reverter and collaborators [START_REF] Reverter | Characterisation of the gill mucosal bacterial communities of four butterflyfish species: A reservoir of bacterial diversity in coral reef ecosystems[END_REF]. Briefly, the bacterial communities of gill mucus of four butterflyfish species (C. lunulatus, C. ornatissimus, C. reticulatus, and C. vagabundus) were studied. Gill mucus DNA was extracted, and DNA samples were sent for 454 pyrosequencing. Multiplex raw SFF (standard flowgram format) files were analyzed using a hybrid analysis pipeline using Usearch5 and Qiime1, and non-chimeric sequences were unweighted and grouped into operational taxonomic units (OTUs) with a cut-off of 98% (Supplementary material 1c); this provided a rarefied table containing the relative abundance (%) of 1041 OTUs from 14 butterflyfish gill mucus samples 4.6. Data analysis.

Multidimensional matrices obtained from the metabolome and microbiome data were analyzed equally. Principal coordinate analysis (PCoA, library ape for R) was used to visualize the differences between the parasitized and non-parasitized fish metabolome and microbiome. Analysis of similarities (ANOSIM, 10,000 permutations, library vegan for R) was used to detect significant differences between the PCoA groups. Partial least squares discriminant analysis (PLS-DA, library mixOmics for R) was used to select the metabolites and OTUs driving the differences between the two fish groups. PLS-DA model accuracy was calculated using two-fold cross-validation (2CV, function MVA.cmv from package RVAideMemoire for R) and the resulting number of misclassifications (NMC) was compared to its permutated null distribution (999 permutations) to test for model significance (P-value < 0.05, function MVA.test from package RVAideMemoire for R). Variable importance in projection (VIP) was obtained (package RVAiedeMemoire for R), and features and OTUs with VIP scores higher than 1 were retained. Kruskal-Wallis test was used to test for a significant difference in the intensity of metabolites and relative abundance of OTUs between parasitized and non-parasitized fish. In order to explore the correlation between VIP metabolites and OTUs, a correlation network was constructed using the software Cytoscape v3.4.0 [START_REF] Shannon | Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks[END_REF]. Spearman correlations between the VIP metabolite and OTUs were computed, and lines were drawn between pairs of metabolites (circular nodes) and OTUs (diamond nodes) when their correlation was higher than 0.5.

Supplementary Materials:

The following are available online at www.mdpi.com/2218-1989/10/6/227/s1, Figure S1: PCoA analysis of gill mucus metabolome (apolar fraction) of parasitized (other, orange triangles) and nonparasitized (C. lunulatus, CL, blue circles) butterflyfish. ANOSIM P-value > 0.05, Figure S2 Funding: This research was funded by the LabEx Corail project (MECANO, to Pierre Sasal). This research is part of an EPHE thesis supported by a LabEx "Corail" doctoral grant awarded to Miriam Reverter. The mass spectrometry analyses performed by S.V. and P.B. were funded by the R&D budget from the Plateforme BioPark d'Archamps.

Figure 1 .

 1 Figure 1. PCoA analyses of gill mucus metabolome of susceptible (other, orange triangles) and nonsusceptible (Chaetodon lunulatus, CL, blue circles) butterflyfish.

Figure 2 .

 2 Figure 2. (a) Bubble plot displaying the molecular masses in Daltons (Da) and retention time in seconds (s) of significantly over-expressed VIPs in Chaetodon lunulatus. Bubble size indicates the foldchange between C. lunulatus and susceptible butterflyfishes, different colors show different identified (CLHbβ-1, CLHbβ-2, and CLHbβ-3 have been characterized) and unidentified peptides (b) Integrated areas of VIP peptides (over-expressed in C. lunulatus). For uncharacterized peptides M is their molecular mass (Da) and T their retention time in seconds (s). Median, first, and third quartile are plotted and the dots indicate outliers.

Figure 3 .

