Intracellular bacteria in New Zealand shellfish are identified as Endozoicomonas species

Kaimoana (shellfish, seafood) is an important food source and a significant social and cultural component of many New Zealand communities, especially the indigenous Māori. Over the past decade a decline has been detected in shellfish health and an increase in mortality events around New Zealand. Intracellular bacteria termed Rickettsia -like organisms (RLOs) have been observed in New Zealand bivalve molluscs during shellfish mortality events. Affected bivalves include cockles Austrovenus stutchburyi , ringed dosinia Dosinia anus , green-lipped mussels Perna canaliculus , pipi Paphies australis , toheroa Paphies ventricosa , tuatua Paphies subtriangulata , deepwater tuatua Paphies donacina and scallops Pecten novaezelandiae . RLOs are an informal morphology-based classification of intracellular bacteria, with the exact identification often unknown. Using shellfish collected during mortality events from 2014 to 2019 and apparently healthy samples collected in 2018 and 2019, we aimed to identify RLOs in New Zealand shellfish. Bacterial 16S rRNA gene sequences from RLO-infected shellfish showed >95% identity to published Endozoicomonas species. In situ hybridization confirmed the presence of the sequenced gene in the gill epithelium and digestive epithelium of all study species. A genus-specific quantitative PCR, targeting the 16S rRNA gene was developed to detect Endozoicomonas spp. in shellfish tissue. Prevalence of Endozoicomonas spp. in samples from mortality events and healthy shellfish analysed by quantitative PCR was high. Samples collected from mortality events, however, had a significantly higher load of Endozoicomonas spp. than the healthy samples. These results give us a greater understanding of these intracellular bacteria and their presence in populations of New Zealand shellfish.