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ORIGINAL RESEARCHpublished: 20 September 2016

doi: 10.3389/fmicb.2016.01473

Edited by:Hongyue Dang,

Xiamen University, China

Reviewed by:Melissa Garren,

California State University, USAMarie-Agnès Travers,

French Research Institutefor Exploitation of the Sea, France

*Correspondence:Rodrigo Rojasrrojas@ucn.cl

Jaime Romerojromero@inta.uchile.cl

Specialty section:This article was submitted to

Aquatic Microbiology,a section of the journal

Frontiers in Microbiology

Received: 23 June 2016Accepted: 05 September 2016Published: 20 September 2016

Citation:Rojas R, Miranda CD, Santander J

and Romero J (2016) First Reportof Vibrio tubiashii Associated with

a Massive Larval Mortality Event in aCommercial Hatchery of ScallopArgopecten purpuratus in Chile.

Front. Microbiol. 7:1473.doi: 10.3389/fmicb.2016.01473

First Report of Vibrio tubiashiiAssociated with a Massive LarvalMortality Event in a CommercialHatchery of Scallop Argopectenpurpuratus in ChileRodrigo Rojas1,2,3*, Claudio D. Miranda2,3, Javier Santander4 and Jaime Romero1,3*

1 Laboratorio de Biotecnología, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Chile,2 Laboratorio de Patobiología Acuática, Departamento de Acuicultura, Universidad Católica del Norte, Coquimbo, Chile,3 Centro Aquapacífico, Coquimbo, Chile, 4 Laboratorio de Patogénesis Microbiana y Vacunación, Facultad de Ciencias,Universidad Mayor, Santiago, Chile

The VPAP30 strain was isolated as the highly predominant bacteria from an episodeof massive larval mortality occurring in a commercial culture of the Chilean scallopArgopecten purpuratus. The main aims of this study were, to characterize and identifythe pathogenic strain using biochemical and molecular methods to demonstrate itspathogenic activity on scallop larvae, to characterize its pathogenic properties and todescribe the chronology of this pathology. The pathogenic strain was identified as Vibriotubiashii based on its phenotypic properties and the sequence analysis of its 16S rRNAand housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA). Whentriplicate cultures of healthy 10–day–old scallop larvae were challenged with 1 × 105

colony forming units (CFU) mL−1 of the VPAP30 strain, percentages of larval survivalof 78.87 ± 3.33%, 34.32 ± 4.94%, and 0% were observed at 12, 24, and 36 h,respectively; whereas uninfected larval cultures showed survival rates of 97.4 ± 1.24%after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30strain include the accumulation of bacteria around the scallop larvae, velum disruptionand necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and48 h was 1.3 × 104 and 1.2 × 103 CFU mL−1, respectively. The invasive pathogenicactivity of the VPAP30 strain was investigated with staining of the bacterial pathogenwith 5-DTAF and analyzing bacterial invasion using epifluorescence, and a completebacterial dissemination inside the larvae at 24 h post-infection was observed. Whenscallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30,the larval survival rate was 59.5 ± 1.66%, significantly (P < 0.001) lower than thecontrol group (97.4 ± 1.20%) whereas larvae treated with heat-treated ECPs exhibiteda survival rate of 61.6 ± 1.84% after 48 h of exposure. This is the first report of theisolation of V. tubiashii from the diseased larvae of the scallop A. purpuratus, occurringin a commercial culture in Chile, and it was demonstrated that the VPAP30 strain exhibitshigh pathogenic activity on scallop larvae, mediated both by bacterial invasion and theproduction of toxigenic heat-stable compounds.

Keywords: Vibrio tubiashii, Scallop larvae, Vibriosis, Shellfish pathology, Argopecten purpuratus

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INTRODUCTION

The culture of the Chilean scallop Argopecten purpuratus(Lamarck, 1819) is the second most important industry inChilean mariculture and is primarily concentrated in the northregion of the country (von Brand et al., 2006); however,recurrent episodes of larval mortalities mainly due to bacterialinfections have been observed, causing high economical lossesand consequently precluding the sustainability of this industry.These bacterial infections are commonly characterized by asudden cessation of larval motility leading to massive mortalitiesof reared larvae. Previous studies demonstrated the pathogenicactivity of bacterial strains identified as Halomonas sp. (Rojaset al., 2009), Vibrio anguillarum-related (Riquelme et al., 1995),Vibrio splendidus (Rojas et al., 2015a) and the association ofAeromonas hydrophila and Vibrio alginolyticus (Riquelme et al.,1996) on scallop larvae.

Vibrio species have been described worldwide as the mainaetiological agents of bacterial pathologies affecting reared larvaeof various shellfish species (Waechter et al., 2002; Anguiano-Beltrán et al., 2004; Estes et al., 2004; Gay et al., 2004; Gómez-León et al., 2005; Prado et al., 2005, 2014, 2015; Labreucheet al., 2006; Garnier et al., 2007; Rojas et al., 2015a; Dubertet al., 2016b). Clinical symptoms commonly exhibited by rearedshellfish larvae affected by vibriosis include the reduction of larvalmotility, erratic swimming, closing of valves, velum detachment,and bacterial swarming inside and around the larvae (Pradoet al., 2005; Beaz-Hidalgo et al., 2010; Rojas et al., 2015a). Mostof these clinical signs were described in larval cultures of theclam species Ruditapes decussatus (Gómez-León et al., 2005) andRuditapes philippinarum (Dubert et al., 2016a), oyster speciesCrassostrea virginica (Gómez-León et al., 2008) and Crassostreagigas (Estes et al., 2004; Gay et al., 2004; Garnier et al., 2007;Elston et al., 2008), and scallop species Pecten maximus (Nicolaset al., 1996; Torkildsen et al., 2005), Argopecten ventricosus(Sainz et al., 1998; Luna-González et al., 2002) and Patinopectenyessoensis (Liu et al., 2013). The pathogenicity of Vibrio strainscausing vibriosis outbreaks is mediated by bacterial invasion(Rojas et al., 2015a; Dubert et al., 2016a) as well as the productionof toxigenic extracellular products (ECPs) (Elston and Leibovitz,1980; Labreuche et al., 2006; Binesse et al., 2008; Hasegawa et al.,2008; Labreuche et al., 2010; Rojas et al., 2015a).

