First description outside Europe of the emergent pathogen Vibrio europaeus in shellfish aquaculture

1 First description outside Europe of the emergent pathogen Vibrio europaeus 1 in shellfish aquaculture 2 3 Rodrigo Rojas1,2, Andrés Blanco-Hortas3, Hannah Kehlet-Delgado4, Alberto Lema5, Claudio 4 D. Miranda1,2, Jaime Romero6, Paulino Martínez3, Juan L. Barja5 and Javier Dubert5* 5 6 1Laboratorio de Patobiología Acuática, Departamento de Acuicultura, Universidad Católica del 7 Norte, Coquimbo, Chile 8 2Centro AquaPacífico, Coquimbo, Chile 9 3Departamento de Genética, Facultad de Veterinaria, Universidad de Santiago de Compostela, 10 Lugo, Spain 11 4Department of Microbiology, Oregon State University, Corvallis, OR, United States 12 5Departamento de Microbiología y Parasitología, CIBUS - Facultad de Biología, Universidad 13 de Santiago de Compostela, Santiago de Compostela, Spain 14 6Laboratorio de Biotecnología, Instituto de Nutrición y Tecnología de los Alimentos, 15 Universidad de Chile, Santiago de Chile, Chile 16 17 DOI: 10.1016/J.JIP.2021.107542 18 https://www.sciencedirect.com/science/article/abs/pii/S0022201121000094?via%3Dihub 19 20 21 Journal: Journal of Invertebrate Pathology (short communication) 22 23 24 *Correspondence to be sent to: 25 [email protected] 26 27 2 Abstract 28 Vibrio europaeus is an emergent pathogen affecting the most important bivalve species reared 29 in Spanish and French hatcheries. Using a genomic approach, we identified V. europaeus 30 outside Europe for the first time from massive larval mortalities of scallop (Argopecten 31 purpuratus) in Chile and from seawater near a shellfish hatchery in the US West Coast. Results 32 show the worldwide spreading and potential impact of V. europaeus for aquaculture; these four 33 countries are among the 10 major producers of mollusks. Pathogenicity of V. europaeus was 34 demonstrated for the first time towards scallop, the second most important species for Chilean 35 mariculture. 36 37 Keywords: Vibrio, virulence, vibriosis, shellfish aquaculture, scallop larvae, hatchery. 38 39 Manuscript 40 Hatcheries constitute an important resource to provide the high number of seed required by 41 shellfish farmers because seed recruitment in wild beds has decreased in recent years. Various 42 factors are involved, such as overfishing, climate change and diseases (da Costa et al., 2020). 43 Vibriosis, caused by some Vibrio species, is the most important bacterial disease affecting the 44 bivalve production process in hatcheries (Dubert et al., 2017a). During recent years V. 45 europaeus has been responsible for mass mortalities in outbreaks that affected different 46 shellfish hatcheries in France and Spain (Dubert et al., 2016, 2017b; Mersni-Achour et al., 2014, 47 2015; Prado et al., 2005, 2015; Saulnier et al., 2010; Travers et al., 2014). 48 Pathogenicity of V. europaeus has been demonstrated by experimental infections over a wide 49 host range. The most important bivalve species reared in the European hatcheries are affected, 50 including Pacific oyster (Crassostreae gigas) and Manila clam (Ruditapes philippinarum), 51 ranked in the top 10 major species produced in world aquaculture (FAO, 2018). V. europaeus 52 is pathogenic to different stages of the life cycle of bivalves such as larvae of oysters (C. gigas 53 3 and Ostrea edulis) and clams (R. decussatus, R. philippinarum, Donax trunculus, Ensis 54 arcuatus and Polititapes romboides), early spat in Manila clam (R. philippinarum) and later 55 spat (juveniles) in Pacific oyster (C. gigas) (Dubert et al., 2016, 2017b; Mersni-Achour et al., 56 2014, 2015; Prado et al. 2005, 2015; Travers et al., 2014). V. europaeus has been also associated 57 with mortalities in important cultured gastropods such as abalones (Haliotis spp.) (Saulnier et 58 al., 2010). 59 In this study, we characterised two bacterial strains isolated from aquaculture environments in 60 Chile and United States, NPI-1 and 071316F, respectively, and the potential impact for shellfish 61 aquaculture was evaluated. The Chilean strain was associated with massive mortality of scallop 62 larvae (Argopecten purpuratus) and we demonstrated its virulence in the original host by 63 experimental infections. 