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Integrative proteomic analysis of digestive tract glycosidases from the invasive golden apple snail, Pomacea canaliculata Sophia Escobar-Correas, Omar Mendoza-Porras, Federico Dellagnola, Michelle L Colgrave, and Israel A. Vega J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00282 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 22, 2019

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Journal of Proteome Research

INTEGRATIVE PROTEOMIC ANALYSIS OF DIGESTIVE TRACT GLYCOSIDASES FROM THE INVASIVE GOLDEN APPLE SNAIL, Pomacea canaliculata Sophia Escobar-Correasa,b, Omar Mendoza-Porrasc, Federico A. Dellagnolaa,b,d, Michelle L. Colgravec*, Israel A. Vegaa,b,d*

a IHEM,

CONICET, Universidad Nacional de Cuyo, Mendoza, Argentina.

b Universidad

Nacional de Cuyo, Facultad de Ciencias Médicas, Instituto de Fisiología,

Mendoza, Argentina. c CSIRO,

Agriculture & Food, 306 Carmody Road, St. Lucia, Queensland 4067, Australia

d Universidad

Nacional de Cuyo, Facultad de Ciencias Exactas y Naturales, Departamento de

Biología, Mendoza, Argentina.

Authors for correspondence: Michelle Colgrave E-mail: [email protected] Israel A. Vega E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT The freshwater snail Pomacea canaliculata, an invasive species of global significance, possesses a well-developed digestive system and diverse feeding mechanisms enabling the intake of a wide variety of food. The identification of glycosidases in adult snails would increase the understanding of their digestive physiology and potentially generate new opportunities to eradicate and/or control this invasive species. In this study, liquid chromatography coupled to tandem mass spectrometry was applied to define the occurrence, diversity and origin of glycoside hydrolases along the digestive tract of P. canaliculata.. A range of cellulases, hemicellulases, amylases, maltases, fucosidases and galactosidases were identified across the digestive tract. The digestive gland and the contents of the crop and style sac yield a higher diversity of glycosidase-derived peptides. Subsequently, peptides derived from 81 glycosidases (46 proteins from the public database and 35 uniquely from the transcriptome database) that were distributed amongst 13 glycoside hydrolase families were selected and quantified using multiple reaction monitoring mass spectrometry. This study showed a high glycosidase abundance and diversity in the gut contents of P. canaliculata which participate in extracellular digestion of complex dietary carbohydrates. Salivary and digestive glands were the main tissues involved in their synthesis and secretion.

KEYWORDS Ampullariids, Liquid chromatography-mass spectrometry (LC-MS), Digestion, Cellulases, Hemicellulases, Amylases, Maltases, Fucosidases, Galactosidases.


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1. INTRODUCTION Apple snails (Caenogastropoda, Ampullariidae) are naturally distributed in humid tropical and subtropical ecosystems of Africa, America, and Asia1. The International Union for Conservative of Nature and the Invasive Species Specialist Group have included three American apple snails, Pomacea canaliculata, Pomacea maculata, and Marisa cornuarietis, in the list of the top “100 world’s worst invasive alien species”2. Pomacea species have also been introduced anthropogenically in South East Asia, China, Japan, USA, and Europe3-5 where they alter ecosystem dynamics4 affecting the abundance and richness of native species6, and producing economic losses in agricultural industries, in particular of rice7. The invasiveness of P. canaliculata has been associated with different biological features, including their resistance to environmental stressors8, pollutants9, 10, tissue regeneration in adult individuals11, and suitability to reproduce in different environments12,