Vibrio tubiashii was recognized 50 years ago as an importantpathogen of hard clam and oyster larvae (Tubiash et al., 1965,1970), causing the pathology “bacillary necrosis”, characterizedby disruption and loss of cilia of the larval velar apparatus,high bacterial colonization of the larval shell and mantle, andabnormal swimming behavior. Later, Elston et al. (2008) reporteda re-emergence of vibriosis episodes caused by V. tubiashii ina shellfish hatchery in North America producing an importantloss of the intensive production of Pacific (Crassostrea gigas) andKumamoto (C. sikamea) oysters and the geoduck clam Panopeabrupta. However, this may be controversial because more recentstudies re-classified (Wilson et al., 2013; Richards et al., 2014)some of these pathogenic strains as V. coralliilyticus, as well asother bivalve pathogenic Vibrio strains previously identified asV. tubiashii (Hada et al., 1984; Estes et al., 2004), demonstrating

a high genomic similarity between both species (Ben-Haim et al.,2003; Ushijima et al., 2014).

Despite that efficient rearing techniques for scallop larvaeproduction that have been developed, Chilean commercialhatcheries are currently suffering recurrent episodes of highmortalities of reared larvae, mainly associated with high levelsof vibrio (Miranda et al., 2014; Rojas et al., 2015a). Theidentification of bacterial strains causing epizootics in larvalcultures and understanding their pathogenic activity are essentialfor the development of adequate and efficient protocols of larvalmanagement, as well as for implementing proper bacteriologicmonitoring strategies to prevent and control bacterial outbreaksoccurring in commercial hatcheries of scallop larvae.

Considering that knowledge of the identity and pathogenicmechanisms of bacterial pathogens causing massive mortalitiesof scallop larvae reared in commercial hatcheries in Chileremains scarce, the aims of this study were to characterize andidentify a highly pathogenic Vibrio strain recovered from massivelarval mortality event that occurred in a commercial hatchery,to characterize its pathogenic properties and to describe thechronology of the pathology.

MATERIALS AND METHODS

Bacterial IsolationThe pathogenic strain VPAP30 was recovered from a massivemortality event of reared-larvae of the scallop A. purpuratusoccurring in a commercial hatchery located in Tongoy Bay in thenorth of Chile. Triplicate samples of settled dead and moribundlarvae were aseptically collected from the bottom of the rearingtank during its water exchange using a sterile glass flask and weretransported to the laboratory for immediate processing. Larvalsamples were centrifuged at 960 g for 2 min using an EppendorfModel 5415D centrifuge (Hamburg, Germany) and the waterexcess was discarded. Settled larvae were ground by hand usinga sterile glass digester containing 2 mL of sterile physiologicalsaline (0.85% NaCl; PS) to obtain a homogenate according to themethod of Nicolas et al. (1996). The homogenate was inoculatedin triplicate onto Tryptic Soy Agar (Difco, NJ, USA) with 2% ofNaCl (Oxoid, Hants, UK) (TSA2), and plates were incubated at20◦C for 48 h. The predominant colony grown almost as a pureculture in plates seeded with triplicate larval samples was isolatedusing TSA2 and the bacterial strain was preserved at −85◦C inCryoBank (Mast Diagnostic, Merseyside, UK) vials prior use.

Biochemical and PhysiologicalCharacterizationThe phenotypic characteristics suggested by Noguerolaand Blanch (2008) to identify Vibrio species, including cellmorphology, Gram stain, oxidation/fermentation of glucose,and resistance to the vibriostatic agent O129 (2,4-diamino-6,7-diisopropylpteridine) (10 and 150 µg per disk) were determinedaccording to the protocols described in Barrow and Feltham(1993). In addition, other phenotypic properties of VPAP30strain were determined. Production of luminescence wasdetected in absence of light by using Marine agar 2216, whereas

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β-haemolysis of red cells was determined by using ColumbiaBlood agar (Oxoid, Hants, UK). Production of Møller’s lysine andornithine decarboxylases and Thornley’s arginine dihydrolasewere detected according to Hansen and Sörheim (1991). Growthat 4, 20, 30, 35, and 40◦C was tested on Tryptic Soy brothsupplemented with 2% NaCl (TSB2) and growth at 0, 3, 6,8, and 10% of NaCl was assayed using peptone broth (BD,Sparks, USA). Additional phenotypic characteristics of VPAP30strain were determined by using the API 20E (bioMérieux,Marcy-l’Etoile, France) and the Biolog (Biolog Inc., Hayward,CA, USA) systems. For the API system the VPAP30 strain wasinoculated according to the manufacturer’s instructions with themodifications suggested by MacDonnell et al. (1982) and the APIstrip was incubated at 20◦C for 48 h. For the Biolog system thestrain was inoculated by using a solution containing 2.5% NaCl,0.8% MgCl2, and 0.05% KCl, according to the instructions ofthe manufacturer and the microplate was aerobically incubatedin the dark at 20◦C for 72 h. For the API 20E and Biologmulti-inoculation tests, readings were made after 48 and 72 h ofincubation, respectively.

Enzymatic AnalysisThe enzymatic activities of VPAP30 strain were determined byusing the API ZYM system (bioMérieux, Marcy-l’Etoile, France)according to the manufacturer’s guidelines. Briefly, VPAP30strain was cultured overnight in TSB2, centrifuged at 4,200 gat 4◦C and resuspended in a NaCl 0.85% solution (bioMérieux,Marcy-l’Etoile, France) to obtain a turbidity of 5 McFarland(1.5 × 109 bacteria mL−1), and 65 µL of this suspension wereadded to each cupule. Test strips were incubated for 4 h at20◦C and following incubation, 1 drop of ZYM A (API; tris-hydroxymethyl-aminomethane, hydrochloric acid, sodium laurelsulfate, H2O) and ZYM B (API; fast blue BB, 2-methoxyethanol)were added to each cupule. Test strips were read after 5 minand the results were scored using the following classification: 0,negative reaction; 1−2 weak activity; 3−5 strong activity. Theassay was performed twice to ensure reproducibility.

