Collect of Amoebophrya parasite (free-living stage) for genomic and transcriptomic analyses v1
Authors: Bigeard Estelle 
Published: December 2018

Amoebophrya (Syndiniales or MALVII, Alveolata) is an eukaryotic parasite which infect and kill its host. Parasitic cultures are therefore particularly rich in bacteria which develop by degrading the organic matter released during the infection. Strains of hosts and parasites come from the Penzé estuary (North-West of France, English Channel, 48°37’N; 3°56’W). A culture of the non-toxic dinoflagellate Scrippsiella acuminata (previously known as S. trochoidea, Kretschmann et al. in 2015) was previously established from the germination of a single cyst collected in 2005 from sediment. A culture of the non-toxic dinoflagellates Heterocapsa triquetra was established after isolation of a single vegetative cell using a glass micropipette from water collected on July 6th 2007 at 27 of salinity. A monoclonal strain was subsequently obtained of each culture after the re-isolation of a single vegetative cell (names of strains: ST147 for S. acuminata and HT150 for H. triquetra). From natural samples, Amoebophrya-like parasites infecting dinoflagellates were detected by their natural bright green autofluorescence using an epifluorescence microscope (BX51, Olympus) equipped with the U-MWB2 cube (450- to 480-nm excitation, 500-nm emission, Coats and Bockstahler 1994). Two reference strains (belonging to MALVII-Clade 2, nomenclature after Guillou et al. 2008) were selected : - A25 : a specialist parasite belonging to sub-clade 1 originated from Penzé. A single infected Scrippsiella sp. cell in the late-stage of infection was picked from water sample collected on June 15th 2009 and incubated into exponentially growing S. acuminata (primary host ST147). This strain is currently maintained in culture with the host ST147 and was purified 6 times by isolating one infected host in late-stage replace into 5 ml exponentially growing healthy host strain into 24 well plates.This parasite is additionally maintained concomitantly with ST161 (S. acuminata) since the 26th of March 2012 . - A120 : a generalist parasite belonging to sub-clade 4 originated from Penzé. A single infected Heterocapsa triquetra cell in late-stage of infection was picked from water sample collected on June 13th 2011 and incubated into exponentially growing H. triquetra (primary host HT150). This strain is currently maintained in culture with the host HT150 (parasite is named A120HT) and was purified 3 times (same protocol as described previously). It is also maintained with S. acuminata ST147 (parasite is named A120ST) since the 23th of April 2012.After a series of cycles in their primary hosts, the strain maintain in H. triquetra was successfully transferred and maintained in S. acuminata (ST147). All strains have been deposited at the Roscoff Culture Collection, http://roscoff-culture-collection.org/, with the following ID numbers: ST147=RCC1627, HT150= RCC3596, A25= RCC4383, A120= RCC4398). Principle / objective: - We should synchronise the infection of the Scrippsiella hosts to have a mass production of dinospores at the same time - We should remove bacteria on the beginning of the infection and limit their growth after - We should collect the free-living stage of the parasite (dinospores) without hosts cells or large cells debris released from hosts (especially genomic material) - We should extract genomic DNA for genomic analyses - We should extract total RNA without residual DNA for transcriptomic analyses