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Remarkably, this

polyphagous species has diverse feeding mechanisms1, 14 including the intake of a wide variety of food types, such as submerged and emergent macrophytes, biofilms, periphyton and animal carrion15, 16. Additionally, it has a well-developed digestive system specialised for the digestion of complex polymers1, 17, 18. However, knowledge of the mechanisms of carbohydrate digestion by P. canaliculata remains incomplete. Cellulose-digestion enzymes have been associated with the gut microbiome and stomach of this snail19, 20. Endogenous cellulases from glycoside hydrolase families (GHFs) 9, 10 and 45 have been previously reported from Ampullaria crossean in Xiamen, China21-23, and from P. canaliculata collected in Bangkok, Thailand19; A. crossean should be a misidentification of P. canaliculata1. Also, various glycosidase activities (endo-1,4-β-D-xylanase, α- and β-mannosidase, α-fucosidase and cellulase) have been found in whole extracts of P. canaliculata24-26, but there is


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no available information about the spatial and temporal framework where the digestion of complex dietary polymers occurs. The study of the set of digestive enzymes responsible for supplying macronutrients to this snail, may allow the identification of molecular targets for control strategies aiming to stop the spread of this invasive pest. In this study the occurrence, diversity and origin of glycosidases detected along the digestive tract of P. canaliculata were resolved by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Subsequently, the abundance of each enzyme was followed by quantitative proteomics using multiple reaction monitoring mass spectrometry (MRM-MS).

2. MATERIALS AND METHODS

2.1. Snail and culture conditions Adult snails (shell length = 33–46 mm) from a cultured strain of P. canaliculata were used. Room temperature was regulated (24-26 °C) and artificial lighting was provided 14 h per day. Room relative humidity was 80%. The animals were maintained in aquaria containing 6 L of tap water. Aquarium water was changed thrice per week. Unless otherwise indicated, animals were fed ad libitum with lettuce from Monday through Friday. This diet was supplemented with fish food pellets with a 40% of total protein (Peishe Car Shulet, Argentina) on Thursday and with toilet paper on Friday. The original stock of snails was collected at the Rosedal Lake (Palermo, Buenos Aires, Argentina; neotype locality). Animals from the original population and from the laboratory strain have been deposited at the collection of Museo Argentino de Ciencias Naturales (Buenos Aires, Argentina; lots MACN-In 35707 and MACN-In 36046, respectively). Sacrifice procedures and


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sampling were carried out in agreement with the policies of the Institutional Animal Care and Use Committee of the Facultad de Ciencias Médicas of the Universidad Nacional de Cuyo (Approval Protocol N° 55/2015).

2.2. Animal acclimation, sampling, and protein extraction A first experiment was aimed at determining the glycoside hydrolases involved in carbohydrate digestion at different sites along the digestive tract of P. canaliculata. Five sampling sites were collected from four replicate snails: (a) the medial portion of the oesophagus i.e. the crop (Cr), which retains food and salivary gland juices that are originated in the buccal cavity; (b) the final portion of the stomach, i.e. the style sac (SS); (c) the coiled gut (CG), which receives contents of the small intestine; (d) both salivary glands (SG); and (e) the digestive gland (DG). The animals were acclimated to a feeding scheme, which has been described elsewhere18. Briefly, animals were fed ad libitum and then fasted for 24 h. After that, each animal was isolated in a beaker containing 70 mL water with three fish food pellets. Once the first pellet was swallowed, fresh lettuce was added into the beaker. Animals consumed all the food that was given. After 90 min, each animal was immersed in an ice-water bath for 10 min to minimise pain and then the shell was cracked and removed. Autostatic forceps were fixed on the posterior oesophagus to prevent the passage of contents between the crop and the stomach during sampling. The crop and the style sac contents were collected using a 1 mL syringe with an 18-gauge needle and were thoroughly dispersed in Protein Extraction Buffer (PEB = 100 mM Tris-HCl pH 7.4, 10 mM NaCl, 0.25%, Triton X-100). Samples from salivary glands, digestive gland, and coiled gut were obtained and homogenised on ice (five cycles of 15 s) with an Ultraturrax homogeniser in 500 μL of PEB. All samples were centrifuged at 12,350 xg for 15 min at 4°C; the pellets were discarded, and the


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protein concentration of the supernatant was quantified according to Bradford protein assay. Aliquots of 330 μg protein were fixed in 70% ethanol (for shipment to Australia) and stored at −80°C until use.

