Comparative Time-Scale Gene Expression Analysis Highlights the Infection Processes of Two Amoebophrya Strains

Abstract

Understanding factors that generate, maintain, and constrain host-parasite associations is of major interest to biologists. Although little studied, many extremely virulent micro-eukaryotic parasites infecting microalgae have been reported in the marine plankton. This is the case for Amoebophrya, a diverse and highly widespread group of Syndiniales infecting and potentially controlling dinoflagellate populations. Here, we analyzed the time-scale gene expression of a complete infection cycle of two Amoebophrya strains infecting the same host (the dinoflagellate Scrippsiella acuminata), but diverging by their host range (one infecting a single host, the other infecting more than one species). Over two-thirds of genes showed two-fold differences in expression between at least two sampled stages of the Amoebophrya life cycle. Genes related to carbohydrate metabolism as well as signaling pathways involving proteases and transporters were overexpressed during the free-living stage of the parasitoid. Once inside the host, all genes related to transcription and translation pathways were actively expressed, suggesting the rapid and extensive protein translation needed following host-cell invasion. Finally, genes related to cellular division and components of the flagellum organization were overexpressed during the sporont stage. In order to gain a deeper understanding of the biological basis of the host-parasitoid interaction, we screened proteins involved in host-cell recognition, invasion, and protection against host-defense identified in model apicomplexan parasites. Very few of the genes encoding critical components of the parasitic lifestyle of apicomplexans could be unambiguously identified as highly expressed in Amoebophrya. Genes related to the oxidative stress response were identified as highly expressed in both parasitoid strains. Among them, the correlated expression of superoxide dismutase/ascorbate peroxidase in the specialist parasite was consistent with previous studies on Perkinsus marinus defense. However, this defense process could not be identified in the generalist Amoebophrya strain, suggesting the establishment of different strategies for parasite protection related to host specificity.

Dinoflagellate Host Chloroplasts and Mitochondria Remain Functional During Amoebophrya Infection

“…The second experiment (hereafter "experiment 2") was used for transcriptomics analyses and has been previously described by Farhat et al (2018). Briefly, 21 culture flasks (600 mL) were inoculated by mixing 150 mL of dinospores of Amoebophrya sp.…”

“…ST147 (dinospore:host ratio of 30:1) cultures. Every 6 h, three flasks were completely harvested (7 times from 0 to 36 h) for mRNA extraction and sequencing, as detailed in Farhat et al (2018). Three flasks containing 450 mL of uninfected hosts were additionally prepared to be used as control and completely harvested at 0 h (T0).…”

“…We downloaded and reanalyzed a recently published transcriptomics dataset from the internal development of two Amoebophrya strains (ENA database project ID PRJEB26803) following a full infection cycle (Farhat et al, 2018). We reconstructed the host organellar energy-related metabolic pathways for which part of the genes are encoded by either the mitochondrial (mt) or the chloroplast (cp) DNA, and monitored their expression.…”

“…We created a modified transcriptome for the host (host-T0new) by identifying and replacing contigs from the host-T0 transcriptome that matched the curated energy-related gene sets identified above (Step 3 in the bioinformatics workflow; Supplementary Figure S1) using tblastn (e-value = 1e −5 ). An artificial "hybrid" reference transcriptome (hybtrans) was created by combining the modified host transcriptome host-T0-new and the parasite predicted mRNAs (that include both the 5 and 3 UTR regions) for the Amoebophrya A120 (Farhat et al, 2018). We used the DESeq2 differential expression analysis tool from the Trinity v2 package (Haas et al, 2013) and custom scripts to monitor the abundance of host and parasite genes in hybtrans throughout the infection.…”

“…Amoebophyra sp. A120 is an aerobic phagotrophic endoparasite (Cachon, 1964) that uses oxygen throughout its life cycle (Farhat et al, 2018) and requires light in order to produce high numbers of infective dinospores (Figures 1A,B). Moreover, our results suggest that the parasite has access to oxygen, ATP, NADPH and other metabolites such as carbohydrates, from the host mitochondrion and chloroplast which remain functional during most of its intracellular stages of development.…”