Spatiotemporal analysis of sporozoite maturation and infectivity

Spatiotemporal analysis of sporozoite maturation and infectivity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Spatiotemporal analysis of sporozoite maturation and infectivity View ORCID Profile Lisa H Verzier , Jean-Michel Thiberge , View ORCID Profile Eduardo Aliprandini , Vanessa Lagal , View ORCID Profile Pauline Formaglio , View ORCID Profile Olivier Silvie , View ORCID Profile Rogerio Amino doi: https://doi.org/10.1101/2025.07.21.665974 Lisa H Verzier 1 Institut Pasteur, Inserm U1347, Université Paris Cité, Malaria Infection and Immunity, ParasitInnov, BioSPC , F-75015, Paris, France 2 Department of Immunology and Infectious Disease, Harvard T. H. Chan School of Public Health , Boston, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lisa H Verzier Jean-Michel Thiberge 1 Institut Pasteur, Inserm U1347, Université Paris Cité, Malaria Infection and Immunity, ParasitInnov, BioSPC , F-75015, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eduardo Aliprandini 1 Institut Pasteur, Inserm U1347, Université Paris Cité, Malaria Infection and Immunity, ParasitInnov, BioSPC , F-75015, Paris, France 3 Affinity Reagents Team, Biologics Engineering, AstraZeneca , Cambridge, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Eduardo Aliprandini Vanessa Lagal 1 Institut Pasteur, Inserm U1347, Université Paris Cité, Malaria Infection and Immunity, ParasitInnov, BioSPC , F-75015, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pauline Formaglio 1 Institut Pasteur, Inserm U1347, Université Paris Cité, Malaria Infection and Immunity, ParasitInnov, BioSPC , F-75015, Paris, France 4 Sorbonne Université, CNRS, Inserm, Centre d’Immunologie et des Maladies Infectieuses, CIMI , F-75013 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pauline Formaglio Olivier Silvie 4 Sorbonne Université, CNRS, Inserm, Centre d’Immunologie et des Maladies Infectieuses, CIMI , F-75013 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Olivier Silvie Rogerio Amino 1 Institut Pasteur, Inserm U1347, Université Paris Cité, Malaria Infection and Immunity, ParasitInnov, BioSPC , F-75015, Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rogerio Amino For correspondence: roti{at}pasteur.fr Abstract Full Text Info/History Metrics Preview PDF Abstract Plasmodium sporozoites must undergo tightly regulated developmental transitions to become infectious and be successfully transmitted from the mosquito vector to a mammalian host. While transcriptomic studies have revealed stage-specific changes across sporozoite populations, the functional consequences of these transitions remain unclear. Here, using P. berghei , we characterised over time the infectivity of sporozoite forms collected from the midgut, haemolymph, salivary glands and saliva. We show that salivary gland invasion is required but not sufficient for sporozoite optimal infectivity, with the acquisition of hepatocyte cell traversal and invasion progressively increasing until a plateau from 18 days post-infection onwards. Using a stage-specific fluorescent reporter as maturation marker, we correlated its high expression with time and infectivity for each compartment but only salivary gland sporozoites acquired maximal infectivity. Notably, our data suggest that salivated sporozoites—the natural transmission form—exhibit enhanced infectivity relative to gland-resident forms both in vitro and in vivo early after salivary gland invasion. This difference decreases following optimal maturation inside the glands over time. These observations show a crescent gradient of sporozoite maturation and infectiveness from the midgut to the saliva when isolated at the same time of infection, which is mainly regulated by the sporozoite invasion of salivary glands. Introduction Plasmodium parasites have evolved to transition between two radically different environments as they cycle between vertebrate and invertebrate hosts. Transiting from a warm-blooded mammal to the mosquito and vice-versa, they must adapt to hosts with distinct metabolic demands and immune landscapes. While transmission to the mosquito triggers the rapid differentiation of gametocytes into gametes, return to the mammalian host depends entirely on a single, multi-invasive specialised form, the sporozoite (SPZ). SPZ are formed within oocysts nestled under the basal lamina of the mosquito midgut. Upon maturation, they egress into the haemolymph—a process thought to involve protease secretion and activation of gliding motility ( 1 – 3 ). They are then passively carried through the haemolymph until they encounter the salivary glands, where they specifically recognise and invade acinar cells ( 4 – 7 ). Salivary gland invasion is a complex, multi-step process. SPZ must breach the basal lamina, traverse acinar cells via transient vacuoles or membrane rupture, and ultimately enter the secretory cavities ( 8 – 11 ). While most parasites accumulate in a