Molecular AnalysisDNA was extracted and purified from a pure culture usingthe Wizard Genomic DNA Purification Kit (Promega, Madison,WI, USA). PCR was performed as described in Romero andNavarrete (2006) with a reaction mixture (30 µL) containing0.25 mM of each deoxynucleoside triphosphate, 0.05 U µL−1

Platinum Taq DNA polymerase (Invitrogen, San Diego, CA,USA), 1 × polymerase reaction buffer, 2 mM MgCl2, and0.25 pmol µL−1 of each primer. To identify the bacterialstrain, amplification of the 16S rRNA gene from positions 28 to1,492 was performed using the primer pair 27F and 1492R aspreviously described (Navarrete et al., 2010). The housekeepinggenes encoding for cell-division protein (ftsZ), glyceraldehyde-3-phosphate dehydrogenase (gapA), gyrase beta subunit (gyrB),rod shape-determining protein (mreB), uridine monophosphatekinase (pyrH), recombinase A (recA), RNA polymerase alphasubunit (rpoA) and topoisomerase I (topA) were used to performa Multilocus Sequence Analysis (MLSA). Amplification of thegenes was performed as previously described (Sawabe et al.,

2013). PCR mixtures were identical to those previously usedfor the 16S rRNA gene, and the specific primers are listed inSupplementary Table S1. The thermal program consisted of 5 minat 95◦C, 25 cycles of 30 s at 95◦C, 30 s at 55◦C, and 30 s at72◦C, and a final 5 min extension at 72◦C. All PCR productswere verified as described in Romero et al. (2002). PCR productswere sequenced and analyzed as described in Romero et al.(2002) using the Ribosomal Database Project II (Cole et al.,2007) or were compared to those available in the National Centerfor Biotechnology Information (NCBI) Reference Sequencedatabase by using a BLAST search to ascertain their closestrelatives. Sequences were identical to those reported in thegenome sequence LBLS00000000 and they were included as aSupplementary Data Sheet 1. Phylogenetic analysis based on theindividual and concatenated sequences were performed usingthe MEGA 6.0 software, after multiple alignments of data byClustalW tool (BioEdit software). Distances and clustering withthe Neighbour Joining (NJ), Maximum Likelihood (ML) andMaximum Parsimony (MP) algorithms were determined usingbootstrap values based on 1,000 replications.

Chronology of the PathologyThe lethality and pathogenic activity time course for the VPAP30strain were studied using an in vitro challenge assay. Healthy10 day-old scallop larvae were added to each well of a 12-welltissue culture plate (Orange Scientific, Braine-l’Alleud, Belgium)containing 4 mL of 0.22 µm-filter sterilized seawater to obtain afinal concentration of 20 larvae mL−1 and were challenged witha final approximate concentration of 8.0 ± 1.0 × 105 colonyforming units (CFU) mL−1 of the VPAP30 strain. The pathogenVibrio pectenicida A365 (Lambert et al., 1998) was included as apositive control using identical conditions. Plates were incubatedat 18◦C for 48 h in the dark. The proportion of live and deadlarvae was determined at 6, 12, 18, 24, 30, 36, 42, and 48 h, andthe symptoms of the pathology were recorded using the invertedmicroscope Olympus, Model CKX41 (Tokyo, Japan). Larvae wereconsidered dead when no movement was observed within thevalves. Larvae not inoculated with bacteria were use as negativecontrol. The pathogenic activity of the V. tubiashii VPAP30 strainon scallop larvae was demonstrated by reisolating the VPAP30strain from moribund experimentally infected larvae, therebyfulfilling Koch’s postulates.

Estimation of LD50 (50% Lethal Dose)The virulence of the VPAP30 strain was estimated bydetermining its 50% lethal dose (LD50) values after 24 and48 h of exposure, according to Reed and Muench (1938). TheLD50 was defined as the dose of the VPAP30 strain required to kill50% of infected scallop larvae. The VPAP30 strain was tested forits pathogenicity in triplicate using 12-well tissue culture plates(Orange Scientific, Braine-l’Alleud, Belgium). Scallop larvaewere added to each well of the tissue culture plate containing4 mL of 0.22 µm-filter sterilized seawater at a concentrationof 20 larvae mL−1, and the VPAP30 strain was added to thewells to obtain final concentrations of 1.37 ± 0.43 × 102,1.37 ± 0.43 × 103, 1.37 ± 0.43 × 104, and 1.37 ± 0.43 × 105

CFU ml−1, using six wells per plate for each concentration. The

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inverted microscope Olympus, Model CKX41 (Tokyo, Japan)was used to determine the numbers of live and dead larvaeat 24 and 48 h post-inoculation. A group of larvae were alsoinoculated with filtered seawater and considered the negativecontrol.

Pathogenic Activity of ExtracellularProducts (ECPs)The ECPs produced by the VPAP30 and V. pectenicida A365strains were obtained using the cellophane overlay plate method(Liu et al., 2001). Briefly, a volume of 0.2 mL of a 36 h cultureof each bacterial strain grown in TSB2 was spread onto sterilecellophane films placed onto TSA2 plates and incubated at 20◦Cfor 36 h. Cellophane overlays were transferred to empty Petridishes and bacterial cells were washed off from the cellophanesheet using phosphate buffered saline (PBS, pH 7.4) and removedby centrifugation at 13,250 g for 20 min at 4◦C. Supernatantswere sterilized by filtration through a 0.22 µm filter (SartoriousStedim Biotech, Germany) and stored at −85◦C until use. Totalprotein concentrations of supernatants were measured using thePierce BCA Protein Assay Kit (Thermo Scientific, Rockford,USA) and were read at 562 nm using a Microplate Reader AsysUVM 340 (biochrom, Cambridge, United Kingdom). Ten day-old scallop larvae were added at a concentration of 20 larvaemL−1 to each well of a 12-well microplate (Orange Scientific,Braine-l’Alleud, Belgium) containing 3.8 mL of microfilteredseawater and then inoculated in triplicate with 0.2 mL of thecell-free supernatant to obtain a final concentration of 4 µgprotein mL−1. Larval cultures inoculated with 0.2 mL of PBSwere used as controls. Microplates were incubated at 18◦Cfor 48 h in the dark and the proportion of dead larvae wasdetermined at 12, 24, 36, and 48 h using the inverted microscopeOlympus model CKX41 (Tokyo, Japan). In addition, supernatantsamples of V. tubiashii VPAP30 and V. pectenicida A365 strainswere heated at 125◦C for 15 min, and the pathogenic activityof treated supernatants was assayed in triplicate, as previouslydescribed.