 3 Figure 3. UHPLC-HRMS/MS analysis in positive mode of the peptide enriched fraction of Chaetodon lunulatus mucus. CLHbβ-1 and CLHbβ-2 are identified in the chromatogram, and their characteristic peptide spectra displaying multicharged ions are shown.

Figure 4 .

 4 Figure 4. LC-ESI-HRMS analysis of the peptide-enriched fraction of Chaetodon lunulatus mucus (A), C. ornatissimus (B) and C. reticulatus (C) in positive mode, displaying the peptides CLHbβ-1, CLHbβ-2, CLHbβ-4, and CLHbβ-5 (CLHb β-3 was not observable on TIC but was identified in ion extraction mode; see Supplementary Figure4).

Figure 5 .

 5 Figure 5. Amino-acid sequences of Chaetodon austriacus hemoglobin precursor. Residues 2 to 31, 32 to 41, 10 to 31, 2 to 32, and 2 to 33 correspond to CLHbβ-1, CLHbβ-2, CLHbβ-3, CLHbβ-4, and CLHbβ-5, respectively.

Figure 6 .

 6 Figure 6. PCoA analyses of gill mucus microbiome of susceptible (other, orange triangles) and nonsusceptible (C. lunulatus, CL, blue circles) butterflyfish.

Figure 7 .

 7 Figure 7. VIP bacterial orders identified between susceptible (orange) and non-susceptible (blue, C. lunulatus) fish. * indicates a significant difference (Kruskal-Wallis, P-value < 0.05).

  variance: 35.8% PCoA 2explained variance: 14% correlated to the three C. lunulatus peptides (CLHbβ-1, CLHbβ-2, CLHbβ-3) (Figure

Figure 8 .

 8 Figure 8. Correlation network (Spearman correlation) between VIP metabolites (circles) and VIP OTUs (diamonds) (A) and VIP peptides (circles) and VIP OTUs (diamonds) (B). Lines show ρ > 0.5.

  : LC-HRMS ESI+ analysis of the synthetic peptide CLHbβ-1 and the acidic enriched peptidic fraction of C. lunulatus mucus, Figure S3: LC-HRMS ESI+ analysis of the synthetic peptide CLHbβ-2 and the acidic enriched peptidic fraction of C. lunulatus mucus, Figure S4: LC-HRMS ESI+ analysis of the synthetic peptide CLHbβ-3 and the acidic enriched peptidic fraction of C. lunulatus mucus in ion extraction mode, Figure S5: LC-HRMS ESI+ analysis of the synthetic peptide CLHbβ-4 and the acidic enriched peptidic fraction of C. lunulatus mucus, Figure S6: LC-HRMS ESI+ analysis of the synthetic peptide CLHbβ-5 and the acidic enriched peptidic fraction of C. lunulatus mucus, Figure S7: MALDI-MS spectrum of the peptides extracted from C. lunulatus gill mucus, Figure S8: MS-MS spectrum of the peptides CLHbβ #1 to 5, Table S1: Acid-extract-nanoLC-MS-MS protein identification report. Supplementary material 1: Details on the material and method section related to previously published work. Author Contributions: M.R., P.S. N.T-B., and B.B. conceived the study. M.R. and P.S. obtained the samples. M.R. and N.T-B. performed the metabolome analysis. M.R. and M.S. performed the bacterial community analysis. N.T-B., D.R., N.I., and A.P. performed peptide synthesis. S.V. and P.B. performed the mass spectrometry analyses of the peptide characterization. M.R. performed the statistical analysis. M.R. and N.T-B. wrote the first manuscript draft. P.S., N.T-B., and M.S. secured funding. All authors commented on the manuscript at different stages and accepted the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Table 1 .