quiescent state within the secretory cavities, a minority reach the salivary ducts, becoming candidates for injection into the mammalian dermis during the next bloodmeal ( 12 – 14 ). These anatomical distinctions have led to the operational classification of SPZ into oocyst or midgut (MG), haemolymph (HL), and salivary gland (SG) forms. The SPZ infectivity—defined as the ability of a pathogen to enter, survive, and replicate within a host ( 15 )—entails the parasite’s capacity to navigate host tissues, traverse cells, and invade and develop intracellularly inside a targeted cell. Following deposition by an infected Anopheles mosquito, typically in the dermis, SPZ display rapid motility (>1 µm/s) that enables them to leave the injection site and locate blood vessels ( 16 – 18 ). Once in circulation, SPZ are transported to the liver where they cross the sinusoidal barrier via endothelial or Kupffer cell traversal, or via a cell traversal independent mechanism, ultimately reaching the hepatic parenchyma ( 4 , 19 – 22 ). After traversal of hepatocytes, yet-unknown factors trigger invasion and subsequent differentiation into the exoerythrocytic form (EEF) ( 23 – 28 ). Motility, cell traversal, and hepatocyte invasion are therefore the principal cellular determinants of SPZ infectivity. The question of when and how sporozoites acquire infectivity has been the subject of long-standing investigation since the 1930’s ( 29 – 31 ). In 1975, Vanderberg demonstrated that SG SPZ established infection in mice, while those from HL and MG were less infectious. He then proposed that infectivity acquisition was time-dependent rather than tissue-dependent ( 32 ). A subsequent study in 1992 challenged this view, showing that midgut SPZ could colonise SG in naïve mosquitoes, whereas SG SPZ could not re-invade ( 32 ), pointing to tissue-specific developmental programming. Corroborating the later study, transcriptomics comparing MG and SG SPZ identified genes “up-regulated in infective sporozoites” (UIS) and “up-regulated in oocyst sporozoites” (UOS) ( 34 – 37 ). Genes included in the former list include UIS3 and UIS4 which are essential in liver stage development, while TREP (UOS3), CRMP2 and CRMP4 are essential for egress from oocyst and SG invasion ( 35 , 38 – 41 ). Additional transcriptomic and proteomic profiling of SPZ populations has revealed significant differences across forms, including the discovery of translational repression mechanisms during SPZ maturation ( 14 , 28 , 36 , 37 , 42 ). Critical liver-stage proteins such as UIS3 and UIS4 were identified through such comparative approaches. More recently, single-cell RNA-sequencing studies have provided fine-grained resolution of SPZ transcriptional heterogeneity across forms ( 43 – 45 ). Notably, Real and colleagues reported a major transcriptional shift between P. falciparum oocyst and HL SPZ, while Bogale and co-workers observed a larger shift between HL and SG SPZ in P. berghei . Whether these discrepancies reflect species differences, collection techniques, or biological variation remains unresolved. Despite these molecular insights, how transcriptomic and proteomic changes translate into functional infectivity remains poorly defined. A detailed understanding of how sporozoites acquire infectivity is not only fundamental to malaria transmission biology but also critical for the rational development of axenic sporozoites, which has yet to replicate this maturation process outside the mosquito vector ( 46 , 47 ). Moreover, most datasets represent single timepoints and do not capture the temporal dimension of SPZ maturation. The role of salivation itself—the physiological route of transmission—also remains understudied. To address these gaps, we examined the infectivity of P.berghei SPZ collected across different compartments and timepoints using complementary in vitro and in vivo assays. Our findings reveal that both tissue and duration of residence influence SPZ competence and identify a transcription marker as a proxy for maturation. Additionally, we uncover that salivated SPZ—the natural form transmitted to mammals—display enhanced infectivity relative to SG SPZ, particularly during early mosquito infection, highlighting the biological relevance of this neglected population. Results Sporozoites acquire optimal infectivity following salivary gland invasion Plasmodium SPZ are formed within oocysts located between the basal lamina surrounding the mosquito MG and the epithelial layer. Upon maturation, they egress into the HL and subsequently invade the SG, where they reside quiescent, possibly for several days, prior to transmission to a mammalian host during a blood meal ( Fig. 1a ). To correlate transcriptomic insights with phenotypic evidence, we systematically characterised the infectivity of these SPZ populations by quantifying their capacity for hepatocyte invasion and cell traversal, measured respectively by scoring the percentage of fluorescent SPZ inside