Forgotten reservoirs: an attempt to revive field research on rodent malaria parasites in their main natural host, the thicket rat Grammomys poensis (formerly Thamnomys rutilans)

Technical Summary: Forgotten Reservoirs – Reviving Field Research on Rodent Malaria Parasites

Problem Statement
Rodent malaria parasites (Plasmodium spp.) have served as critical experimental models for human malaria research since the mid-20th century. However, the laboratory lines currently in use (e.g., P. berghei, P. yoelii) were isolated decades ago (the most recent in 1972) and have undergone continuous serial blood passage in domestic mice. This long-term laboratory maintenance acts as a potent selective regime, potentially altering virulence, transmission costs, and gametocyte investment, thereby reducing their representativeness of contemporary wild parasites. Furthermore, there is a significant gap in understanding the ecology and evolution of these parasites in their natural hosts and vectors. The thicket rat, Grammomys poensis (formerly Thamnomys rutilans), is considered the primary natural host for murine Plasmodium in Central Africa. Despite historical reports of high infection prevalence in this species, recent attempts to establish long-term field sites to study these interactions have been hindered by a lack of recent field isolates and difficulties in capturing the host.

Methodology
The study aimed to establish a long-term field site in the Lekoni region of southeastern Gabon (Batéké Plateau), an area previously documented to harbor rodent malaria. The research team conducted an intensive 18-month field campaign involving eight 15-day missions.

• Sampling Strategy: Two complementary capture methods were employed across twelve forest fragments:
  1. Tomahawk Trapping: 150 traps were set daily at 1.5–2 meters above ground (within the liana/branch lattice near nests) for 3–4 consecutive nights per session. Bait consisted of pineapple, banana, peanut paste, and palm nut. Total effort: 12,150 trap-nights.
  2. Direct Nest Collection: A dedicated team visually located G. poensis nests (typically 2–4 meters high) and collected rodents directly. A total of 1,380 nests were inspected.
  3. Nest Boxes: An attempt was made to install 50 artificial wooden nest boxes to monitor arboreal rodents, though this method was largely unsuccessful due to termite damage and lack of occupancy verification.
• Molecular Analysis: Captured individuals (n=20) were euthanized, and samples (blood, brain, lung, intestine, liver) were collected.
  • Host Identification: Morphological identification was confirmed via Sanger sequencing of the mitochondrial Cytochrome b (Cytb) gene for two representative specimens.
  • Parasite Detection: Two sensitive nested-PCR assays targeting the Plasmodium Cytb gene were used: the protocol by Boundenga et al. (2019) and the protocol by Duval et al. (2009). Both assays were validated with positive controls and internal DNA quality checks.

Key Results

• Host Capture Efficiency: Despite the extensive effort, only 20 adult Grammomys poensis were captured over the 18-month period. Crucially, zero individuals were captured using Tomahawk traps; all successful captures (100%) resulted from direct collection at nests. Approximately one in eight nests was occupied, and capture success within occupied nests was roughly 20%.
• Parasite Prevalence: None of the 20 adult G. poensis tested positive for rodent Plasmodium using either of the two PCR assays across all five tissue types sampled.
• Comparison with Historical Data: These findings contrast sharply with historical surveys from the 1960s and 1980s in Nigeria, the Central African Republic, and the Republic of Congo, which reported infection prevalences ranging from 33% to 100% in G. poensis.
• Contextual Findings: Previous surveys in the same Lekoni region (Boundenga et al., 2019) detected murine Plasmodium (P. yoelii and P. vinckei) in sympatric ground-dwelling rodents (Praomys and Mastomys) but found no infections in the few G. poensis sampled (0/4).

Significance and Claims
The paper posits that the absence of Plasmodium in the sampled G. poensis population, despite the presence of the parasite in sympatric ground-dwelling hosts, suggests that rodent malaria transmission in this specific system is more spatially and ecologically constrained than previously assumed. The authors propose several non-exclusive hypotheses for this pattern:

1. Local Extinction: Habitat fragmentation in the Batéké Plateau may have reduced host population connectivity, driving local extinction of specific Plasmodium lineages within G. poensis.
2. Vector Constraints: The arboreal nature of G. poensis may limit exposure to mosquito vectors that primarily feed at ground level on terrestrial rodents.
3. Host Resistance or Shift: Local populations may have evolved resistance, or the parasite may have shifted its primary host preference to other rodent species in the region.

Conclusion
The study highlights the significant practical and ecological challenges in reviving field research on the Plasmodium–Grammomys system. The failure of standard trapping methods and the lack of infection in captured hosts indicate that establishing a longitudinal field site in this region is currently unfeasible without new methodologies (e.g., termite-resistant nest structures, camera traps, and parallel vector surveys). The authors conclude that while the recovery of new field isolates is vital for updating experimental models and understanding natural parasite diversity, the current ecological reality in the Batéké Plateau may differ fundamentally from historical models, necessitating a re-evaluation of the host–parasite dynamics in fragmented tropical landscapes.