64 NPI-1 was isolated as a predominant strain during an episodic outbreak of disease in which 65 farmers detected high mortalities (70%) in larval cultures of A. purpuratus (veliger larvae; 10 66 days old; 126,80 ± 4,42 µm). The larvae were reared in conical tanks (8000 L) at 18ºC in a 67 commercial hatchery located in Tongoy Bay (Chile) (30°15′27″S 71°29′33″W). 68 Microbiological sampling and bacterial isolation were performed following Rojas et al. (2019). 69 The genome of the strain NPI-1 was sequenced by SNPsaurus using PacBio Sequel II (Pacific 70 Biosciences) and HiSeq 4000 (Illumina) sequencers. Quality control of Illumina reads was 71 performed using Trimmomatic (Bolger et al., 2004), filtering poor quality reads and trimming 72 poor quality bases. Assembly of PacBio reads was performed using de novo long-read 73 assembler Flye (Kolmogorov et al., 2019) and polished by Racon (Vaser et al., 2017). Finally, 74 the resulting assembly was curated by the Illumina reads using Pilon tool (Walker et al., 2014). 75 In a previous study, Gradoville et al. (2018) reported that Netarts Bay (Oregon, United States) 76 (45°24'52"N 123°56'05''W) had a high prevalence of oyster-pathogenic Vibrio spp., such as V. 77 coralliilyticus, especially during the summer and low tide. Strain 071316F was isolated in 78 Netarts Bay in July 2016 from seawater near a shellfish hatchery following the procedures 79 4 described by Kehlet-Delgado et al. (2020) for bacterial isolation. Assembly of the draft genome 80 was obtained from Miseq (Illumina) sequencing. Final genome assemblies were annotated with 81 Rapid Annotations using Subsystems Technology (RAST Server) (Aziz et al., 2008) and 82 deposited at DDBJ/EMBL/GenBank (Table S1). 83 Workflow described by Chun et al. (2018) was followed to classify strains NPI-1 and 071316F 84 at the species level. First, the 16S rDNA sequence was retrieved from genome annotation and 85 used in a 16S-based search on EzBioCloud database (Yoon et al., 2017). Average Nucleotide 86 Identity (ANI) calculations were performed using OrthoANI algorithm and digital DNA-DNA 87 hybridization (dDDH) genomes by means of the Genome-to-Genome Distance Calculator using 88 Formula 2 (identities/HSP length) (Meier-Kolthoff et al., 2013; Lee et al., 2016). An up-to-date 89 bacterial core gene set (UBCG) was used to infer phylogenomic relationship between strains 90 NPI-1 and 071316F and the related taxa using a set of 92 core genes (Na et al., 2018). The 91 pipeline uses Prodigal and hmmsearch for gene finding and identification from genome 92 assemblies, respectively (Eddy, 2011; Hyatt et al., 2010). The individual UBCGs were aligned, 93 concatenated and alignment positions showing more than 50% gap characters were excluded. 94 The final nucleotide alignments were used to infer the maximum likelihood (ML) phylogenetic 95 trees using FastTree with the GTR model by bootstrapping over 1000 replications and Gene 96 Support Indices (GSI) (Price et al., 2010). The phylogenetic tree generated from the 97 concatenated alignment of 92 UBCGs was visualized and edited with MEGA7 (Tamura et al., 98 2013). On the basis of 16S rDNA sequences, V. europaeus and V. bivalvicida, species belonging 99 to the Orientalis clade, were the closest relatives to NPI-1 (99.93% and 98.98%, respectively) 100 and 071316F (100% and 99.00%, respectively) (Table S2). Remaining results were below the 101 98.70% cut-off proposed to delineate bacterial species by Stackebrandt and Ebers (2006) (Table 102 S2). The Orientalis clade has relevant significance for bivalve aquaculture because it includes 103 well-known bivalve pathogens such as V. europaeus, V. bivalvicida and V. tubiashii (Dubert et 104 al., 2017a). 