RNA-Seq of Phenotypically Distinct Eimeria maxima Strains Reveals Coordinated and Contrasting Maturation and Shared Sporogonic Biomarkers with Eimeria acervulina

Abstract

Strains of Eimeria maxima, an enteric parasite of poultry, vary in virulence. Here, we performed microscopy and RNA sequencing on oocysts of strains APU-1 (which exhibits more virulence) and APU-2. Although each underwent parallel development, APU-1 initially approached maturation more slowly. Each strain sporulated by hour 36; their gene expression diverged somewhat thereafter. Candidate biomarkers of viability included 58 genes contributing at least 1000 Transcripts Per Million throughout sporulation, such as cation-transporting ATPases and zinc finger domain-containing proteins. Many genes resemble constitutively expressed genes also important to Eimeria acervulina. Throughout sporulation, the expression of only a few genes differed between strains; these included cyclophilin A, EF-1α, and surface antigens (SAGs). Mature and immature oocysts uniquely differentially express certain genes, such as an X-Pro dipeptidyl-peptidase domain-containing protein in immature oocysts and a profilin in mature oocysts. The immature oocysts of each strain expressed more phosphoserine aminotransferase and the mature oocysts expressed more SAGs and microneme proteins. These data illuminate processes influencing sporulation in Eimeria and related genera, such as Cyclospora, and identify biological processes which may differentiate them. Drivers of development and senescence may provide tools to assess the viability of oocysts, which would greatly benefit the poultry industry and food safety applications.

Keywords: Cyclospora; Eimeria; coccidia; control; development; differential expression; oocyst; sporulation; transcription; vaccine.

Figure 1

Differential oocyst development in E. maxima strains APU-1 and APU-2. (A). Percent sporulation of strains during oocyst development. Strain APU-2 reached maximum sporulation sooner. Strains reached maximum sporulation at 30 h. (B). Representative photomicrographs of developing E. maxima oocysts at each time point. Additional time points T30 and T42 are included. Unsporulated oocysts (T0) are diploid and go through meiosis. Four haploid sporoblasts are visible by 12 h (T12) and these become sporocysts (T18–T48). Two sporozoites form in each for the four sporocysts.

Figure 2

Pearson’s correlation matrix of transcription between strains APU-1 and APU-2. Mean log₂ of mapped reads from three replicates per time point were compared for each strain every 6–12 h during oocyst sporulation. Individual squares represent Pearson’s product correlations between (and within) strains at each time point. Correlations generally decreased with time in each strain. The boxed in area focuses on APU-1 vs. APU-2 correlations, specifically. Note that contemporaneous correlations were generally strongest at the same time point, with exceptions.

Figure 3

Transcript bias during oocyst sporulation in E. maxima strains APU-1 and APU-2. The bias in transcription (when a subset of genes most disproportionately contribute to transcripts) is greatest mid-sporulation and peaks at hour 18 (T18). Parallel temporal patterns in transcriptional bias hold whether the analysis is performed on all genes (black, APU-1; dark gray, APU-2) or restricted to those each contributing at least 100 transcripts per million (>100 TPM) (blue, APU-1; gray, APU-2). Transcriptional bias was calculated at each time point (based on mean TPM from three replicates per time point) for each strain.

Figure 4

Temporal trend of constitutively expressed genes in E. maxima strains APU-1 and APU-2. Mean TPM of these 58 genes is displayed over the time course of sporulation. Their mean TPM peaked at 12–18 h. These data support the conclusion that most transcripts important to the continuous development of one strain are also important to the other strain.

Figure 5

Differentially expressed genes (DEGs) during sporulation in E. maxima strains APU-1 and APU-2. Expression during sporulation was compared between strains using DESeq2. Threshold criteria of >1.5 or <−1.5 log₂ fold change (FC) with adjusted p < 0.05 (Adj. p-value) were utilized. By these criteria, 204 significantly DEGs were identified (in red). This plot depicts expression of E. maxima APU-2 vs. APU-1 (143 upregulated, 61 downregulated genes in APU-2). NS indicates non-significant.

Figure 6

Log₂ FC differential expression between time points T36 and T0 for strains APU-1 and APU-2. Much directional similarity between expressed genes is evident, concordance in top-right and bottom-left quadrants. The blue line indicates the regression line. Note: for 75 of 6057 annotated genes, differential expression could not be determined between time points in strains; therefore, the graph depicts differential expression of 5982 genes.

Figure 7

Relative expression of selected genes correlates highly between RNA-Seq and RT-qPCR assays. (A). Temporal changes in expression of genes selected for validation using RT-qPCR at time points T18 and T36 during oocyst sporulation. The log₂ FC using RT-qPCR and RNA-Seq (differential expression by DESeq2) are compared. Gene expression at each time point is depicted relative to initial transcription level (T0). Bars with solid fill represent RT-qPCR and bars with stripe fill represent RNA-Seq. Error bars depict variance among RT-qPCR estimates from three replicate experiments that each employed three replicates per time point. (B). Correlation plot of RT-qPCR vs. RNA-Seq log₂ FC data shown in (A). This incorporates data from all three genes and time points T18, T36 (both strains). Red dashes indicate regression line.