Cosmopolitan Distribution of Endozoicomonas-Like Organisms and Other Intracellular Microcolonies of Bacteria Causing Infection in Marine Mollusks.

Irene Cano,Stein Mortensen,David Ryder,Isabelle Arzul,Evelyn Collins,Bruno Chollet,Francesca Carella,Lyndsay Christie,Eduardo Bustos,Matthew Green,Ana L Valdivia,Cara L Brosnahan,Stephen W Feist,Ionan Marigómez,Deborah Cheslett,Karin B Lohrmann,Barbara Bodinier,Tobia Pretto,Dolors Furones,Marı́a J Carballal ,Stephen C Webb ,Brian Jones ,Georgia M Ward ,Kevin W Christison ,António Villalba ,Wakeman C Kevin ,Noèlia Carrasco

doi:10.3389/fmicb.2020.577481

Abstract

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia-like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae, the genera Mycoplasma and Francisella, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens.

Highlights

The global production of marine bivalves is in a steady increase, with an estimated 15 million tonnes harvested per year including both mariculture and fisheries (Wijsman et al, 2018)
Inclusions that appeared as spherical microcolonies with densely packed basophilic bacteria-like organisms were interpreted as intracellular microcolonies (IMC)
Of those samples which fell below the lower quartile (9,752 reads per a sample), a majority corresponded with samples treated with formalin (n = 16), though samples treated or prepared in other ways were represented within that group (n = 9)

Summary

Introduction

The global production of marine bivalves is in a steady increase, with an estimated 15 million tonnes harvested per year (average data from 2010 to 2015) including both mariculture and fisheries (Wijsman et al, 2018). Infections related to IMC in economically important marine molluskan species have been reported in Spain, infecting the common cockle Cerastoderma edulis (Carballal et al, 2001), the grooved razor shell Solen marginatus, the pod razor clam Ensis siliqua (Ruiz et al, 2013, 2015) and the Mediterranean mussel Mytilus galloprovincialis (Villalba et al, 1997); in France, infecting the Mediterranean mussel (Comps and Tigé, 1999), the European flat oyster Ostrea edulis (Comps et al, 1977; Comps, 1985b) and the wedge clam Donax trunculus (Comps, 1985a); in Chile, infecting the Chilean mussel Mytilus chilensis (Lohrmann et al, 2019) and the Peruvian scallop Argopecten purpuratus (Lohrmann et al, 2002; Lohrmann, 2009); in Norway, infecting the European flat oyster (Mortensen, 1993); in Italy, infecting the wedge clam (Carella et al, 2019); and in New Zealand, infecting the Pacific oyster Crassostrea gigas and the dredge oyster Ostrea chilensis (Hine, 1997, 2000; Diggles et al, 2002). IMC infections directly or indirectly associated with mortality have been reported in the United States, infecting the sea scallop Placopecten magellanicus (Gulka et al, 1983; Walter et al, 2007), the Pacific razor clam Siliqua patula (Elston, 1986) and the abalone Haliotis rufescens (Moore et al, 2000); in France and the United Kingdom (UK) infecting the king scallop Pecten maximus (Le Gall et al, 1988; Cano et al, 2018); in the Philippines and the Federated States of Micronesia infecting the giant clam Hippopus hippopus (Norton et al, 1993); in China infecting the Suminoe oyster Crassostrea ariakensis and the blood clam Tegillarca granosa (Wu and Pan, 2000; Zhu et al, 2012); in Spain, infecting the banded carpet shell Polititapes rhomboides (Villalba et al, 1999); and in New Zealand infecting the toheroa Paphies ventricosa and the tuatua Paphies subtriangulata (Taylor, 2017a,b)

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Results

Conclusion