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Manuscript Summary

Berthomieu et al. address a critical gap in avian malaria research by developing molecular tools to quantify and distinguish between male and female gametocytes in Plasmodium relictum, the transmissible sexual stages that determine disease spread. Avian malaria parasites pose significant threats to bird populations worldwide, particularly in vulnerable island ecosystems, yet progress in understanding transmission dynamics has been hampered by the lack of molecular markers equivalent to those available for human and rodent malaria systems. The nucleated nature of avian erythrocytes, which contain overwhelming amounts of host genetic material, has historically prevented the development of such tools. The authors employed single-cell RNA sequencing on P. relictum-infected canary blood, adapting protocols to overcome host cell contamination challenges. Using orthology-guided mapping to establish Plasmodium berghei reference datasets, they identified stage-specific gene expression patterns and selected molecular markers for asexual stages, male gametocytes, and female gametocytes. These markers were validated through experimental infection of canaries with two P. relictum lineages and compared against traditional microscopic detection methods. The molecular markers demonstrated superior sensitivity, detecting gametocytes in 21-32% of samples that tested negative by microscopy and maintaining detection for extended periods during infection. However, the markers revealed consistently higher male-to-female gametocyte ratios (53% vs 33% male) compared to microscopic estimates. The high conservation of selected markers across Plasmodium species suggests potential broad applicability to other avian malaria lineages, providing essential tools for studying transmission dynamics and evolutionary ecology of host-parasite interactions under changing environmental conditions.

MAJOR REVISIONS

Marker Selection and Validation Methodology Marker Redundancy and Primer Design Validation Critique

The selection of only two markers per developmental stage provides insufficient redundancy for robust molecular assay development, particularly given the critical importance of accurate gametocyte quantification in epidemiological studies. Standard protocols in diagnostic marker development typically employ 3-5 candidate markers per target to account for potential primer failures, amplification efficiency variations, and biological variability across parasite populations (Bustin et al., 2009). Additionally, the incomplete implementation of intron-spanning primer design, described as done " whenever possible," introduces a significant risk of genomic DNA contamination affecting quantification accuracy. The substantial variation in amplicon sizes (114-267 bp, representing a 2.3-fold difference) creates differential amplification kinetics that can systematically bias quantitative comparisons, particularly problematic for sex ratio calculations where PCR efficiency differences compound over 45 amplification cycles.

Relevant section: Table 1 shows DNA/cDNA amplicon sizes and primer sequences, with methods stating intron-spanning design was implemented "whenever possible." 

Framework for Addressing: Expand marker selection to minimum 4-5 candidates per stage with comprehensive primer efficiency validation showing 90-110% efficiency and R2 >0.99 for all primer pairs. Redesign primers to ensure uniform amplicon sizes (±20 bp) and mandatory intron-spanning design for all markers. Conduct computational specificity analysis using BLAST against complete P. relictum genome to confirm single target amplification. Perform amplicon sequencing validation for all primer pairs to verify specific target amplification. Include DNase treatment step in RNA extraction protocol and validate absence of genomic DNA contamination through no-RT controls for all samples.

Single-Cell Transcriptomics Technical Implementation - Host Cell Lysis Protocol and Contamination Control Critique: 

The host cell lysis protocol adaptation requires strengthening to address potential systematic biases in parasite transcript recovery and stage representation. The saponin-based lysis approach (0.15% for 5 minutes) may introduce differential cell permeabilization affecting parasite integrity across developmental stages, while the substantial single-cell loss during quality control (63/92 cells retained, 32% loss) combined with only five identified clusters suggests insufficient statistical power for robust marker identification (Reid et al., 2018). Furthermore, the scmap similarity threshold of 0.4 for cross-species stage assignment lacks empirical justification and may contribute to the observed 30% false-negative rate, where AP2 -AP2-G-positive samples lack detectable gametocyte markers. The single-time-point sampling (10 days post-infection) for marker development may inadequately capture expression variability, particularly given that pDELURB4 shows later temporal dynamics (peak days 14-19) than the sampling time point. 

