Background: The apicomplexan parasites Eimeria spp. are the causative agents of coccidiosis, a disease with a significant global impact on the poultry industry. The complex life cycle of Eimeria spp. involves exogenous (sporogony) and endogenous (schizogony and gametogony) stages. Unfortunately, the genetic regulation of these highly dynamic processes, particularly for genes involved in specific developmental phases, is not well understood. Methods: In this study, we used RNA sequencing (RNA-Seq) analysis to identify expressed genes and differentially expressed genes (DEGs) at seven time points representing different developmental stages of Eimeria tenella. We then performed K-means clustering along with co-expression analysis to identify functionally enriched gene clusters. Additionally, we predicted apicomplexan AP2 transcription factors in E. tenella using bioinformatics methods. Finally, we generated overexpression and knockout strains of ETH2_0411800 to observe its impact on E. tenella development. Results: In total, we identified 7329 genes that are expressed during various developmental stages, with 3342 genes exhibiting differential expression during development. Using K-means clustering along with co-expression analysis, we identified clusters functionally enriched for oocyte meiosis, cell cycle, and signaling pathway. Among the 53 predicted ApiAP2 transcription factors, ETH2_0411800 was found to be exclusively expressed during sporogony. The ETH2_0411800 overexpression and knockout strains did not exhibit significant differences in oocyst size or output compared to the parental strain, while the resulting ETH2_0411800 knockout parasite showed a relatively small oocyst output. Conclusions: The findings of our research suggest that ETH2_0411800 is not essential for the growth and development of E. tenella. Our study provides insights into the gene expression dynamics and is a valuable resource for exploring the roles of transcription factor genes in regulating the development of Eimeria parasites. Graphical

Background: Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. Methods: Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. Results: SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. Conclusions: We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control. Graphical

Background: Eimeria parasite infection occurs via ingestion of oocysts. The robust, bilayer oocyst wall is formed from the contents of wall-forming bodies (WFBs), WFB1 and WFB2, located exclusively in macrogametocytes. Eimeria necatrix gametocyte proteins 22 and 59 (EnGAM22 and EnGAM59) have been found to localize to WFBs and the oocyst wall. However, the exact localization of these two proteins is not clear. Methods: WFBs of E. necatrix were extracted from purified gametocytes using a cutoff filter and the extracts of purified WFBs and gametocytes were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Then, the localization of EnGAM22 and EnGAM59 proteins was determined using an indirect immunofluorescence assay. Finally, the development of macrogametocytes and the oocyst wall of E. necatrix was analyzed using laser confocal microscopy and scanning electron microscopy. Results: Purified WFBs had the same shape and size as those observed in macrogametocytes. EnGAM22 protein localized to WFB1, whereas EnGAM59 protein localized to WFB2. Both EnGAM22 and EnGAM59 native proteins were detected in the extracts of WFBs and gametocytes. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface of the macrogametocyte to form an anti-EnGAM22 positive layer. WFB2 then appeared to give rise to the inner layer, which was anti-EnGAM59 positive. Conclusions: EnGAM22 and EnGAM59 proteins localized to WFB1 and WFB2 and were involved in the formation of the outer and inner layers of the oocyst wall of E. necatrix, respectively. The processes of macrogametogenesis and oocyst wall formation of E. necatrix are similar to other Eimeria parasites. The anti-EnGAM22 antibody could be used as a tool to track the transport and secretion of proteins in WFB1 during oocyst development. Graphical