2.3. Protein preparation and tryptic digestion for mass spectrometry After removing ethanol, the proteins were lyophilised and reconstituted in 165 μL urea buffer (8M Urea, 0.1M Tris-HCl pH 8.5). The samples were vortexed (30 s, five times), and sonicated (2 min; once); these procedures were repeated twice. Proteins were quantified by Bradford protein assay using the manufacturers’ instructions yielding protein concentrations: salivary gland = 1.11.3 μg of protein/μL; crop content = 3.2-9.2 μg of protein/μL; digestive gland = 3.3-9.6 μg of protein/μL; style sac content = 1.6-10.3 μg of protein/μL; coiled gut = 6.4-12.2 μg of protein/μL. Soluble proteins (100 μg) from each sample extract were transferred to a 10 kDa MWCO filter unit (Millipore) and centrifuged at 20,800 xg for 10 min at room temperature. Three technical replicates were prepared from each sample. The retained proteins (>10 kDa) were subsequently reduced, alkylated and digested using a modified version of the filter-assisted sample preparation (FASP) workflow27 that is compatible with reagents (detergents, chaotropes) and downstream analysis by LC-MS/MS. The filters were washed three times with urea buffer and then centrifuged (20,800 xg, 10 min). Cysteine residues were reduced with 100 mM dithiothreitol (DTT) for 60 min at room temperature. Excess DTT was removed by centrifugation (20,800 xg, 10 min). Free sulfhydryl groups were alkylated by incubation with 50 mM iodoacetamide for 20 min at room temperature followed by centrifugation (20,800 xg, 10 min). After reduction and alkylation, each filter unit was washed twice with 200 μL of urea buffer and then twice with 200 μL of 50 mM ammonium


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bicarbonate (AmBic, pH 8.5); each washed was followed by centrifugation (20,800 xg, 10 min). Proteins were digested overnight at 37°C with sequencing grade trypsin (V5111, Promega; protein/trypsin ratio = 50/1) in AmBic (pH 8.5). After digestion, the resulting tryptic peptides (99% confidence, with one or more unique peptides (>95% confidence) using the false discovery rate (FDR) analysis provided within the ProteinPilot software.


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Peptide summaries (Supporting Table 1) generated by ProteinPilot were analysed. Peptides with low peak intensity ( DG > SG > CG). The fucosidases were mainly found in tissues (SG, DG and CG, range 13-15), with the style sac content yielding only two representatives and none


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detected in the crop. The number of galactosidases (range 11-22) were variable between contents and tissues (Cr = DG > SS > CG > SG). A comparison of the tissue-specific expression was undertaken examining unique and shared peptides of the identified glycosidases (Figure 3) in order to avoid issues with protein inference arising from the use of a non-redundant protein database. The crop content showed approximately four times more unique peptides than the style sac content (410 versus 102). The digestive gland showed the highest number of unique peptides compared to salivary gland and coiled gut (165 > 95 > 30). Thirty-nine peptides were shared among digestive contents and tissues of the digestive tract of this snail. Crop and style sac contents had the highest number of peptides uniquely detected (438 peptides) while coiled gut, salivary and digestive glands shared 96 peptides.

3.2. Peptide quantification and glycoside hydrolase families A total of 250 peptides belonging to 81 glycosidases (46 from the public databases and 35 uniquely from the transcriptome database) were selected (Supporting Table 3) for LC-MRM-MS relative quantitation. Figure 4 shows an analysis of the relative percent of each peptide in relation to glycosidase type and their distribution along the digestive tract. Peptides of cellulases and amylases (Figure 4A, C) showed higher values for both contents (crop and style sac) as opposed to the digestive gland. Also, peptides from cellulases and amylases showed lower or null percent values in samples taken from salivary glands and coiled gut. Some peptides of these enzymes (cellulases Pc125214 and A0A2T7PT81; amylases A0A2T7NPM3, A0A2T7PLX3 and Pc109575) showed an idiosyncratic behavior for which lower values in crop and higher values in style sac were found (Supporting Figure 1).