Invasive Pathogenic ActivityThe methodology of Sherr et al. (1987) to label bacteria with5-([4,6-dichlorotriazin-2-yl]amino) fluorescein hydrochloride(5-DTAF, Sigma–Aldrich, D-0531, St. Louis, MI, USA) wasmodified to obtain the best labeling conditions for the Vibriostrain. The pathogenic strain was cultured in TSB2 (Difco) at20◦C for 24 h with shaking at 100 rpm using an orbital shaker(WiseShake SHO- 2D, Daihan Scientific, Gangwon-do, Korea).The broth was centrifuged at 5,725 g for 8 min, then thebacterial pellet was resuspended in 10 mL of sterile seawaterand the optical density was adjusted to 0.8–1.3 at 610 nmin a spectrophotometer (PG Instruments T70, Leicestershire,UK) under aseptic conditions. The 5-DTAF was dissolved in0.22 µm-filtered PBS (pH 7.4) to obtain a final concentrationof 0.5 mg mL−1. A 0.5 mL aliquot of the 5-DTAF solutionwas added to 9.5 mL of the bacterial suspension, and themixture was incubated at 20◦C for 1 h in total darkness withshaking at 90 rpm. After incubation, the bacterial culture was

pelleted by centrifugation (5,725 g for 6 min) and resuspendedin 0.22 µm-filtered seawater and the procedure was repeateduntil an unstained suspension was observed. Healthy 10 day-oldscallop larvae of A. purpuratus maintained in 12-well microplates(Orange Scientific, Braine-l’Alleud, Belgium) at a density of20 larvae mL−1 were inoculated in triplicate with the stainedVPAP30 strain to obtain a final concentration of 1 × 105 CFUmL−1 and were observed at 0.5, 1, 4, 6, 12, 18, and 24 husing the Nikon fluorescence microscope Eclipse 50i. Bacterialconcentrations were confirmed by a standard dilution platingtechnique as previously described (Rojas et al., 2015a). Larvalcultures inoculated with unstained pathogenic strain as wellas larval cultures not inoculated with the assayed strain wereincluded as controls. The bioassay was performed twice toconfirm reproducibility.

Production of Virulence FactorsProduction of the virulence factors caseinase, gelatinase, lipase,β-haemolysin, and phospholipase were determined as describedby Natrah et al. (2011). For the lipase and phospholipaseassays, marine agar 2216 (Difco, NJ, USA) (MA) plates weresupplemented with 1% Tween 80 (Sigma-Aldrich, St. Louis,MO, USA) or 1% egg yolk emulsion (Oxoid, Hants, UK),respectively. The development of opalescent zones around thecolonies after 2 days of incubation at 20◦C was considereda positive result. The caseinase assay plate was prepared bymixing double strength MA with a 4% skim milk powdersuspension (Oxoid, Hants, UK), and sterilized separately at121◦C for 5 min. Clearing zones around the bacterial coloniesgrown after 2 days of incubation at 25◦C were considereda positive result. Gelatinase assay plates were prepared bymixing 0.5% gelatine (Sigma−Aldrich, St. Louis, MO, USA)into MA. After incubation for 4 days, saturated ammoniumsulfate (80%) in distilled water was poured over the plates andafter 2 min, clearing zones around the colonies were considereda positive result. β-haemolytic activity was determined usingColumbia Blood agar (Oxoid, Hants, UK), and clearing of theagar around the colony after 2 days of incubation at 25◦Cwas considered a positive result. All assays were performed intriplicate.

Statistical AnalysisLarval survival percentages were transformed to arcsin (squareroot [survival rate ration]) and were compared using one-wayANOVA. When overall differences were significant, a posterioriTukey’s multiple range test was used to determine significantdifferences (P < 0.05). Furthermore, the log-rank test was used tocompare the survival rates of larval groups not infected, infectedwith V. tubiashii VPAP30 and infected with V. pectenicida A365using the Kaplan-Meier procedure. All statistical analyses wereperformed using SigmaStat 3.1 (Systat Software Inc.).

Biological Safety ProceduresAll material contaminated with microorganisms, as well asall used bacterial cultures were discarded after sterilization byautoclaving.

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RESULTS

Phenotypic CharacterizationThe pathogenic strain showed phenotypic propertiescharacteristic of representatives of the genus Vibrio (Thompsonet al., 2004). The VPAP30 strain was a Gram-negative, motileshort rod, producer of oxidase and catalase, susceptible to O/129and unable to grow in the absence of NaCl (Table 1). TheVPAP30 strain was able to produce arginine dihydrolase, indoleand gelatinase, acid, and gas from glucose, and degradationof amygdalin, whereas it was unable to produce the enzymestryptophan deaminase, lysine decarboxylase, and ornithinedecarboxylase, as well as acetoin, H2S from glucose, andacid from the sugars arabinose, inositol, mannitol, mannose,melibiose, rhamnose, and sorbitol. Additionally, the VPAP30strain was positive for citrate production, growth on TCBSmedium and acid production from sucrose, whereas it wasunable to grow at 4, 35 and 40◦C. The API ZYM profile of theVPAP30 strain is presented in Table 2 showing the capacity toproduce the enzymes alkaline phosphatase, leucine arylamidase,trypsin, and naphthol-AS-BI-phosphohydrolase, as well as weakproduction of valine arylamidase.