HepG2 hepatoma cells and by the percentage of wounded and resealed HepG2 cells after 2 hours of incubation with SPZ collected over the course of mosquito infection (12-28 days post-infection (pi)). Download figure Open in new tab Figure 1. Sporozoite (SPZ) formation in the midgut (MG) & infectivity acquisition inside salivary glands (SG). a. Scheme showing the different forms of SPZ, forming inside oocysts on the mosquito MG, egressing in the haemolymph (HL) and inside SG. b. Percentage of MG, HL and SG SPZ inside HepG2 cells (cell invasion) according to the day SPZ were collected. c. Percentage of wounded and resealed HepG2 cells (cell wounding) according to the day and tissue SPZ were collected. d . Percentage of MG, HL and SG SPZ isolated 14 days pi able to glide in 2D measured as complete a full circle. e . Comparison of the log parasitaemia across time after intravenous injection of 5,000 HL or SG SPZ isolated 14 days pi in C57Bl/6 mice. As shown in Fig. 1b-c , MG SPZ exhibited minimal infectivity, with fewer than 0.25% successfully invading HepG2 cells. Consistently, the proportion of wounded HepG2 cells remained exceedingly low (<1.5%), corroborating the lack of expression of proteins involved in cell traversal in MG sporozoites ( 43 , 45 , 48 ). Upon egress into the HL, SPZ demonstrated a modest gain in invasive ability, reaching a maximum of 7.4% invasion by three weeks pi; however, traversal capacity remained poor, with wounded cells never exceeding 3% in average. By contrast, SG SPZ displayed a significantly greater capacity for both invasion and traversal, with 35% of parasites invading HepG2 cells and wounding 20% of cells, consistent with transcriptional reprogramming and acquisition of infectivity following SG invasion as described in prior studies ( 36 , 43 – 45 ). Both anatomical location and time post infection influenced sporozoite functionality ( Fig. 1b-c ). HL and SG SPZ harvested at days 12 and 15 pi were significantly less competent in both invasion and traversal compared to those collected at day 18 pi or later. Logarithmic modelling of invasion and traversal rates for SG SPZ revealed a plateau phase around day 18 pi (R² = 0.99 and 0.94, respectively), indicative of a progressive acquisition of infectivity. Considering that in our experimental setup the number of SPZ within the SG rises until approximately 18 days pi before reaching a near steady state ( Supplementary Fig. 1a ), and that the proportion of invasive SPZ (∼35%) observed at day 18 pi approximates the number present at day 15 pi, it is likely that sporozoites require several days within the glands to achieve full maturation. Download figure Open in new tab Supplementary Figure 1. Dynamics of the different SPZ forms number and morphology. a . Number of SPZ from each compartment throughout mosquito infection. B. SPZ expressing GFP constitutively under the eef1a promoter were gated according to their green fluorescent; the resulting profile by side scatter area (x axis, logged) and forward scatter width (yaxis) is displayed in green and show a reduction in the spread of forward scatter values, with SG and SPZ having higher values. To complement these observations, we assessed SPZ motility, a function critical for both invasion and traversal ( 49 , 50 ). Motility assays performed at 14 days pi revealed that MG SPZ were essentially incapable of performing circular gliding under two-dimensional substrate, consistent with their poor infectivity. HL SPZ exhibited limited motility, with only 7.3% completing a full circle. In contrast, SG SPZ demonstrated robust, albeit variable, motility, with on average 65% of parasites executing at least one complete circle in two minutes of analysis ( Fig. 1d ). Finally, to evaluate in vivo infectivity, we intravenously injected mice with 5,000 SPZ from day 14 pi, focusing on the two stages that have shown infectivity (HL and SG) and monitored subsequent parasitaemia. Consistent with in vitro observations, SG SPZ were highly infectious, with all mice displaying detectable parasitaemia by day 3 pi. By contrast, HL SPZ exhibited a one-day delay in patency, and by day 4 pi, parasitaemia in this group was approximately 1.5 logs lower than in mice infected with SG SPZ. Together, these findings demonstrate that SPZ acquire full infectivity only after SG invasion, with MG SPZ remaining largely non-infectious, HL SPZ gaining limited infectivity, and SG SPZ progressively maturing into fully invasive forms over several days, reaching optimal infectivity around 18 days pi. Sporozoites’ transcriptional maturation marker Uis4 , the most abundant transcript in P.berghei SG SPZ, displays increasing expression along pseudotime trajectories in single-cell RNA-seq analyses ( 43 , 45 ). To dynamically monitor SPZ maturation, we used a fluorescent reporter parasite line, which expresses mCherry under the control of the uis4 promoter ( 51 ). This line allows measurement of uis4 promoter activity via