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The distribution of peptides from hemicellulases (Figure 4B), maltases (Figure 4D) and galactosidases (Figure 4F) were more variable across the different samples taken from the P. canaliculata digestive tract. Unexpected findings were the different profiles (Supporting Figure 1) found for peptides of maltases A0A2T7PY79 (VTASSLTK and QNYPVTLDTVR) and A0A2T7NPM8 (QGGVDLPTADSCQK). On the other hand, fucosidases were exclusively found in tissues (Figure 4E), with a significantly higher proportion in the digestive gland compared to the salivary gland and coiled gut. An analysis of amino acid sequence similarities in the carbohydrate-active enzymes database (CAZy)37 showed 13 glycoside hydrolase families. A total of thirty cellulases in three families: sixteen GHF9 (Figure 5A), thirteen GHF10 (Figure 5B) and one GHF45 (Figure 5C), were highly expressed in crop and style sac contents. Nineteen hemicellulases were distributed into six families of glycoside hydrolases: two GHF2 (Figure 5D), five GHF3 (Figure 5E), four GHF5 (Figure 5F), three GHF10 (Figure 5G), three GHF16 (Figure 5H), and two GHF38 (Figure 5I). These GHFs showed different patterns of distribution along the digestive tract of P. canaliculata. One peptide with a different pattern of expression was found in hemicellulase A0A2T7NWR9 of GHF10 (Supporting Figure 1). GHF2 and GHF3 showed higher values in digestive gland and in crop and style sac contents. Specifically, GHF5, GHF10, and GHF16 hemicellulases showed higher relative abundance in the crop and style sac with a pattern similar to that observed for the GHF9, GHF10 and GHF45 cellulases. In contrast, GHF38 hemicellulases were exclusively found in coiled gut and both salivary and digestive glands. GHF13 comprised six amylases (Figure 5J) that were highly expressed in the crop and style sac contents. Three maltases of GHF13 (Figure 5K) were detected in salivary glands and had low percent values in samples taken from digestive contents. GHF31 maltases (Figure 5J) were highly expressed in crop and style sac contents and lower in


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digestive gland, with the exception being the maltase A0A2T7NWB7 expressed at tissue level (SG > DG = CG). Eight galactosidases were grouped within either the GH1 (Figure 5N) and GH35 (Figure 5O) families. The five GHF1 proteins were highly expressed in crop and style sac contents, while the three GHF35 proteins were exclusively expressed in digestive tissues, wherein the expression levels followed the pattern DG > SG > CG. The six monitored fucosidases of the GHF29 (Figure 5M) showed an expression pattern similar to that of the GH35 galactosidases. An analysis of variance for glycoside hydrolase peptides across the digestive contents and tissues from P. canaliculata was undertaken (GLMMs - LSD of Fischer; Supporting Table 6). Statistically significant differences in the variance were often found when GLMMs was run using the individual as the random factor. For some peptides (grey; Supporting Table 6) the differences may also be explained by the individual factor. Generally, all peptides of the same hydrolase showed similar abundance and distribution patterns. Figure 6 shows some examples of each type of glycosidase. For maltase A0A2T7NWB7 and hemicellulase Pc127144, the salivary glands showed a significantly higher abundance than digestive gland and coiled gut (Figure 6A and B, respectively). For fucosidase Pc125661, hemicellulase A0A2T7P4T1, and galactosidase A0A2T7PBX1 (Figure 6C-E), the digestive gland showed often significantly higher peptides abundance than salivary glands and coiled gut, but the fucosidase Pc125661 was not found in either of digestive contents, crop or style sac. Peptides of maltase Pc10364, cellulase A0A2T7PPN2, hemicellulase Pc123712, amylase Pc109575 and galactosidase A0A2T7PCL9 (Figure 6F-J) were mainly found in digestive contents, with the crop content significantly higher than digestive gland.