Further phenotypical characterization by using the Biologsystem, demonstrated that VPAP30 strain was able to use asa sole carbon source, dextrin, glycogen, tween 40, tween 80,N-acetil-D-glucosamine, D-cellobiose, D-fructose, D-galactose,α-D-glucose, maltose, D-mannose, D-melibiose, sucrose, aceticacid, β-hydroxy butyric acid, α-keto butyric acid, D,L-lactic acid,succinic acid, bromo succinic acid, L-alanine, L-alanyl glycine,L-asparagine, L-aspartic acid, L-glutamic acid, glycyl-L-asparticacid, glycyl-L-glutamic acid, L-histidine, L-ornithine, L-proline,D-serine, L-threonine, inosine, uridine, thymidine, and glycerol(Supplementary Table S2).

Identification by 16S rRNA Gene andMLSA AnalysisMolecular classification of the VPAP30 strain was determined by16S rDNA sequence analysis (1,350 bp) as shown (SupplementaryData Sheet 1). The phylogenetic tree constructed fromevolutionary distances of 15 representative strains using theneighbor-joining method is shown in Figure 1, showing thatthe VPAP30 strain was close to V. orientalis (98.4% of identity).Furthermore, the 16S rRNA sequence was aligned with referencesequences using the Sequence Match tool from the RibosomalDatabase Project II (RDP II) website, indicating that the closestrelative for the VPAP30 strain corresponded to Vibrio sp.(Accession number HF568951) with a 99.4% similarity.

For a more accurate identification of the VPAP30 strain, theMLSA scheme was designed and eight genes encoding varioushousekeeping proteins associated with different functions (ftsZ,gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) were used (Sawabeet al., 2013). Partial sequences of the eight loci were comparedto 15 loci of the Vibrio strains previously studied by Prado et al.(2015) as well as the Vibrio coralliilyticus RE98 strain isolatedfrom diseased oyster larvae (Richards et al., 2014), and previouslymiss-classified as V. tubiashii (Elston et al., 2008). The resulting

TABLE 1 | Phenotypic characteristics of Vibrio tubiashii VPAP30.

Characteristic Characteristic

Morphology Rod Citrate +

Motility + Gelatinase production +

Gram stain − Gas from glucose +

Growth on TCBS + Indole production +

O/F Glucose F Reduction of NO3 to NO2 +

Oxidase + β-galactosidase (ONPG) −

Catalase + Swarming on solid media −

Arginine dihydrolase + Urease −

Luminiscence − Voges – Proskauer −

Lysine decarboxylase − Acid from:

Ornithine decarboxylase − Arabinose −

Growth at 0% NaCl − Inositol −

Growth at 3% NaCl + Manitol −

Growth at 6% NaCl + D – mannose −

Growth at 8% NaCl − Melibiose −

Growth at 10% NaCl − Rhamnose −

Growth at 4◦C − Sorbitol −

Growth at 20◦C + Sucrose +

Growth at 30◦C + Susceptibility to:

Growth at 35◦C − O/129 (10 µg) +

Growth at 40◦C − O/129 (150 µg) +

F, Fermentative; O/F, oxidation-fermentation; ONPG, o-nitrophenyl-β-galactosidase.

TABLE 2 | Enzymatic properties of V. tubiashii VPAP30 strain by using theAPI ZYM system (Biomerieux).

Activity

Control Negative

Alkaline phosphatase Strong

Esterase (C4) Negative

Esterase lipase (C8) Negative

Lipase (C14) Negative

Leucine arylamidase Strong

Valine arylamidase Weak

Cystine arylamidase Negative

Trypsin Strong

α-Chymotrypsin Negative

Acid Phosphatase Negative

Naphthol-AS-BI-Phosphohydrolase Strong

α-Galactosidase Negative

β-Galactosidase Negative

β-Glucoronidase Negative

α-Glucosidase Negative

β-Glucosidase Negative

N-Acetyl-β-glucosaminidase Negative

α-Mannosidase Negative

α-Fucosidase Negative

tree is shown in Figure 2, in which the Orientalis clade, definedby Sawabe et al. (2013), is shown in color. Within this group, theVPAP30 strain was closest to V. tubiashii and clearly separatedfrom the other compared vibrios. Sequence comparisons revealed

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FIGURE 1 | Phylogenetic tree based on 16S rRNA gene sequence constructed by the neighbor-joining method. Photobacterium damselae was used as anoutgroup. Horizontal branch lengths are proportional to evolutionary divergences. Bootstrap values from 1,000 replicates appear next to the corresponding branch.

that the VPAP30 shared an 86.0% identity with V. tubiashiiin the concatenated sequences, whereas V. orientalis was thesecond nearest sequence with 67.9% identity, and only exhibitinga similarity of 60.8% with the compared V. coralliilyticus strain.

Bacterial Pathogenic ActivityPathogenic activity of the VPAP30 strain was demonstratedby infecting healthy scallop larvae with VPAP30 and V.pectenicida A365 strains, demonstrating that both pathogenicstrains produced high levels of larval mortality. However, theVPAP30 strain produced significantly (P < 0.001) higher levels oflarval mortality than those produced by the V. pectenicida strainduring all challenge assays. After 24 h of exposure, larval survivalof larvae challenged with the VPAP30 strain was 34.32 ± 4.94%,significantly (P < 0.001) lower than that observed in larvaechallenged with V. pectenicida A365 (77.85 ± 4.62%) and notchallenged larvae (100%). Larval survival at 36 h post-inoculationwith the VPAP30 strain was 0%, significantly (P < 0.001) lowerthan that observed in larvae challenged with V. pectenicida(61.01 ± 3.57%). Negative control larval exhibited a larvalsurvival of 97.4 ± 1.24% after a period of 48 h (Figure 3). TheLD50 for the VPAP30 strain at 24 and 48 h was 1.3 × 104 and1.2× 103 CFU mL−1, respectively.

