First characterization of the parasite Haplosporidium costale in France and development of a real‐time PCR assay for its rapid detection in the Pacific oyster, Crassostrea gigas

Abstract

The Pacific cupped oyster Crassostrea gigas is one of the most "globalized" marine invertebrates and its production is predominant in many parts of the world including Europe. However, it is threatened by mortality events associated with pathogenic microorganisms such as the virus OsHV-1 and the bacteria Vibrio aestuarianus. C. gigas is also a host for protozoan parasites including haplosporidians. In contrast with Haplosporidium nelsoni previously detected in Europe, H. costale was considered exotic although its presence in French oysters was suggested in the 1980s based on ultrastructural examination. Here, a combination of light and transmission electron microscopy, PCR and sequencing allowed characterizing the presence of the parasite in the context of low mortality events which occurred in 2019 in France. Histological observation revealed the presence of uninucleated, plasmodial and spore stages within the connective tissues of some oysters. Ultrastructural features were similar to H. costale ones in particular the presence of axe-shaped haplosporosomes in spore cytoplasms. Three fragments of the genome including partial small subunit rRNA gene, the ITS-1, 5.8S and ITS-2 array and part of the actin gene were successfully sequenced and grouped with H. costale homologous sequences. This is the first time that the presence of H. costale was confirmed in C. gigas in France. Furthermore, a TaqMan real-time PCR assay was developed and validated (DSe = 92.6% (78.2-99.8) and )) to enable the rapid and specific detection of the parasite. The application of the PCR assay on archived samples revealed that the parasite has been present in French oyster populations at least since 2008. Considering the little information available on this parasite, the newly developed TaqMan assay will be very helpful to investigate the temporal and geographic distribution and the life cycle of the parasite in France and more generally in C. gigas geographic range.

Haplosporidium pinnae Parasite Detection in Seawater Samples

“…Alignments with 18S–ITS1–5.8S–ITS2–28S partial sequences of Haplosporidium costale from Crassostrea virginica (KF790901–2 [ 50 ]) and from C. gigas (MZ666374 [ 51 ]), of Haplosporidium sp. from Saccostrea glomerata (KF790894–900 [ 50 ]), and of Haplosporidium littoralis (KJ150289 [ 52 ]) were used to compare and to allocate the start and stop nucleotide locations of rRNAs.…”

Identification and Characterization of Infectious Pathogens Associated with Mass Mortalities of Pacific Oyster (Crassostrea gigas) Cultured in Northern China

“…[ 88 , 89 ], Haplosporidium spp. [ 90 ], and Mikrocytos mackini [ 91 ]. The susceptibility of Pacific oyster to Marteilia refringens , Bonamia ostreae , and Bonamia exitiosa is still undetermined [ 92 , 93 ].…”

Loop-Mediated Isothermal Amplification for the Fast Detection of Bonamia ostreae and Bonamia exitiosa in Flat Oysters

“…Control DNA for B. exitiosa , B. ostreae , and other species of parasites were sourced from the collection of the Ifremer ASIM Laboratory (France). The presence of the parasite on infected molluscs was confirmed by conventional PCR combined with sequencing [ 21 , 39 , 40 ], except for the samples infected with M. mackini and the one co-infected with H. nesloni and H. costale , which were only tested by real-time PCR. However, the status of those two samples was confirmed by the donor laboratory.…”