4. DISCUSSION


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This work reports the diversity, occurrence, and origin of intracellular and extracellular enzymes involved in the digestion of carbohydrates along the digestive tract of the apple snail P. canaliculata, using a proteomic approach and database searching employing a custom database, comprising the public protein database of Molluscan origin (UniProt) and the transcript-derived database of P. canaliculata29. This proteomic approach revealed six different types of glycosidases that are involved in the digestive processes of this invasive snail, i.e. the cellulases, hemicellulases, amylases, maltases, fucosidases and galactosidases.. The digestive gland and contents of the crop and style sac revealed a higher diversity of glycosidase-derived peptides (Figures 2 and 3). Peptides representing 81 glycosidases (46 from the public databases and 35 uniquely from the transcriptome database) distributed across 13 GHFs were selected and quantified in digestive contents and glands of this snail (Figures 4-6). Conserved expression patterns of each type of glycosidase and their families were found in the digestive tract of the apple snail P. canaliculata, despite the high biological variability of some peptides (Supplementary Table 2).

4.1. Diversity, occurrence and origin of glycosidases 4.1.1. Cellulases Cellulose is an energetically-valuable carbohydrate biopolymer found in algae and plant cell walls38. In this work, thirty cellulases from families GHF9, 10 and 45 were found in crop and style sac contents and to a lesser extent within the digestive gland. They were comparatively lower or null in samples taken from salivary glands and coiled gut. These findings indicate that cellulases are mainly originated in the digestive gland and released through their two main ducts into the stomach vestibule and in consequence they are found downstream in the style sac. Cellulases that were found upstream in the crop content can be explained by the absence of a sphincter separating


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the stomach and posterior oesophagus and the backward flux driven by pumping action of the muscular gizzard18. The genome of P.canaliculata has revealed expanded cellulase gene families39, which may give rise to increased gut enzyme versatility found in this study. It has also been shown that cellulose expression is influenced by the feeding state (i.e. satiated or starved) of apple snails40. In addition, cellulases A0A2T7PPN6 (GHF9), A0A2T7NYY1 (GHF10), A0A2T7NZR0 (GHF10) and Pc89752 (GHF10) were uniquely found in the digestive contents indicating a possible origin in unicellular glands of the gut or in commensal symbiotic organisms41. To date, a bacterial cellulase secreted by Bacillus sp. from A. crossean has been characterised at molecular and biochemical levels41, 42. Together, these findings could explain why P. canaliculata is able to survive on an exclusive cellulose diet for 60 days43.

4.1.2. Hemicellulases Hemicelluloses are complex polysaccharides present in the plant diet of apple snails and they are putative sources of diverse monosaccharides (glucose, xylose, mannose, or galactose) bound by different linkages44. In this work, hemicellulases with different sugar-specificities were detected along the digestive tract of P. canaliculata. Almost all hemicellulases (except two mannosidases from GHF38) originated in the digestive gland fulfilling extracellular functions at the crop and stomach levels. Two different quantitative profiles were found in digestive gland: representatives from GHF2 (glucuronidases and mannosidases) and GHF3 (xylosidases) showed higher tissue values than contents while representatives from GHF5 (mannosidases), 10 (xylanases) and 16 (β1,3-glucanases) families showed lower tissues values than contents. It is possible that the digestive gland expressed different hemicellulose isoforms which are associated with the ingested food types or the feeding state of the snails40; however, future research will have to clarify this point.


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Mannosidases from GHF38 were detected in the salivary glands, but these peptides might be overestimated since this tissue showed about 7x less soluble proteins compared to other tissues and the samples were normalized by protein load prior to digestion and analysis. Even though α- or β-mannosidases, and β-xylosidases activities have been reported from viscera of P. canaliculata24-26 and β-1,3-glucanases from digestive fluids of marine gastropods45-47, it is still possible that these enzymes originate in the digestive gland because it is the most voluminous visceral organ43.

