Abstract
The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation was previously achieved for Perkinsus spp. but with few tools for transgene expression and limited selection efficacy. We sought to expand the power of experimental genetic tools for Perkinsus using P. marinus as a model. We constructed a modular plasmid assembly system for expression of multiple genes simultaneously. We developed efficient selection systems for three drugs, puromycin, bleomycin and blasticidin, that are effective in as little as three weeks. We developed eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination but with transgene fragmentation including deletion of some elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide great new capacity to genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.
Highlights
Perkinsus species are major marine parasites that cause disease and mortalities in commercially and environmentally important marine molluscs including oysters, abalone, clams and other shellfish (Choi and Park 2010; Villalba et al 2004)
The parasite is taken up by the filter-feeding host animal and phagocytosed by host hemocytes mediated by a galectin receptor (Lau et al 2018; Tasumi and Vasta 2007)
There are currently no practical treatments available to limit the spread of the parasite and Perkinsus infection is listed on the World Organisation of Animal Health (OIE) as a reportable disease of molluscs
Summary
Perkinsus species are major marine parasites that cause disease and mortalities in commercially and environmentally important marine molluscs including oysters, abalone, clams and other shellfish (Choi and Park 2010; Villalba et al 2004). Upon replication and egress from the host cell, the released parasites continue to infect more hemocytes or proliferate directly within the host haemolymph. The host tissues are overwhelmed, and parasites are released by the moribund animal into the water column where they can infect further shellfish. There are currently no practical treatments available to limit the spread of the parasite and Perkinsus infection is listed on the World Organisation of Animal Health (OIE) as a reportable disease of molluscs. Other members of the Phylum Perkinsozoa are parasites of microeukaryotes and are responsible for controlling major algal blooms (Alacid et al 2017; Noren et al 1999), and in recent years others have been recognised as parasites of other animals including fish and amphibians (Chambouvet et al 2015; Freeman et al 2017)
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