Genomics and transcriptomics of nervous system from Rhodnius prolixus, and its modulation after a blood meal in an immature stage

Technical Summary: Genomics and Transcriptomics of the Rhodnius prolixus Nervous System and Modulation After a Blood Meal

Problem Statement
Rhodnius prolixus is an obligatory blood-feeding insect and the primary vector of Trypanosoma cruzi, the causative agent of Chagas disease. While the species has served as a physiological model since the 1930s, genomic resources have historically been limited. A genome sequence published over a decade ago contained significant gaps and annotation errors, particularly regarding neuropeptide signaling components. Furthermore, previous transcriptomic studies on blood feeding in triatomines have focused primarily on peripheral tissues (e.g., ovaries, fat body), leaving a gap in understanding the central nervous system (CNS) response to hematophagy. The regulation of physiological processes triggered by blood ingestion—such as osmotic regulation, diuresis, and development—is coordinated by the nervous system, yet the specific transcriptional dynamics in the CNS shortly after feeding remain largely uncharacterized.

Methodology
The study employed a multi-faceted approach combining genomic mining, transcriptomics, and bioinformatic analysis:

1. Genomic Re-annotation: The authors mined a recently released, chromosome-level assembly of the R. prolixus genome (GenBank accession: GCA_049639745.1). They performed targeted homology-based searches (BLASTP and TBLASTN) using protein sequences from previous literature to identify neuropeptide precursors, receptors, processing enzymes, and biogenic amine signaling components. Manual reconstruction was performed for genes that were absent, fragmented, or incorrectly annotated in the previous VectorBase assembly (VectorBase-68).
2. Experimental Design: Fifth-instar nymphs were used, representing an immature stage where blood feeding triggers molting and ecdysis. Two conditions were established: unfed (starved) and fed (13 hours post-blood meal).
3. RNA Sequencing: Nervous systems (brain, ganglia, and glands) were dissected from pooled insects (3 per replicate) to generate 8 biological replicates (4 per condition). Total RNA was extracted, and libraries were sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end reads).
4. Bioinformatic Analysis:
   • Mapping: Reads were quality-filtered and mapped to the new GCA_049639745.1 genome and compared against the older VectorBase-68 assembly to assess improvements in mapping metrics.
   • Differential Expression: After filtering for low-count and stable genes, 6,638 genes were retained. Differential expression analysis was conducted using DESeq2 with negative binomial generalized linear models. Significance was defined as an s-value < 0.05 and an absolute Log2 fold-change > 1.
   • Functional Enrichment: Gene Ontology (GO) enrichment analysis was performed using the ermineR package to identify overrepresented biological processes and molecular functions among differentially expressed genes (DEGs).

Key Contributions and Results

• Genomic Improvements: The study demonstrates that the new chromosome-level genome assembly represents a significant improvement over the previous version. Mapping rates increased from ~74–78% (VectorBase-68) to >87% with the new assembly. The authors successfully identified and manually annotated numerous neuropeptide precursor genes (e.g., CCHa1, CCHa2, PDF, EH, Elevenin-1) and receptors that were missing or incomplete in the automated annotations of the older database.
• Baseline CNS Transcriptome: In the nervous system of unfed fifth-instar nymphs, the authors characterized the expression of 46 predicted neuropeptide/peptide hormone genes and 44 receptor genes. Neuropeptide-like precursor 1 (NPLP1) was the most highly expressed neuropeptide precursor, followed by Proctolin, Allatostatin CCC, and Myoinhibitory peptide. The AST-C receptor was the most highly expressed receptor.
• Transcriptional Response to Feeding:
  • Magnitude and Direction: 13 hours after a blood meal, 96 genes (1.46% of the expressed transcriptome) were differentially expressed. Contrary to some findings in other hematophagous insects where upregulation dominates early responses, 77.2% of the DEGs in R. prolixus were downregulated after feeding.
  • Functional Categories:
    • Immunity and Stress: Immune-related genes showed mixed regulation. While some defensins and a C-type lectin (slf) were upregulated, several other immune genes (including usp2, serp, bt, and sls) were downregulated. The authors suggest this downregulation may prevent an immune attack on beneficial gut microbiota that expand after feeding. Stress response genes included upregulated chaperones (cct1, cct5, stip1) and downregulated redox-related proteins.
    • Endocrine Regulation: Genes related to insulin signaling (chico), ecdysteroid metabolism, and the GPA2/GPB5 receptor (LGR1) were downregulated. This aligns with the hypothesis that the system remains "sensitized" during starvation to rapidly respond to nutrient abundance, shifting to a less responsive state post-feeding.
    • Muscular Physiology: Eleven transcripts related to muscle structure and contraction (e.g., Actinin, Myosin, Troponin) were downregulated, reflecting the physiological shift toward molting processes.
    • Energetic Metabolism: Lipid biosynthesis genes (FAS1, FAD) were upregulated, while enzymes involved in gluconeogenesis (PCB) and trehalose transport (TRET) were downregulated. Notably, α-tubulin was significantly upregulated, challenging its utility as a stable housekeeping gene in feeding-state experiments.
  • GO Enrichment: Significantly affected biological processes included protein folding (heat shock proteins), fatty acid metabolism, and monocarboxylic acid metabolic processes. Molecular functions affected included ATP-hydrolysis and ligase activities.

Significance and Claims
The authors claim this work provides the first transcriptomic characterization of the rapid (13-hour) response to blood intake specifically within the nervous system of a triatomine insect. The study highlights that the nervous system undergoes significant transcriptional reprogramming shortly after feeding, characterized predominantly by gene downregulation.

The paper posits that the "starved" state represents a physiological configuration prepared for an immediate response to feeding, whereas the post-feeding state shifts to a "less responsive" mode. This central regulation likely triggers downstream physiological changes in peripheral organs, such as diuresis and molting. By providing a corrected and more complete genomic annotation for neuropeptide signaling, the study offers a more robust foundation for future bioinformatic pipelines and functional studies in R. prolixus, contributing to the broader understanding of vector biology and the control of neglected tropical diseases.