mCherry fluorescence, while expressing GFP under the control of the constitutive eef1a promoter, facilitating detection and flow cytometric analysis of SPZ ( Fig. 2a ). Download figure Open in new tab Figure 2. Measurement of SPZ maturity in tissues over time using a stage-specific fluorescent reporter. a. Fluorescence analysis of transgenic SPZs expressing GFP under the control of a constitutive promoter ( eef1a ) and mCherry under the control of a stage-specific promoter ( uis4 ). b. SPZs were collected from MG, HL and SG in different days after the mosquito infectious feeding and cytometric analysis shows tissue and time-dependent increase of uis4 -mcherry fluorescence. c-d . Percentage of SPZ expressing high levels of mCherry was correlated with the percentage of SPZ that invaded HepG2 cells (left) or the percentage of wounded cells (right) for each compartment ( c ) or altogether ( d ). Pearson coefficient and their significance are shown on each graph (Pearson correlation, * p < 0.05,** p < 0.01). Using this line, we isolated MG, HL, and SG SPZ from the same infected mosquitoes and quantified GFP and mCherry fluorescence by flow cytometry from 11 to 27 days pi. GFP fluorescence remained stable across all stages, enabling consistent gating of SPZ based on GFP intensity and side scatter properties ( Supplementary Fig. 1b ). On the other hand, transcriptional activity measured by mCherry fluorescence in mCherry@ uis4 parasites increased as SPZ progressed from MG to HL and SG forms in the same mosquito as well as in each compartment over time ( Fig. 2 ). We further characterised the reporter line across the course of infection, stratifying mCherry fluorescence into three categories of intensity: mCherry high (10□-10□, red), mCherry low (10³-10□, yellow), and mCherry neg (<10³, green). MG SPZ consistently failed to reach high levels of mCherry, whereas HL and, more prominently, SG SPZ showed progressive increase in mCherry expression over time. This observation reinforces the major transcriptional distinction between MG SPZ and the HL/SG SPZ populations, as highlighted in previous single-cell studies, and highlights a slow but steady and time-dependent transcription activation or de-repression ( 43 , 44 ). Given the apparent temporal trend, we examined the relationship between the proportion of mCherry high SPZ and their capacity to invade HepG2 cells. As shown in Fig. 2b , despite the small number of points, significant correlations were observed when analysing MG and SG SPZ individually ( Fig. 2c , left panels, r > 0.98, p < 0.05* for MG and SG SPZ). However, when stages were pooled, the overall correlation weakened ( Figure 2d , r = 0.731; p = 0.02*). Moreover, mCherry expression in SG SPZ at day 13 pi was comparable to HL SPZ at day 18-19 pi, despite displaying ∼7 times more invasion ( Fig. 1b-c ; Fig. 2c , left panels). Similar results were observed regarding cell traversal, except that only SG SPZ showed significant correlation between cell wounding and mCherry expression (r = 0.995, p = 0.01**; Fig. 2c , right panels) corroborating that only SG SPZ express the main proteins involved in cell wounding. The linear regressions analysis also shows that MG and HL SPZ share a similar profile of mCherry/infectivity, with MG SPZ being proportionally less mature and infectious than HL SPZ. Acquisition of optimal motility, cell traversal and cell invasion activities is only observed after invasion of SG. This finding highlights fundamental functional differences between different SPZ forms, even if they express similar levels of uis4 -driven mCherry. Therefore, time-dependent uis4 transcriptional maturation occurs in each mosquito compartment, but the maximal estimated infectivity of MG and HL SPZ (100% of mCherry high , <10% of invaders and 20% of invaders and ∼10% of wounded cells). Together, these data suggest that while uis4 promoter activity serve as a useful marker of sporozoite maturation within each SPZ form, it does not reliably predict infectivity across different SPZ populations. In particular, late-stage HL SPZ can express high levels of uis4-driven mCherry yet remain markedly much less infectious than relatively immature SG SPZ. Salivated sporozoites display enhanced infectivity during early mosquito infection We next turned our attention to salivated (SAL) SPZ. Although SG SPZ are routinely used in experimental infections, only SAL SPZ have the physiological opportunity to initiate infection in a mammalian host. Furthermore, single-cell transcriptomic analyses have revealed distinct transcriptional profiles between SG and SAL SPZ in P. berghei ( 43 ), which may help explain why mosquito bite-mediated infections are more efficient than direct intradermal injections of SG SPZ. Indeed, a small number of mosquito bites—delivering only a few tens to hundreds of SPZ—can establish blood-stage infections comparable to those resulting from intradermal injection of 5,000 SG SPZ ( 52 ). To investigate