The VPAP30 strain produced, on challenged scallop larvae,the classical signs of vibriosis affecting mollusc larvae. Thesesigns were identical to those observed in the larval culturesuffering a vibriosis outbreak that developed in the commercialhatchery when the VPAP30 strain was recovered. The mainclinical symptoms exhibited by larvae infected with the VPAP30

strain were disruption of the velum, ciliary cells detached fromthe velum and necrosis of the digestive gland tissue (Figure 4).Erratic swimming was the first clinical sign and appeared at 6 hpost-infection. At 12 h post-infection, the majority of challengedlarvae showed destruction of the velum and necrosis of thedigestive gland, whereas bacterial swarms around the larvae wereobserved after 24 h.

Invasive Pathogenic ActivityThe VPAP30 strain was efficiently stained with 5-DTAF andfluorescence was maintained for at least 36 h (Figure 5A),permitting the use of stained bacterial cells to visualize theinvasive ability of the pathogenic strain along the time. Thestained VPAP30 strain was detected at a low concentration inthe digestive gland of challenged scallop larvae after 30 minof infection (Figure 5B), increasing to high levels after 1 h ofinfection (Figure 5C). Later, at 24 h post-infection, cells of theVPAP30 strain were detected at high concentrations in all larvaltissues as well as around the larval shells (Figure 5D).

Pathogenic Activity of ECPsWhen scallop larvae were exposed to ECPs produced byVPAP30 and V. pectenicida A365 strains, they exhibited identicalsymptoms to those observed during bacterial challenges. At 12 hpost-inoculation with the ECPs of VPAP30 and V. pectenicidastrains, percentages of larval survival were 88.56 ± 2.67% and90.32 ± 2.47%, respectively. Survival rates of larvae infectedwith VPAP30 and V. pectenicida strains remained at levels notsignificantly different (P < 0.05) until 36 h post-inoculation

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FIGURE 2 | Phylogenetic tree based on concatenated sequences of the housekeeping genes ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topAobtained by the neighbor-joining method. Horizontal branch lengths are proportional to evolutionary divergences. Bootstrap from 1,000 replicates appears nextto the corresponding branch.

FIGURE 3 | Survival of 10-day-old scallop larvae not challenged (control) and challenged with 1 × 105 CFU mL−1 of Vibrio tubiashii VPAP30 andVibrio pectenicida A365. Values are a mean (±SD) of three replicates. Asterisks indicate significant differences.

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FIGURE 4 | Main clinical signs exhibited by Argopecten purpuratus larvae infected with V. tubiashii VPAP30 after 24 h of exposure. (A) Bacterialswarms of bacteria on the margins of the larvae, (B) velum disruption, (C) detachment of ciliary cells of the velum (black arrows), and (D) necrosis of digestive tissueof scallop larvae stained with trypan blue. Scale bars: 30 µm.

(69.06 ± 0.74% and 67.03 ± 5.01%, respectively). However, at48 h post-inoculation, ECPs of V. pectenicida produced a larvalsurvival of 41.90± 5.39%, significantly (P < 0.05) lower than thatproduced by the ECPs of the VPAP30 strain (59.50 ± 1.66%),whereas larval survival of the control group was 97.40 ± 1.20%(Figure 6). When heat-treated ECPs of the VPAP30 strain wereassayed, their pathogenic activity remained present, and treatedlarvae exhibited survival rates of 74.3 ± 2.02% and 61.6 ± 1.84%after 36 and 48 h of exposure, respectively. Otherwise, theenzymatic activity of the untreated and heat-treated ECPs showedthat only naphthol-AS-BI-phosphohydrolase activity remains inthe heat-treated ECPs of V. tubiashii and V. pectenicida strains(Table 3).

DISCUSSION

Several phenotypic tests differentiated the VPAP30 strain fromthe closest related Vibrio species belonging to the Orientalis clade,such as being negative for lysine decarboxylase activity and nogrowth at 8% NaCl. The phenotypic profile of the VPAP30 strainis consistent with the characteristics described for V. tubiashiiATCC 19109 (Hada et al., 1984), except for the lack of acidproduction from melibiose and arabinose. In addition, the onlyphenotypic difference of the VPAP30 strain with the V. tubiashiistrain described by Noguerola and Blanch (2008) is the inabilityto produce the enzyme o-nitrophenyl-β-galactosidase (Table 4).

A phylogenetic study based on analysis of the 16S rRNAgene sequence indicated that the VPAP30 strain formed a cluster

within the genus Vibrio as expected, but an accurate identificationat the species level requires the application of complementarygenomic analyses, such as MLSA (Thompson et al., 2004). Thephylogenetic tree based on concatenated sequences (5,130 bp)of the housekeeping genes ftsZ, gapA, gyrB, mreB, pyrH, recA,rpoA, and topA, confirmed the close relationship between theVPAP30 strain and V. tubiashii species. The MLSA analysisprovided a similarity of 86.0% with V. tubiashii ATCC 10919.Therefore, based on the similarity of concatenated sequences, theidentification of VPAP30 corresponded to a V. tubiashii strain.Otherwise, the genomic similarity of the VPAP30 strain withrepresentative strains belonging to other related clades, such asV. coralliilyticus and V. rotiferianus, was only 60.8 and 66.7%,respectively.

The high concentration of the VPAP30 strain in the deadand moribund scallop larvae samples, recovered almost as apure culture in a non-selective bacteriological medium, the highpathogenic activity on experimentally infected scallop larvae,resemblance of the chronology of the pathology as well as thecapacity to produce the same pathological symptoms exhibitedby the sampled scallop larvae, strongly support the hypothesisthat the mortality event that occurred in the commercialscallop hatchery in Chile was primarily caused by this strain.Notably, the high density and predominance of the colonymorphotype of the VPAP30 strain was not observed in agar platesseeded with rearing tank water and swimming larvae samples.Furthermore, in a similar study, the primary pathogenic roleof 3 V. splendidus strains in hatchery-reared scallop larvae waspreviously demonstrated in a similar manner, fulfilling Koch’s

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FIGURE 5 | Invasive activity of V. tubiashii VPAP30 strain on larvae of scallop A. purpuratus determined by epifluorescence microscopy. Bacterial cellsof V. tubiashii stained with 5-DTAF (A); Stained V. tubiashii in the digestive gland at 30 min post-infection (B); Bacterial cells of V. tubiashii in the digestive gland at1 h post-infection (C); Bacterial cells invading completely the larval body cavity and surrounding shell at 24 h post-infection. (Dg) Digestive gland; (Ve) Velum; (Mo)Mouth. Scale bars: 10 µm (A); 50 µm (B−D).

postulates (Rojas et al., 2015a). These recent studies suggest thatdespite good sanitary conditions and preventive measures, suchas efficient influent water treatment strategies, a high diversityof Vibrio species exhibiting important virulence frequently enterlarval rearing tanks, thus exposing Chilean scallop hatcheries torecurrent vibriosis outbreaks.

