Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location

Abstract

Mikrocytos mackini is a microcell parasite of Pacific oysters only known to occur on the Pacific coast of North America. It is the only described species in the genus, although a genetically divergent Mikrocytos sp. organism has been reported once in both the Atlantic Ocean and China. We developed methods for sequencing the internal transcribed spacer (ITS) of rDNA for the purpose of characterizing extant diversity within M. mackini throughout its known geographic range, and surveying for other evidence of Mikrocytos sp. organisms. Our specific aims were to examine relatedness of M. mackini among sites to make inferences about its recent evolutionary history, and to provide baseline data for future development of a species-specific molecular detection method. We found a total lack of genetic variation within M. mackini across the complete ITS1-5.8S-ITS2 array in over 70 samples collected throughout its range. We hypothesize that this could be a result of a founder effect if the parasite had been introduced into its known range alongside its host, which was imported from Asia beginning around 1914 to about 1961. We detected a single divergent sequence at a short stretch of 18S that was identical to the Mikrocytos sp. detected elsewhere, which adds to the recent and growing body of evidence that Mikrocytos is much more broadly distributed than the limited range of M. mackini suggests. A 1903 bp section of rDNA from Mikrocytos sp. was generated that contained regions of high divergence from M. mackini (in ITS1 and ITS2) that could be exploited for molecular diagnostics.

Denman Island disease (causative agent Mikrocytos mackini) in a new host, Kumamoto oysters Crassostrea sikamea

“…4) (Farley et al 1988). Whether it is truly endemic only to this region or whether it may have been introduced to this region with Pacific oysters is not known (Abbott et al 2011). In the currently reported case, the discovery of DID in Kumamoto oysters was made during routine surveillance and testing, not as a result of morbidity and mortality reports.…”

“…PCR primers Mm18S_1435F and pro28S-R were used to amplify and sequence a ca. 700 bp product following methods in Abbott et al (2011). This sequence is known to be invariant within M. mackini but is substantially divergent from the only other Mikrocytos species that has been sequenced at this region (Mikrocytos sp.-BC; Abbott et al 2011).…”

“…700 bp product following methods in Abbott et al (2011). This sequence is known to be invariant within M. mackini but is substantially divergent from the only other Mikrocytos species that has been sequenced at this region (Mikrocytos sp.-BC; Abbott et al 2011). Sequence data were viewed, edited, and compared against the published M. mackini sequence (GenBank HM563060) using Sequencher v4.8 (Gene Codes).…”

“…from multiple geographic regions (Gagné et al 2008, Wang et al 2010, Abbott et al 2011. PCR primers Mm18S_1435F and pro28S-R were used to amplify and sequence a ca.…”

“…The causative agent is an amitochondriate protistan parasite of undetermined taxonomic affinity (Abbott et al 2011, cf. Carnegie et al 2003, commonly referred to as a 'microcell' based on its histological presentation.…”