this phenomenon, we used the mCherry@ uis4 line to compare mCherry fluorescence between SG and SAL SPZ. Saliva was collected by placing gel-loading tips containing 5 µL of 1% fatty acid-free BSA in DPBS around the mosquito proboscis and allowing salivation at 37°C for 15-20 minutes. As shown in Fig. 3a , SAL SPZ exhibited markedly higher mCherry fluorescence compared to SG SPZ 13 days pi, with no detectable mCherry neg parasites, indicating elevated uis4-driven transcription. Control experiments confirmed that incubation at 37°C did not alter mCherry expression in SG SPZ ( Supplementary Fig. 2a ), suggesting that either mCherry high SPZ are preferentially expelled or that the process of salivation itself induces transcriptional or translational changes. Download figure Open in new tab Supplementary Figure 2. Temperature does not induce a shift in uis4 transcription while salivation protocol can lead to loss of infectivity a . SG and SAL mCherry@ uis4 SPZ were collected and gated as show in Supplementary Fig. 1a . Difference mCherry fluorescence profiles were observed (top) and which were unchanged after incubation at 37°C for 20 min, condition of salivation (bottom), confirming the higher mCherry expression in SAL SPZ was not due to the higher temperature they were exposed to. b . Infected mosquitoes were left to feed on 1% fatty acid-free BSA/DPBS for 10 min before count and injection into 3 four-week-old C57Bl/6 mice. No mice developed a blood stage infection when injected with SAL SG while control mice who received SG SPZ were all positive 3 days pi. Download figure Open in new tab Figure 3. Salivated (SAL) SPZ are more infectious in vivo early in mosquito infection. a . Fluorescence analysis of MG, HL, SG and SAL SPZ (blue, red, green, and black respectively in histogram) collected 13 days post-infection (pi) summarised in a heatmap with percentage fo SPZ expressing no (green; 10 5 ) levels of mCherry. b-c . 50 SG or SAL SPZ were isolated 15 days pi ( b ) or 250 SG or SAL SPZ were isolated 21 days pi ( c ) and injected IV in C57Bl/6 mice. Parasitemia was followed from 3 days pi and compared by two-way ANOVA (left graph). Parasitemia and prevalence of infection on day 6 pi were also compared (right graph, unpaired t-test and Fisher’s exact test). We next assessed whether the differences in transcriptional maturity correlated with infectivity in vivo . Infections were performed at 15 days pi and, due to the limited numbers of SAL SPZ available, only 50 SPZ were injected per C57BL/6 mouse intravenously. Attempts to increase SPZ output by adapting feeder-based methods described in other studies proved unsuccessful as the isolated SAL SPZ were not infectious ( Supplementary Fig. 2b ). Parasitaemia post-IV injection was monitored daily from day 3 until mice reached 0.5% parasitaemia or day 10. As shown in Fig. 3b , mice infected with SAL SPZ exhibited significantly higher parasitaemia trajectories compared to those infected with SG SPZ (Two-way RM ANOVA, p = 0.02**). While prevalence of infection did not differ significantly— possibly due to the limited number of mice—parasitaemia at day 6 pi was significantly higher in SAL SPZ-infected animals (Mann-Whitney test, p = 0.048*). To determine whether this advantage persisted at later stages, infections were repeated at 21 days pi, when mCherry high population is close to its maximum value in SG SPZ ( Fig. 2b ) and SG SPZ have reached their optimal infectivity ( Fig. 1b-c ). Owing to increased SPZ yields, 250 SPZ were injected per mouse. As shown in Fig. 3c , although a trend towards greater infectivity of SAL SPZ remained, the differences did not reach statistical significance (Two-way RM ANOVA, p = 0.09). Salivated sporozoites form larger and more transcriptionally active liver stages in vitro To assess whether the enhanced infectivity of SAL SPZ observed in vivo is reflected during liver-stage development and whether the trend observed after day 21 pi could be physiologically relevant, we performed in vitro infections of HepG2 cells using SAL and SG SPZ harvested at 19 days pi. Owing to this bottleneck, SAL and SG SPZ infections were performed at equivalent multiplicities of infection (MOI), with an additional SG control infection at a standard MOI of 1:4 (SPZ:HepG2 cell, high MOI). Quantification of EEFs at 48 hours pi revealed a ∼2.5 times more infected HepG2 cells in the SAL group relative to SG control ( Fig. 4a, p = 0.045*), suggesting either increased efficiency of productive invasion or enhanced intracellular survival. Download figure Open in new tab Figure 4. SAL SPZ are more efficient at infecting HepG2 in vitro , forming larger exo-erythrocytic forms (EEFs) a . Higher percentage of HepG2 cells are infected by SAL than SG SPZ when measured 48h pi. Four infections were performed with 100 to 500 SPZ isolated 19 days pi depending of the rate of salivation (paired t-test). b . Comparison of linear regressions of GFP and mCherry fluorescence