4.1.3. Amylases and maltases As expected, amylases and maltases were distributed along the digestive tract of P. canaliculata. These enzymes work together to hydrolyse starch and glycogen arising from the consumption of plants, algae, periphyton and animal carrion, respectively16, 48. In this study, six amylases of the GHF13 (Figure 5J) were highly expressed in the crop and style sac contents indicating that these enzymes are working within the gut extracellular medium. The presence of six amylases may be due to the structure of the amylase gene and their differential tissue expression as was reported in marine bivalves Crassostrea gigas49 and Pecten maximus50. Although the tissue origin of amylases cannot be assigned in this study (Figures 4C-5J), previous research has reported the occurrence of a powerful amylase in the saliva of P. canaliculata17. It is possible that the disaccharides hydrolysis in P. canaliculata begin in the snail mouth since the salivary glands showed a high abundance from peptides of three maltases (A0A2T7NZL2 and A0A2T7PNS7 from GHF13; A0A2T7NWB7 from GHF31). In addition, the maltose released from dietary starch is presumably hydrolysed downstream of the digestive tract since nine maltases (one GHF13 member; eight GHF31 members) were often found in the crop and style sac contents,


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and digestive gland. Amylase, maltase and other glycosidases activities have been reported in the salivary gland from adult individuals of the freshwater gastropod Biomphalaria straminea51. Also, an α-D-glycosidase, with high maltase but null amylase enzymatic activity, was isolated and characterised in the visceral mass from the marine gastropod Aplysia fasciata52; however, the tissue origin was unknown.

4.1.4. Fucosidases Fucosidases are known to occur in various gastropods (Haliotis rufescens53; Charonia lampas54; Turbo cornutus55, Aplysia kurodai56), in the bivalve Chamelea gallina57, and in the cephalopod Octopus vulgaris58. The present study identified six fucosidases of the GHF29 which showed tissue-specific expression that were practically null in the crop and style sac contents (Figure 5M). These results agree with previous reports of fucosidase isoforms from haemocytes, digestive gland (hepatropancreas), and viscera of P. canaliculata24, 59-61. Although the physiological role of these enzymes is still unknown, some hypotheses suggest: (a) that fucosidades were associated with lysosomal compartments, where post-translational modifications of glycoproteins occur61, 62; or (b) that dietary algal polysaccharides with high fucose content (as fucoidan) were absorbed by endocytosis from digestive epithelial cells and then hydrolysed in granular and vacuolar compartments63, 64.

4.1.5. Galactosidases In this study, eight galactosidases from GHF1 (Figure 5N) and GHF35 (Figure 5O) were monitored. Five GHF1 were highly expressed in crop and style sac contents and to a lesser extent in digestive gland. This finding indicates that galactosidases GHF1 would participate in the


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extracellular hydrolysis of galactose-rich dietary polysaccharides deriving from plant cell walls (i.e. galactomannan and galactoglucomannan)65. On the other hand, digestive tissues showed exclusively GHF35 galactosidases (Figure 5O) indicating that they may participate in the metabolism of storage carbohydrates. This is the first report of galactosidases in digestive tissues of an adult ampullariid, even though α- and β-galactosidase activities are known to occur during embryo development of Pomacea sp.66. Also, β-galactosidase activity has been reported in juveniles of the freshwater snail, Biomphalaria glabrata67. This enzymatic distribution appears to be associated with the presence of galactan, a storage carbohydrate composed mainly of galactose, which is produced in the albumen glands and accumulates around the eggs of the spawn of various snails68, 69.

4.2. Conclusions and future work The glycosidase diversity found in the luminal contents along the digestive tract indicates that extracellular digestion of complex carbohydrates from a diet is physiologically important in the apple snail P. canaliculata. Also, the consecutive occurrence of glycosidases in contents and glandular tissues ensure complete digestion of varied carbohydrate sources by this polyphagous snail. The glycosidases were notably lower in both diversity and abundance in the coiled gut of P. canaliculata. They likely originate in the salivary and digestive glands. The understanding of the digestive physiology of P. canaliculata has increased in the last decades18, 19, 70, and recent advances in ‘omics’ technologies can contribute to further elucidate key digestive mechanisms of P. canaliculata20, 29, 39. It has been postulated that biological invasions may be prevented with possible applications of physiological knowledge71. The comprehensive investigation of the enzymes involved in the digestive physiology of P. canaliculata provides a


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foundation for the development of strategies aiming to eradicate and/or control this invasive species.


