A longitudinal transcriptomic analysis of Rhipicephalus microplus midgut upon feeding

Abstract

Rhipicephalus microplus, a highly host-specific tick that primarily feeds on cattle, posing a significant threat to livestock production. The investigation of tick physiology is crucial for identifying potential targets in tick control. Of particular interest adult female ticks undergo a significant expansion of the midgut during feeding, leading to an over 100-fold increase in body weight. Beyond the functions of storing and digesting blood meals, the tick midgut plays a crucial role in acquiring and transmitting pathogens. However, our understanding of tick midgut physiology remains limited. In this study we conducted a comprehensive longitudinal transcriptome analysis of the midgut from adult female R. microplus ticks collected at various feeding stages, providing an overview of the transcriptional modulation in this organ as feeding progress. By employing a de novo assembly approach followed by coding-sequences (CDS) extraction, 60,599 potential CDS were identified. In preparation for functional annotation and differential expression analysis, transcripts that showed an average transcript per million (TPM) ≥ 3 in at least one of the biological conditions were extracted. This selection process resulted in a total of 10,994 CDS, which were categorized into 24 functional classes. Notably, our differential expression analysis revealed three main transcriptional profiles. In the first one, representing the slow-feeding stage, the most abundant functional classes were the "protein synthesis" and "secreted" groups, reflecting the highly active state of the tick midgut. The second profile partially accounts for the rapid-feeding stage, in which a high number of differentially expressed transcripts was observed. Lastly, the third transcriptional profile represents post-detached ticks. Notably the highest number of modulated transcripts was observed up to 48 h post-detachment (hpd), however no major differences was observed up to 168 hpd. Overall, the data presented here offers a temporal insight into tick midgut physiology, contributing to the identification of potential targets for the development of anti-tick control strategies.

Keywords: Blood meal digestion; Midgut; RNA-sequencing; Ticks.

Fig. 1.

Overview of the transcriptome profile of Rhipicephalus microplus midgut at different feeding stages. (A): Representative images of the collected ticks by their average weight or hours post-detachment. Each dot represents 1 mm. (B): Multidimensional plot of the transcripts with transcript per million (TPM) ≥ 3 in at least one of the biological groups. (C): Heatmap plot using the normalized TPM values of each transcript. Each column represents a biological replicate. G1 – G4 indicates partially fed ticks that were collected and grouped based on their average weight; (G1) 38.9 ± 4.48 mg, (G2) 60.8 ± 10.1 mg, (G3) 94.7 ± 9.0 mg and (G4) 192.9 ± 12.4 mg. While 0 h – 168 h represents ticks collected at different time points after their natural detachment from its host.

Fig. 2.

Relative quantification of the 24 functional classes over the different biological groups of Rhipicephalus microplus midgut. Dots represent the average TPM (%) of each functional group. Errors bars represent the standard error of the mean. G1 – G4 indicate partially fed ticks that were collected and grouped based on their average weight; (G1) 38.9 ± 4.48 mg, (G2) 60.8 ± 10.1 mg, (G3) 94.7 ± 9.0 mg and (G4) 192.9 ± 12.4 mg. Meanwhile, 0 h – 168 h represent ticks collected at different time points after their natural detachment from their host.

Fig. 3.

Volcano plot panel illustrating the differentially expressed transcripts found between in Rhipicephalus microplus midgut collected at different feeding stages. Statistical difference was considered when a transcript presented a Log₂(Fold change) higher than 2 or lesser than −2 (vertical dotted lines) and a false discovery rate (FDR) ≤ 0.05 (horizontal dotted lines). Up-regulated transcripts are shown in blue, down-regulated transcripts are shown in red and transcripts that were not considered differentially expressed are shown as gray dots. G1 – G4 indicates partially fed ticks that were collected and grouped based on their average weight; (G1) 38.9 ± 4.48 mg, (G2) 60.8 ± 10.1 mg, (G3) 94.7 ± 9.0 mg and (G4) 192.9 ± 12.4 mg. Meanwhile, 0 h – 168 h represent ticks collected at different time points after their natural detachment from their host. The post detachment versus before detachment comparison includes 24–168 hpd versus G1–0-hpd groups.

Fig. 4.

Unsupervised clustering of the transcripts that presented an average transcript per million (TPM) ≥ 3 in the midgut of Rhipicephalus microplus midgut at different feeding conditions. The dots represent the average Z-score of the TPM from the transcripts contained within each cluster at each feeding stage. The errors bars represent the standard deviation of the mean.

Fig. 5.

Relative quantification of the 24 functional classes of Rhipicephalus microplus midgut transcripts within cluster 1. The average TPM, as percentage, is represented by the size of each sphere. G1 – G4 indicates partially fed ticks that were collected and grouped based on their average weight; (G1) 38.9 ± 4.48 mg, (G2) 60.8 ± 10.1 mg, (G3) 94.7 ± 9.0 mg and (G4) 192.9 ± 12.4 mg.