ABSTRACT: Purpose: Parasites of the genus Eimeria, and the closely related human pathogen Cyclospora cayetanensis, cause enteric disease in livestock and people worldwide. These coccidian parasites must undergo maturation (sporulation) before becoming infectious. Understanding oocyst maturation would benefit attempts to reduce the harm they cause to veterinary and human health. No animal model exists to study C. cayetanensis and the parasite cannot be grown in culture. We assessed transcription in maturing oocysts of Eimeria acervulina, a widespread chicken parasite, in order to identify markers of viability and those that may differentiate immature and mature oocysts. We also investigated predicted gene function and determined which of this parasite’s genes also occur in C. cayetanensis. Methods: E. acervulina oocysts were collected from chickens (3 biological replicates) and allowed to sporulate for 24 hours. RNA was extracted from oocysts that were collected every four hours and subjected to RNA-Sequencing. Data were screened for constitutively expressed genes throughout the time course. We also identified differentially expressed genes between immature and mature oocysts. We performed functional characterization of constitutively -expressed and significantly differentially-expressed genes and also biochemical pathway analysis. RT–qPCR validation on selected constitutively and differentially-expressed genes was performed using SYBR Green assays. Results: RNA-Seq yielded ~2 billion paired-end reads, 92% of which mapped to the E. acervulina genome. The ~6,900 annotated genes underwent temporally-coordinated patterns of gene expression. Fifty-three constitutively expressed genes each contributed >1,000 TPM throughout, including those encoding cation-transporting ATPases, an oocyst wall protein, a palmitoyltransferase, membrane proteins, and hypothetical proteins. These genes were enriched for 285 gene ontology (GO) terms and 13 genes were associated with a total of 17 KEGG pathways, defining housekeeping processes and functions important throughout sporulation. Expression of more than 40% of these genes (2,928) differed in sporulated vs. unsporulated oocysts; of these, nearly two-thirds (1,843) increased their expression over time. Eight genes expressed most in unsporulated oocysts, and encoding proteins promoting oocyst maturation and development, could be assigned to 37 GO terms and 5 KEGG pathways. Fifty-six genes underwent significant upregulation in sporulated oocysts, each contributing at least 1,000 TPM. Of these, 40 were annotated by 215 GO assignments and 9 were associated with 18 KEGG pathways, encoding products involved in respiration, carbon fixation, energy utilization, invasion, motility, and stress and detoxification responses. Several RT-qPCR assays confirmed RNA-Seq as a valid means to estimate gene expression. Conclusions: Sporulation orchestrates coordinated changes in the expression of many coccidian genes, most especially those important to metabolic activity. These data elucidate the biology of coccidian development, provide means to evaluate the maturity of parasite cohorts, and may prove useful in predicting infectiousness. Moreover, because many of these genes have homologues in C. cayetanensis, they may prove useful as biomarkers for infectiousness.