105 5 For genomic analyses, we also sequenced the genome of V. europaeus 07/118 T2 (=CECT 106 8426), a strain responsible of massive mortalities of C. gigas spat in a French hatchery (Travers 107 et al., 2014). We followed the same procedure described for NPI-1 and obtained a fully resolved 108 genome for V. europaeus (Table S1). Genomic comparisons (Table S3) showed that only 109 percentages of OrthoANI (>98%) and dDDH (>84%) compared with NPI-1 or 071316F and V. 110 europaeus were higher than the species-delineating thresholds established for ANI (95~96%) 111 and for dDDH (70%) (Meier-Kolthoff et al., 2013; Richter and Rosselló-Móra, 2009). Genome112 based phylogeny (Figure 1), according with OrthoANI and dDDH results, strongly supported 113 (100 bootstrap) the taxonomic affiliation of strains NPI-1 and 071316F with V. europaeus 114 (Figure 1). For strain 071316F, GSI showed that 79 out of 92 UBCGs supported this 115 concatenated alignment and suggested that possible events of lateral gene transfer occurred in 116 twelve genes (Na et al., 2018). In addition, results based on ANI, dDDH and phylogenomics 117 indicated that the French strain (07/118 T2) was the closest relative to NPI-1 (Figure 1 and 118 Table S3). 119 Virulence of the strain V. europaeus NPI-1 was evaluated in its original host by experimental 120 infections as previously described Rojas et al. (2019). Challenges were performed in 12-well 121 microplates for 60 h (18ºC, dark) and in triplicate (Orange Scientific). Healthy A. purpuratus 122 larvae (122.56±4.86 µm) were inoculated at a final concentration of 1.0×105 CFU mL−1 and 123 wells without bacteria were included as negative controls in all experiments. In addition, 124 bacterial concentration required to kill the 50% of infected scallop larvae (LD50) was calculated 125 with R by fitting the mortality curves to a logistic regression model using MASS package 126 (Venables & Ripley, 2002) and visualized with ggplot2 package (Wickham, 2016). Larvae were 127 checked under inverted microscopy (Nikon Eclipse Ts2) and most of them showed the typical 128 signs of vibriosis after 24 h: erratic swimming was the first clinical sign followed by disruption 129 of the velum, necrosis of the digestive gland and bacterial swarming. NPI-1 infection led to 130 high mortality rates (90.03±6.75%) at 48 h and reached the 95.79±5.95% at the end of the 131 6 experiment in all replicates. Mortalities in the negative controls were lower than 4.61±1.34% 132 (Figure 2A). According to Koch´s postulates, strains were re-isolated from inoculated wells 133 after each challenge. Virulence of the strain NPI-1 was estimated by its LD50: 1.16×105 CFU 134 mL-1 at 24 h and 1.59×102 CFU mL-1 at 48 h (Figure S1). Interestingly, strain NPI-1 was less 135 virulent than the strain V. europaeus 07/118 T2, which produced an LD50 value of 2.3×103 CFU 136 ml−1 after 24 h post-infection towards its original host (C. gigas larvae) (Mersni-Achour et al., 137 2015). 138 Finally, the role of the extracellular products (ECP) released by the strain V. europaeus NPI-1 139 in its original host was also evaluated using the protocol described by Rojas et al. (2019) based 140 on cellophane plate technique. Experimental infections were performed as described above and 141 healthy scallop larvae (125.6 ± 4.84 µm) were inoculated with ECP-cell free supernatant to 142 obtain a final protein concentration of 2, 4 and 8 µg mL-1. Percentage of mortalities were 143 directly related with the ECP concentrations and were 32.86±4.27%, 46.30±4.52% and 144 62.50±6.22% at 60 h, respectively (Figure 2B). As previously observed by Mersni-Achour et 145 al. (2015) for the virulent strain V. europaeus 07/118 T2, even the most enriched fraction of 146 ECP was less toxic to larvae than whole bacterial cells, suggesting that multiple virulence 147 factors are required by V. europaeus to induce a complete infection phenotype. 148 Our results demonstrated the global spread of the emergent pathogen V. europaeus. This is 149 particularly relevant for shellfish aquaculture because V. europaeus has been detected in four 150 of the ten major world producers of marine mollusks, including Spain (#6) and France (#9), and 151 now for the first time Chile (#4) and the US (#8) (FAO, 2018). 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