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FIGURES

Figure 1. A schematic view of workflow. Snail acclimation and sampling have been reported previously (18). After protein extraction of samples, tryptic digestion was performed using a filteraided sample preparation. The peptides obtained were analysed by LC-MS/MS and then identified by searching against a custom database, i.e. public protein database of Mollusca (Uniprot) and transcript database of P. canaliculata (29). The unannotated proteins were searched in BLASTp. After manual removal of redundant proteins, a final protein list was obtained.


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Figure 2. Classification and distribution of glycosidases. Proteins from the global proteomic profiling were classified taking account their functional groups. The distribution along the digestive tract is shown: 1, salivary gland; 2, crop content; 3, digestive gland; 4, style sac content; 5, coiled gut.


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Figure 3. Unique and shared peptides of glycosidases from digestive tract of P.canaliculata. Peptides derived from glycosidases that were identified by LC-MS/MS and then compared among them. Each organ can be differentiated by a unique peptide profile. The value in parentheses indicates the number of peptides found in each sampled tissue/content: SG, salivary gland; Cr, crop content; DG, digestive gland; SS, style sac content; CG, coiled gut.


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Figure 4. Peptide distribution and abundance along the digestive tract. A total of 250 peptides from 81 glycosidases were selected for LC-MRM-MS quantitation. The abundance of each peptide was calculated as the sum of MRM peak areas. This value was then compared to the average of all measurements (i.e. 100% represents the mean area for each peptide along the digestive tract). Those peptides that showed a different pattern within each family are shown in grey.


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Figure 5. Peptide distribution and abundance according to glycoside hydrolase families. Peptides were classified in 13 glycoside hydrolase families (GHFs) based on the amino acid sequences similarities in the carbohydrate-active enzymes database (CAZy; http://www.cazy.org/). The abundance of each peptide was calculated as the sum of MRM peak areas. This value was then compared to the average of all measurements (i.e. 100% represents the mean area for each peptide along the digestive tract). Those peptides that showed a different pattern within each family are shown in grey. The lines and error bars represent the mean ± SD for each tissue/content type.


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Figure 6. Peptide expression patterns of selected glycosidases along the gastrointestinal tract of P. canaliculata. The MRM peak area (mean ±SD) for two peptides from each protein across the digestive tract was evaluated statistically using multiple comparisons. Different letters


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indicate statistically significant differences among groups. The significance level was fixed at p < 0.05.

ASSOCIATED CONTENT Supporting Table 1. Peptide identification metrics. Supporting Table 2. BLASTp results for proteins identified as glycosidases. Supporting Table 3. MRM transitions for peptides monitored by LC-MRM-MS. Supporting Table 4. Co-efficient of variance of peptides monitored in MRM-MS from samples of the digestive tract of P. canaliculata. Supporting Table 5. Occurrence of glycosidase peptides in the digestive tract of P. canaliculata. Supporting Table 6. Quantitation and statistical analysis of 81 glycosidases from P. canaliculata. Supporting Figure 1: Quantitation of peptides showing a different behaviour (grey) along the digestive tract.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by grants from Universidad Nacional de Cuyo; Consejo Nacional de Investigaciones Científicas y Técnicas and Fondo Nacional de Ciencia y Técnica of Argentina.


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ABBREVIATIONS AmBic, Ammonium Bicarbonate; CG, Coiled Gut; Cr, Crop; DG, Digestive Gland; DTT, Dithiothreitol; FDR, false discovery rate; GHF, Glycoside Hydrolase Family; GLMMs, Generalized Linear Mixed Models; LSD, Least Significant Difference; MRM, multiple reaction monitoring; PEB, Protein Extraction Buffer; SG, Salivary Gland; SS, Style Sac.

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