signals of 48h EEFs from SG and SAL SPZ using the same MOI, and from standard infection using SG SPZ at MOI 1:4 (SG MOI 1:4). P-values are from slope comparison with the SG MOI 1:4 regression. c-d . Area of EEFs measured 48h pi using mCherry@uis4 parasites ( c ) and GFP @hsp70 parasites ( d ). Adjusted P-values obtained using Holm-Šídák’s multiple comparisons test. To further explore the uis4-driven maturity marker in EEFs, we analysed the relationship between constitutively expressed GFP ( eef1a -driven) and mCherry ( uis4 -driven) fluorescence in individual EEFs. Quantitative fluorescence analysis by flow cytometry indicated that EEFs derived from SAL SPZ exhibited significantly higher mCherry/GFP intensity ratio than SG SPZ at low and high MOI. Accordingly, the slope of the curve of EEFs derived from SG SPZ high MOI significantly differed from that using SAL SPZ (p <0.0001, Fig. 4b ), but was similar to the slope of the linear regression using SG SPZ low MOI (p = 0.285, Fig. 4b ). This indicated that the higher transcription of uis4- driven mCherry in SAL SPZ in comparison to SG SPZ ( Fig. 3A ) translated into a higher expression of this maturity marker also in liver-stages. This increased fluorescence ratio could also be related to an increase in parasite biomass, possibly reflecting more advanced development and/or larger EEFs. Quantification of EEF area at 48 pi indeed revealed significantly larger EEFs in the SAL SPZ-infected cultures ( Fig. 4c ). To check if the observed phenotype was specific to the maturity reporter parasites, we repeated the experiment using hsp70- driven GFP SPZ, obtaining the same result ( Fig. 4d ). Altogether, these findings suggest that SAL SPZ give rise to a higher number of, and transcriptionally more active, liver-stage parasites in vitro, even after SG SPZ have reached full infectivity. The transcriptional variability observed across individual EEFs further underscores distinct developmental dynamics between SG- and SAL-derived infections, warranting deeper investigation. Discussion Sporozoite maturation has long been a subject of study across Plasmodium species and Anopheles mosquitoes, giving rise to discrepancies in reported infectivity and data interpretation. Transcriptomics studies comparing different SPZ forms—MG, HL, SG and occasionally SAL— have recently been published to investigate the transcriptional differences of these developmental stages, though no functional analysis was performed and correlated to the transcriptomics profiles ( 36 , 37 , 43 – 45 ). One of the key contributions of this study lies in its longitudinal approach. By characterising infectivity of all forms of SPZ throughout mosquito infection, we demonstrated that both tissue and time are essential to P. berghei SPZ maturation, reconciling different studies suggesting one or the other factor as a main or only driver for maturation ( 32 , 33 , 53 , 54 ). While HL SPZ reached some degree of infectivity with time, they remained sub optimally infectious, with lower in vitro infectivity and a delayed prepatent period when injected intravenously into mice, sharing a similar profile with MG SPZ, which is quite distinct from that of optimally infectious SG SPZ. Interestingly, SG SPZ also matured with time, reaching their peak infectivity and plateauing 18 days pi. Correlating with this time-dependent maturation, uis4- driven transcripts level can be used as a marker for infectivity, though only within SPZ forms as similar uis4 expression in HL and SG SPZ results in dissimilar motility, invasion and cell traversal activities. Finally, SAL SPZ showed enhanced infectivity compared to SG SPZ, suggestive that either the most mature SPZ are salivated or that salivation itself triggers further maturation. Increase of motility in the mature SG SPZ population has been associated with the colonization of the mosquito salivary ducts and could explain the ejection of this more infectious population during salivation ( 12 ). While the characterisation of this population was limited by the number of SPZ that could be obtained, the preliminary data provided here indicate SAL SPZ are more likely to establish EEFs which are larger and more transcriptionally active. Although the role of UIS4 in evading host clearance suggests that the increase in EEF numbers may result from reduced elimination ( 55 ), whether other mechanisms—such as enhanced invasion or egress of more liver-stage merozoites—contribute to higher parasitaemia in SAL-infected mice remains to be determined. SG invasion remains a poorly understood mechanism, with few vector-parasite interaction identified ( 5 , 35 , 39 , 56 ). The key role of rhoptry, invasive organelles shown to be essential to hepatocyte and red blood cell invasion, has long been studied though only recently was confirmed as SG SPZ are found to have fewer rhoptries than MG and HL SPZ ( 8 , 57 – 61 ) and knockout or knockdown of rhoptry proteins impair SG invasion ( 8 , 58 – 61 ). This clear distinction between SG SPZ to the previous forms of the parasite is likely related to the transcriptional and translational changes observed in SG SPZ such as increased uis4 transcripts levels, as well as the global changes described by others including the high expression of cell traversal proteins ( 36 , 37 , 43 , 45 , 62 ). Although HL SPZ were found infectious and triggered mice immune response in another study ( 63 ), their observations are in accordance to ours as they found HL SPZ to be mostly non motile in 2D, with reduced infectivity in vitro and one day delayed (10-fold reduction)—though not significant in their case—prepatent period in vivo . Interestingly, SG invasion is essential but not sufficient for maturation as P. yoelii SPZ that could invade An. albimanus SG could not establish a liver infection in mice suggesting that other environmental factors are at play than solely rhoptry discharge ( 64 ). Additionally, sporozoite maturation is time-dependent, as was first noted in P. gallinaceum ( 53 , 54 ), but appears to plateau with only ∼30 % of SG SPZ capable of infecting hepatocytes in vitro at any given time, suggesting that not all sporozoites attain full infectivity. Why only a subset of SG SPZ is competent for invasion, and whether this correlates with their spatial positioning within the gland, remains to be determined. Recent advances in expansion microscopy and spatial transcriptomics, combined with our newly characterised fluorescent reporter line, offer promising tools to begin resolving the anatomical and molecular determinants of sporozoite infectivity at single-parasite resolution ( 57 , 65 ). Notably, our data differ from a similar study looking at P . falciparum SG SPZ infectivity ( 66 ). While we found no decrease in P. berghei in vitro infectivity from 18 days pi to 28 days pi, van Schuijlenburg and colleagues found that P. falciparum SPZ slowly “age” and become less motile, infectious and immunogenic. Single-cell transcriptomic analyses support this distinction: while both the Bogale and Real datasets describe stage-dependent transcriptional reprogramming, the timing and nature of these shifts differ between species. P. berghei SPZ appear to maintain a quiescent state within the SG more effectively and are able to activate rapidly upon salivation. This is evident both in scRNA-seq data—reflected in a higher proportion of ribosomal RNA and markers of translational activation—and in our functional assays ( 43 ). On the other hand, Real et al. have found little to no difference in transcription comparing SG SPZ and SAL SPZ ( 44 ). These biological differences are echoed in practical handling: while P. berghei SG SPZ are typically dissected in PBS with dissection lasting for several hours, P. falciparum require maintenance in insect medium and lose infectivity within 1-2 hours. Together, these findings suggest that P. berghei sporozoites are primed for rapid activation and accelerated protein synthesis upon salivation, a feature that may facilitate efficient nutrient acquisition and successful transition to liver-stage development. Further optimisation of the salivation protocol will be critical to enable a more detailed comparison between SG and SAL SPZ and to elucidate how their functional and molecular differences influence liver-stage infection. When mosquitoes were allowed to salivate into feeders, no infectivity was observed—possibly due to excessive dilution, reducing saliva proteins concentration and SPZ-SPZ proximity cues that may help preserve viability. Enhancing SPZ yield and concentration would not only facilitate more robust intravenous infections but also enable intradermal injections, which are more physiologically relevant. Such a protocol would likely exacerbate any phenotype linked to salivation, especially in later timepoints where the functional divergence between SAL and SG SPZ may become more subtle. Beyond infection rates, further characterisation will be needed to determine whether the observed increase in parasitaemia following SAL SPZ injection reflects greater hepatocyte invasion, reduced clearance, or both. Analysis of merosome output and quantification of liver-stage merozoite release could provide mechanistic insight into the basis of this in vivo phenotype. Ultimately, understanding how sporozoites acquire infectivity—and the environmental and molecular triggers that orchestrate this transition—is essential for the rational development of axenic SPZ. Defining the sequence of events required for functional maturation will not only illuminate fundamental aspects of Plasmodium biology but also support efforts to produce transmission-competent SPZ for vaccine and therapeutic applications in the absence of a mosquito vector. Method Ethics statement Experiments involving animals were all approved by the Animal Care and Use Committee of Institut Pasteur (CETEA Institut Pasteur 2013-0093, Ministère de l’Enseignement Supérieur et de