Molecular Characterization of an Endozoicomonas-Like Organism Causing Infection in the King Scallop (Pecten maximus L.)

Abstract

One of the fastest growing fisheries in the UK is the king scallop (Pecten maximus L.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resembling Rickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from “Candidatus Endonucleobacter bathymodioli” and 95% with Endozoicomonas species. In situ hybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences from Endozoicomonas spp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.IMPORTANCE Molluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of an Endozoicomonas-like organism (ELO) associated with an important commercial scallop species.

Shedding and survival of an intracellular pathogenic Endozoicomonas-like organism infecting king scallop Pecten maximus

“…All animals, except 1 (out of 45), showed large bacterial aggregates within gill epithelial tissue. Two colony types of Gram-negative bacteria, Type I and II, were observed as previously described in Lyme Bay MPA scallops (Cano et al 2018) (Fig. 2A).…”

“…This assay was used in quantifying the bacterial load in animals within the Lyme Bay MPA, outside of reported mortality events. Previous mass mortalities were recorded during the summer months, when average water temperatures reached 16.8°C (Cano et al 2018). It was therefore hypothesised that water temperature could affect ELO infection severity, with the inference that higher temperatures facilitate faster bacterial growth and greater transmission (Braid et al 2005).…”

“…Samples were collected in September and October 2016 and January and April 2017. Animals were observed for gross pathology, and a full suite of tissues was sampled for histo pathology, as previously described (Cano et al 2018). Epithelial aggregate counts were measured in 3 separate areas of gill section, and the average value was used as a semi-quantitative indicator of ELO infection severity (minimal, 0 < x ≤ 2; high, 2 < x ≤ 10; severe, 10 < x).…”

“…The reaction then completed 40 cycles, comprising a denaturation step of 95°C for 15 s and an extension step of 60°C for 1 min. Each sample was run in duplicate alongside a standard curve produced from a fragment of 749 bp of the scallop ELO 16S rRNA gene, amplified using the universal prokaryote 16S primer FD1 (5'-AGA GTT TGA TCC TGG CTC AG-3') (Weisburg et al 1991) and the ELO diagnostic primer IMC-R (5'-CTT CGT TAC CAG AAA CTC TAA GGT C-3') (Cano et al 2018).…”

“…Historically, in the ab sence of molecular analysis, these bacteria have been described as a Rickettsiales-like organism (RLO) (Le Gall et al 1991). However, 16S rRNA sequencing showed 95% sequence similarity with species within the genus Endozoicomonas (Gammaproteobacteria: Oceanospiralles) and were therefore designated as an Endozoicomonas-like organism (ELO) (Cano et al 2018).…”