 1 Sequences and mass spectrometric data for peptides CLHbβ-1 to 5.

						m/z LC-MS		m/z MALDI-MS
	Name	Sequence	Calculated MW (Monoisotopic Mass)	Charge	Calculated m/z	(Monoisotopic Mass) Observed for natural	Observed for synthetic	(Average Value) Theoretical Observed
				State	LC-MS	peptides	peptides	MH+	MH+
	CLHbβ-2	LLIVYPWTQR	1287.7339	2	644.8748	644.8738	644.8743	1289.562	1289.682
	CLHbβ-3	AAITSLWGKIDVGEIGPQALTR	2295.2586	3	766.0940	766.0947	766.0927	2297.659	2296.555
	CLHbβ-1	VKWTDAERAAITSLWGKIDVGEIGPQALTR	3280.7567	5	657.1592	657.1599	657.1589	3283.754	3283.980
	CLHbβ-4	VKWTDAERAAITSLWGKIDVGEIGPQALTRL	3393.8408	5	679.7760	679.7758	679.7773	3396.914	3396.935
	CLHbβ-5 VKWTDAERAAITSLWGKIDVGEIGPQALTRLL	3506.9248	5	702.3928	702.3929	702.3931	3510.073	3510.192

  ). ESI-HRMS: Calculated for C63H97N15O14: [M + H] + m/z 1288.74177; [M + 2H] 2+ m/z 644.87479; [M + 3H] 3+ m/z 430.1914; found m/z: 644.8743.

	CLHbβ-2: VKWTDAERAAITSLWGKIDVGEIGPQALTR Crude peptide (268 mg, 71% yield, >
	90% purity). ESI-HRMS: Calculated for C147H237N41O44: [M+H] + m/z 3281.76464; [M + 2H] 2+ m/z
	1641.38623; [M + 3H] 3+ m/z 1094.5934; [M + 4H] 4+ m/z 821.19703; [M + 5H] 5+ m/z 657.1592 found m/z:
	657.1589.
	CLHbβ-3: AAITSLWGKIDVGEIGPQALTR Crude peptide (210 mg, 83% yield, > 90% purity).
	ESI-HRMS: Calculated for C103H170N28O31: [M + H] + m/z 2296.26651; [M + 2H] 2+ m/z 1148.6372; [M +
	3H] 3+ m/z 766.0941; found m/z: 766.0927.
	CLHbβ-4: VKWTDAERAAITSLWGKIDVGEIGPQALTRL Crude peptide (220 mg, 65% yield, >
	90% purity). ESI-HRMS: Calculated for C153H248N42O45: [M + H] + m/z 3394.84871; [M + 2H] 2+ m/z
	1697.92827; [M + 3H] 3+ m/z 1132.28757; [M + 4H] 4+ m/z 849.46802; [M + 5H] 5+ m/z 679.77600 found m/z:
	679.7773.
	CLHbβ-5: VKWTDAERAAITSLWGKIDVGEIGPQALTRLL Crude peptide (257 mg, 83% yield,
	> 90% purity). ESI-HRMS: Calculated for C159H259N43O46: [M + H] + m/z 3507.93277; [M + 2H] 2+ m/z
	1754.47029; [M + 3H] 3+ m/z 1169.98281; [M + 4H] 4+ m/z 877.73906; [M + 5H] 5+ m/z 702.3928 found m/z:
	702.3931.

Acknowledgments:

The spectroscopic experiments were performed using the Biodiversité et Biotechnologies Marines Platform (Bio2Mar, http://bio2mar.obs-banyuls.fr/fr/index.html) facilities at the University of Perpignan via Domitia. The authors would like to thank Karine Escoubeyrou and Alice Rodrigues for assisting with the initial HR-LC-MS analyses that were performed on the Bio2Mar platform facilities at Banyuls-sur-Mer. The authors would also like to acknowledge the assistance of Guillaume Mitta and Delphine Destoumieux-Garzon from IHPE laboratory on the acidic peptide extraction.

Conflicts of Interest: "The authors declare no conflict of interest."