This study demonstrated that this highly pathogenic strainidentified as V. tubiashii can produce total mortality in anexperimentally infected healthy scallop larvae culture after aperiod of 36 h. The clinical signs caused by the VPAP30strain resembled those previously described for larval vibriosisoccurring in various bivalve species, such as clams, oysters andscallops (Lacoste et al., 2001; Gómez-León et al., 2005; Torkildsenet al., 2005; Elston et al., 2008; Prado et al., 2014; Rojas et al.,2015a; Dubert et al., 2016a). Furthermore, the LD50 value of theVPAP30 strain was similar to the values reported for three highlypathogenic Vibrio strains pathogenic to Pacific oyster larvae, withapproximately 104 CFU mL−1 and 103 CFU mL−1 after 24 and48 h, respectively (Estes et al., 2004), but it must be noted thatsome of these strains have been re-classified as V. coralliilyticus(Wilson et al., 2013; Richards et al., 2014).

The observed pathogenic activity of the VPAP30 strain isremarkably higher than that produced by other Vibrio species

FIGURE 6 | Survival of 10-day-old scallop larvae not challenged(control) and challenged with extracellular products of V. tubiashiiVPAP30 and V. pectenicida A365. Values are a mean (±SD) of threereplicates. Asterisks indicate significant differences.

pathogenic to mollusc larvae. For example, Yue et al. (2010)estimated a LD50 of ∼ 6 × 106 CFU mL−1 for a Vibrioparahaemolyticus-related strain pathogenic to larvae of Meredix

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TABLE 3 | Comparative enzymatic activities displayed by whole cells and ECPs of V. tubiashii VPAP30 and V. pectenicida A365.

Enzymatic activity V. tubiashii VPAP30 V. pectenecida A365

ECPs Heat-ECPs Cells ECPs Heat-ECPs Cells

Alkaline phosphatase + − + + − +

Leucine arylamidase + − + − − −

Valine arylamidase + − + − − −

Trypsin + − + − − −

Acid phosphatase − − − + − +

Naphthol-AS-BI-phosphohydrolase + + + + + +

N-Acetyl-β-glucosaminidase − − − + − +

TABLE 4 | Comparative phenotypic characteristics of V. tubiashii (VPAP30)and Vibrio species belonging to Orientalis Clade.

Test 1 2 3 4 5 6

Arginine dihydrolase + + d + + +

Lysine decarboxylase − − + − − −

Growth at 0% NaCl − − − − + −

Growth at 8% NaCl − v + + + −

Growth at 4◦C − − + − + −

Growth at 40◦C − − − v − +

Citrate + + + − + +

Voges-Prokauer − − − − + +

Indole production + + + − + +

ONPG − + v + − +

Use of α-Ketoglutarate as cs − − − − ND −

Use of D-Glucosamine as cs − v v ND + +

Use of Lactose as cs − − − − − −

Fermentation of:

Melibiose − v ND ND − −

Arabinose − ND ND ND − +

1, V. tubiashii (this study); 2, V. tubiashii (Noguerola and Blanch, 2008); 3, V.orientalis (CAIM332T); 4, V. sinaloensis (CAIM 797T); 5, V. hepatarius (Thompsonet al., 2003); 6, V. brasiliensis (Thompson et al., 2003). +, positive; −, negative;ND, no data available; d, discrepancies exist; v, variable results; cs, carbon source.

meredix, 100-fold higher than the LD50 value of the VPAP30strain. In another study, V. parahaemolyticus-related straincaused 100% mortality only after 6 days of challenge (Sainzet al., 1998), contrasting with the high virulence exhibited by theVPAP30 strain, which killed all challenged larvae after 36 h ofexposure.

The V. tubiashii VPAP30 strain was able to invade the scalloplarvae entering through the mouth to the digestive systemand colonizing the body cavity and surface of the shell. Theseresults of invasive activity are consistent with Dubert et al.(2016a) for the pathogenic species V. neptunius PP-145.98, V.tubiashii ssp. europaensis CECT 8136T and V. bivalvicida CECT8855T during their pathogenic activity on Manila clam larvae.These authors defined three stages in the infective processcaused by these pathogenic species as follows: (1) during thefirst 2 h of infection, Vibrio strains were filtered by larvalvellum and entered the digestive system through the esophagusand stomach colonizing the digestive gland and intestine; (2)

then, Vibrio strains spread and proliferated to the surroundingorgans in the body cavity (6−8 h post infection), and (3) after14 h of challenge, the body cavity was completely colonizedby Vibrio strains. Interestingly, 5-DTAF-stained cells of theVPAP30 strain maintained their virulence on scallop larvaeproducing high mortality as well as the clinical signs typicalof vibriosis. Therefore, the use of bacterial cell staining with5-DTAF to study the chronology of infections events causedby Vibrio species is highly recommended for mollusc larvaepathologies.

