Single-cell RNA sequencing of Plasmodium vivax sporozoites reveals stage- and species-specific transcriptomic signatures

Background Plasmodium vivax sporozoites reside in the salivary glands of a mosquito before infecting a human host and causing malaria. Previous transcriptome-wide studies in populations of these parasite forms were limited in their ability to elucidate cell-to-cell variation, thereby masking cellular states potentially important in understanding malaria transmission outcomes. Methodology/Principal findings In this study, we performed transcription profiling on 9,947 P. vivax sporozoites to assess the extent to which they differ at single-cell resolution. We show that sporozoites residing in the mosquito’s salivary glands exist in distinct developmental states, as defined by their transcriptomic signatures. Additionally, relative to P. falciparum, P. vivax displays overlapping and unique gene usage patterns, highlighting conserved and species-specific gene programs. Notably, distinguishing P. vivax from P. falciparum were a subset of P. vivax sporozoites expressing genes associated with translational regulation and repression. Finally, our comparison of single-cell transcriptomic data from P. vivax sporozoite and erythrocytic forms reveals gene usage patterns unique to sporozoites. Conclusions/Significance In defining the transcriptomic signatures of individual P. vivax sporozoites, our work provides new insights into the factors driving their developmental trajectory and lays the groundwork for a more comprehensive P. vivax cell atlas.

• Reviewer #2: Could the number of patients used in this study be indicated?
Was this blood pooled? Which protocol/criteria was used in screening and collection of blood samples • Reviewer #1: Pg 13 lines 392-5. Please explain how mosquitoes containing oocyst were identified and kept/maintained to develop sporozoites.
We have provided additional clarification on the protocol used for P. vivax infected blood-collection and membrane feeding as well as identification of infected mosquitoes and the checking of midgut oocysts of An. dirus in sections "Blood samples, mosquitos and infections" at line 388. The text now reads: Line 389-401: Blood samples from symptomatic patients with P. vivax presenting to local health facilities in Mondulkiri province (Kaev Seima)  • Reviewer #1: Sporozoites were isolated 16-18 days after infectious blood meal.
It is not clear how dissection was done and how the flies were treated before being dissected.
An. dirus dissections were aseptically performed by manual dissection by the expert dissection team to isolate the salivary glands that contain the P. vivax sporozoites used in this study into HBSS (as described at line 403-414). To further clarify this detail we have added to line 406-408: Lines 403-408: P. vivax sporozoites were isolated from the salivary glands of female An. dirus mosquitoes 16-18 days after their infectious blood-meal, where days post blood-meal was dependent on the replicate being processed.
An. dirus were immobilised with 70% Ethanol spray. Each replicate represents one independent feed of a clinical isolate isolated from symptomatic P. vivax patients in Mondulkiri. A team of technicians performed aseptic salivary-gland dissections for a maximum period of one hour. Generally, ~75 to 100 mosquitoes were dissected in the one-hour sitting for each of the three replicates.
• Reviewer #3: What wasn't clear was the total number of unique parasite lines used in the initial mosquito infections and were these mixed infections or clonal.
In our study, the three replicates presented each represent an independent mosquito feed, each isolated from a different P. vivax patient in Mondulkiri. Given the practical limitations imposed when working with field isolates, we focused our studies on defining the transcriptomic signatures of the sporozoites and did not study the genetic background of each isolate. Each Plasmodium spp. infected blood-meal is species-typed using a real-time PCR assay, as previously described (Canier et al. 2013Mal Journal 10.1186/1475-2875 to ensure no multi-species infections are processed. Determining the complexity of infection was not in our experimental plan as it has previously been shown that up to 92% of Cambodian P. vivax infections are polyclonal (Friedrich et al. 2016 PLoS NTD 10.1371/journal.pntd.0004526). We saw minimal batch effects between the three replicates and used current best-practices for batch-correction. We suggest that resolving the effect of P. vivax genetic diversity and transcriptomic signatures be a question of future scientific endeavours, however was beyond the scope of the current study.
To clarify the total number of P. vivax isolates used in our experimental design, we have added the following at line 406-407: Each replicate represents one independent feed of a clinical isolated from symptomatic P. vivax patients in Mondulkiri. 2019, comparing sporozoites to blood-stage forms. We chose to limit these comparisons only to P. vivax available data as opposed to including orthologous comparison to P. cynomolgi. We have therefore included the following text in the methods at line 517-522: Differentially expressed genes found in "hypnozoites" and "mixedLS" (adjusted p-value <0.01) from Gural et al. 2018 [68]; "MotilityActivation", "LiverStageEarly" and "InsectStageFinal" sporozoites from Roth et al. 2018 [21]; and "sporozoite" and "blood-stage" parasites from Vivax Sporozoite Consortium 2019 [19] were detailed in this comparison.
We also draw the reviewer's attention to Figure 1F, where we detailed expression of orthologous upregulated-in infectious sporozoite genes in both the Vivax Sporozoite Consortium 2019 IJP bulk-RNA seq study and Westenberger et al. 2010 PLoS NTD microarray study of P. vivax sporozoites.

Conclusions
• Reviewer #2: The closing statements can be better re-worded to capture the broad public health concern of P. vivax malaria control (IVM) beyond the molecular significance.
We thank the reviewer for allowing us the opportunity to refine our closing message as follows; Line 376-379: It is indeed such knowledge that underpins the success in developing the tools and strategies that are needed to control and ultimately eliminate this globally distributed parasite species thereby freeing endemic countries from the heavy public health burden it exacts. Modified at current lines 280-282 as follows to improve clarity; These findings indicate that despite the uniform morphology and cellular invasion approach used between the species; the underlying transcriptomic signature of individual sporozoites differs between species.
• Reviewer #1: Pg 12 lines 347. The sentence is too long consider shortening it to be clear. What does 'intriguing' mean in this case?
To improve clarity, this sentence now reads at lines 346-349: Noting the role of translational repression in eukaryotic developmental fating via RNA-binding proteins such as PUF2 [57], it is intriguing to speculate on possible links between this population and the bradysporozoites hypothesized to become hypnozoites.
We use the word "intriguing" to emphasise this is an important biological connection between the discussed bradysporozoite hypothesis and the known function of PUF2 in other organisms but to emphasise this is further speculation given the lack of definitive evidence in our current study.
• Reviewer #1: Pg 14 'Hepatocyte infection and liver stage assessment' What is the need of describing a procedure that was described elsewhere? Consider deleting the entire paragraph.
We have removed this paragraph and added an additional reference of the protocol Reviewer #2 suggested some grammatical changes in the PDF attachment that we have modified at Line 29: addition of 'and causing malaria'