Relevant section: Methods describe "40 μl of the sampled blood were placed in a 1.5 ml Eppendorf tube, and the nuclear and mitochondrial DNA were stained" with subsequent saponin lysis and cell sorting protocols. 

Framework for Addressing: Validate lysis protocol through stage-specific recovery efficiency analysis using known parasite mixtures and implement viability staining to assess cell integrity post-lysis. Increase biological replication using multiple infected birds at optimal parasitemia timepoints for each lineage. Justify scmap threshold through systematic analysis of classification accuracy across different similarity values using cross-validation approaches. Expand temporal sampling to capture peak expression for both lineages (days 7, 10, 14, and 17 post-infection). Implement technical validation through spike-in controls and batch effect assessment using principal component analysis to identify systematic sources of variation.

MINOR REVISIONS

Calibration Standards and Quantification Synthetic Plasmid Calibration Methodology Critique: 

The calibration approach using synthetic plasmids requires detailed methodological description and validation to ensure quantitative accuracy. The current methodology mentions using plasmids containing "single copy of each target gene" but provides insufficient detail about plasmid construction, sequence verification, or copy number determination procedures. The use of separate calibration plasmids for female markers due to "complexity of the gene fragment sequences" introduces systematic quantification bias, as male/asexual markers utilize single plasmids containing both targets while female markers require two separate plasmids with different GAPDH normalization standards (Bustin et al., 2009). Furthermore, melting curve analysis is mentioned only for intron-spanning primers, leaving systematic validation of amplicon specificity incomplete across all markers. 

Relevant section: Methods state "Calibration curves were established using a synthetic plasmid (Eurofins) containing a single copy of each target gene" and "For the quantification of female stages, due to the complexity of the gene fragment sequences it was not possible to synthesise a single plasmid." 

Framework for Addressing: Provide detailed description of plasmid construction including vector backbone, insert sequences, and cloning strategy with sequence verification by Sanger sequencing. Implement uniform calibration approach using equimolar mixtures of separate single-target plasmids for all markers to eliminate systematic bias between male and female quantification. Conduct comprehensive melting curve analysis for all primer pairs across both lineages and report melting temperatures with validation of single-peak profiles. Determine absolute plasmid copy numbers through spectrophotometric quantification and digital PCR validation. Include primer efficiency determination (90-110%) and linearity assessment (R2 >0.99) for all calibration curves.

Experimental Controls and Validation Negative Control Implementation Critique: 

The experimental design requires implementation of comprehensive negative controls that are standard in molecular parasitology studies to validate assay specificity and eliminate potential artifacts. The current methodology lacks uninfected control birds to assess background host gene expression that might cross-react with parasite-specific primers, particularly critical given the challenges of detecting parasite transcripts within nucleated avian erythrocytes containing abundant host RNA (Valkiūnas, 2004). Additionally, no mock-infected controls were included to verify that observed signals represent genuine parasite-derived transcripts rather than artifacts from injection procedures or host immune responses. The study also lacks evidence of randomization protocols for bird assignment to lineage groups or control of potential confounding variables such as bird age, sex, or housing conditions that could systematically bias results, given the small sample size (n=3 per group). 

Relevant section: Methods section describing infection protocol but omitting mention of uninfected controls; no description of randomization procedures or covariate control. 

Framework for Addressing: Include a minimum of 2 uninfected control birds sampled at equivalent timepoints to assess baseline host gene expression and potential cross-reactivity of parasite primers. Implement mock-infected controls using sterile PBS injection to validate that signals represent parasite-specific rather than injection-induced responses. Document randomization protocol for bird assignment, including random number generation method and stratification by relevant covariates (age, sex, body weight). Report bird characteristics (age, sex, body condition) in the supplementary table and test for systematic differences between treatment groups using appropriate statistical tests.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they used generative AI to come up with new ideas for their review.