Additionally, the pathogenic activity of the VPAP30 strainmediated by the production of extracellular products (ECPs)was evaluated. Our results demonstrated that cell-free ECPsof the VPAP30 strain are involved in the pathogenic actionon scallop larvae, causing a mortality of 40% of challengedlarvae after 48 h of exposure. The results of this studydemonstrate that the extracellular toxigenic activity exhibitedby this strain is mainly mediated by the production ofheat stable compounds, causing larval necrosis and thedetachment of ciliary cells, consistent with previous reports,which demonstrated that Vibrio strains can produce heat stableciliostatic toxins and proteinases that degrade larval tissue(DiSalvo et al., 1978; Nottage et al., 1989). Furthermore, Traverset al. (2014) demonstrated the toxicity of ECPs produced byFrench V. tubiashii strains on the oyster Crassostrea gigas,producing a mortality of 41% after 2 days of challenge. Inaddition, the enzymatic activities of ECPs released by strainsbelonging to the V. tubiashii ssp. europaensis, V. neptunius,and V. bivalvicida species were determined by Dubert et al.(2016a), primarily describing a protease activity. Riquelmeet al. (1995) reported an episode of larval mortality in rearedlarvae of Argopecten purpuratus identifying the causal agentas V. anguillarum-like and causing 30% mortality at 24 hpost-infection, mainly mediated by the production of anextracellular toxin. Rojas et al. (2015a) isolated 3 pathogenicstrains identified as V. splendidus, which were recoveredfrom different episodes of massive mortalities occurring ina commercial culture of the scallop larvae A. purpuratusin Chile, causing larval mortalities of approximately the80% of challenged larvae at 48 h post-inoculation, andcharacterized by bacterial invasion of larval tissue as well as theproduction of extracellular products toxigenic to A. purpuratuslarvae.

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In contrast to the results obtained by Dubert et al. (2016a),the pathogenic activity of ECPs produced by the VPAP30 strainremained after heat treatment, indicating the activity of thermo-resistant toxins, but only naphthol-AS-Bi-phosphohydrolaseactivity remained in the heat treated ECPs, suggesting thatenzymatic activities detected by the API ZYM system are notinvolved in the pathogenic activity of this strain.

Several proteins are secreted by V. tubiashii strains, includinga low molecular weight ciliostatic toxin, which is a veryimportant virulence factor in shellfish larval vibriosis (Nottageet al., 1989). As previously described, a major trait ofvibriosis is extensive necrosis followed by sudden death,which is consistent with the involvement of proteinases andhaemolysins (Nottage and Birkbeck, 1986), as exhibited byscallop larvae inoculated with ECPs released by the VPAP30strain.

A mollusc larvae pathogenic strain reclassified by Wilsonet al. (2013) as V. coralliilyticus (former V. tubiashii ATCC19105) produces some extracellular compounds, including acytolysin/haemolysin (Kothary et al., 2001) and a protease(Delston et al., 2003). More recently, Mersni-Achour et al.(2015) remarked on the importance of the productionof metalloproteases in the pathogenic activity by this V.coralliilyticus strain (former V. tubiashii ATCC 19105) on oysterlarvae, detecting the production of a metalloprotease encodedby the vtpA gene. It must be noted that a metalloproteaseencoding gene similar to vtpA was detected in the genome of V.tubiashii VPAP30 (Rojas et al., 2015b). Moreover, Hasegawa et al.(2008) demonstrated that metalloprotease inhibitors severelyreduce the toxicity of ECPs produced by two strains recentlyidentified as V. coralliilyticus (Wilson et al., 2013; Richards et al.,2014), but previously miss-identified as V. tubiashii, on Pacificoyster larvae. In addition, the authors reported an inhibitoryactivity on its extracellular haemolysin of the extracellularmetalloprotease produced by these strains (Hasegawa and Häse,2009).

Although various V. coralliilyticus strains were previouslymiss-identified as V. tubiashii, sequence analysis performedon the VPAP30 strain clearly differentiated this strain tothe V. coralliilyticus species, despite the apparent carriageof a metalloprotease-encoding gene similar to the detectedin V. coralliilyticus (Rojas et al., 2015b). Furthermore, manystudies reported V. coralliilyticus as an opportunistic pathogenagainst larvae of various mollusc species, including Greenshellmussel Perna canaliculus, great scallop Pecten maximus andoysters Crassostrea gigas, Crassostrea virginica, and Ostrea edulis(Kesarcodi-Watson et al., 2009, 2012; Genard et al., 2013;Richards et al., 2015), prompting the necessity for adequatedifferentiation between V. tubiashii and V. coralliilyticus specieswhen they are involved in mollusc larval pathologies.

CONCLUSION

We demonstrated for the first time, the pathogenic activity of aV. tubiashii strain recovered from a vibriosis outbreak occurringin a commercial Chilean hatchery on A. purpuratus scallop larvae.Our results demonstrated that this V. tubiashii strain can producetypical clinical signs of vibriosis, and its pathogenicity is mediatedby invasive activity and the production of toxigenic heat-stableextracellular products. In addition, the high pathogenic activity ofthe V. tubiashii VPAP30 strain on experimentally infected scalloplarval cultures producing vibriosis symptoms to those observedduring the massive mortality event in the Chilean commercialhatchery suggest that this strain was the causative agent of themass mortalities occurring in the referred event.

AUTHOR CONTRIBUTIONS

RR designed the study, isolated the bacterial strain, performedall challenge assays, drafted the manuscript, and is thecorresponding author and primary contact during themanuscript submission, review, and publication process. CMcontributed significantly to the design, drafting, revisions, andinterpretation of data. JR designed the study together withRR, supervised the study and advised molecular analysis andinterpretation. JS helped to do and analyze the MLSA analysis.All authors have made intellectual contribution to the work, andapproved it for publication.

FUNDING

This study was financially supported by the Science andTechnology National Council (CONICYT) of Chile by thePostdoctoral Project Grant No. 3150395 and FONDECYT grantNo. 1140734.

ACKNOWLEDGMENTS

The authors thank staff of commercial hatchery Invertec Ostimarfor good collaboration with scallop larval sampling and also aregrateful to Dr. Jean Louis Nicolas for providing the V. pectenicidaA365 strain. The comments and suggestions of the reviewers aregreatly appreciated as they helped to improve the manuscript.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at:http://journal.frontiersin.org/article/10.3389/fmicb.2016.01473

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

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