la Recherche MESR 01324) and performed according to European guidelines and regulations (directive 2010/63/EU). Mice were sourced from Janvier Labs. Parasites and mosquito infections Different reporter parasite lines were used in this study, all generated in the P. berghei ANKA background. A strong fluorescent line that expresses GFP under the hsp70 promoter ( 67 ) was used for in vivo experiments to facilitate the measure of blood parasitaemia. The other reporter line used was a P. berghei ANKA expressing GFP under the eef1a promoter background: a transcription reporter generated by introducing mCherry driven by uis4 5’ and 3’ UTR ( 51 ). All mosquitoes used were Anopheles stephensi mosquitoes (SDA500 strain) that were reared in the Centre for Production and Infection of Anopheles (CEPIA) at Institut Pasteur and maintained on 10% sucrose/water. For production of P. berghei SPZ, 4-week old RjOrl:SWISS were infected by IP injection of infected red blood cells. Parasiteamia was left to establish over 3-4 days until mature gametocytes were present and mice were used to infect 1 to 2-day old mosquitoes. One week post infectious blood meal, mosquitoes were fed on naïve 5-week old RjOrl:SWISS mice. SPZ isolation SPZ isolation was performed in DPBS 1X as early as 11 days post infectious bloodmeal, and up to 28 days pi. MG SPZ were obtained by crushing infected MG with a pestle, releasing SPZ from the oocysts. HL SPZ were isolated by cutting the last segments of a mosquito’s abdomen and washing the HL out by flushing 10-20 µL of DPBS with an insulin syringe introduced into the mosquito thorax. SG SPZ were isolated from infected glands and freed after crushing them with a pestle. In all cases, mosquito debris were removed by filtrating through a 35 µm filter before counting and further processing. Mosquito salivation Mosquitoes were aspirated, knocked down on ice and sorted under a fluorescent stereomicroscope to select insects with infected SG. They were then attached on a microscope slide by gluing their wings to tape. Their proboscises were encased into a gel loading tip containing 5µL of 1% fatty acid-free BSA/DPBS (Sigma-Aldrich A6003). Mosquitoes were left to salivate 20 min at 37°C in a humidified chamber and tips’ content was pooled before counting on Kova or by flow cytometry using CountBright™ Absolute Counting Beads (Invitrogen C36950). To account for different handling, all SPZ used in head-to-head comparison to SAL SPZ were dissected in 1% fatty acid-free BSA/DPBS, diluted to the same concentration as the SAL SPZ, and incubated at 37°C for 20 min before proceeding. Cell maintenance and in vitro assays HepG2 cells (ATCC HB-8065) were maintained on DMEM high glucose containing GlutaMAX (Gibco 10566016) completed with 10% heat-inactivated foetal bovine serum, 1X of non-essential amino acids, 2% penicillin-streptomycin-neomycin. Cells were passaged when reaching confluency or every 3-4 days. In vitro assays were performed as described before ( 68 ). Briefly, they were seeded at 40,000 cells per well into 96-well plates a day prior to infection. SPZ—isolated as described above—were added to cells before gently centrifugating parasites onto them at 100 x g for 3 min. For cell wounding and hepatocyte invasion, media was collected 2h pi and cells were washed in DPBS, collected as well. Cells were trypsinised and pooled with the recovered media before analysis by flow cytometry (CytoFLEX S, Beckman). Percentage of wounded cells was determined by gating HepG2 cells followed by dextran-rhodamine + HepG2. Cell invasion, or the percentage of SPZ that have invaded, was calculated by dividing the number of intracellular parasite (GFP + HepG2) by the total number of SPZ recovered (sum of GFP + HepG2 and GFP + SPZ detected). In the case of EEF development assays, cells were washed 2h pi and incubated another 46h with daily media change. Two days pi, cells were washed and trypsinised before flow cytometry analysis. SPZ challenge As described previously ( 68 ), four- to five-week-old C57Bl/6 mice were used for in vivo infections. SPZ were collected as described above and injected in the tail vein. 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OpenUrl CrossRef PubMed Web of Science 68. ↵ Aguirre-Botero MC , Pacios O , Celli S , Aliprandini E , Gladston A , Thiberge JM , et al. Late killing of Plasmodium berghei sporozoites in the liver by an anti-circumsporozoite protein antibody . eLife . 2025 Feb 14; 14 : RP105291 . OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted July 21, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Spatiotemporal analysis of sporozoite maturation and infectivity Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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Share Spatiotemporal analysis of sporozoite maturation and infectivity Lisa H Verzier , Jean-Michel Thiberge , Eduardo Aliprandini , Vanessa Lagal , Pauline Formaglio , Olivier Silvie , Rogerio Amino bioRxiv 2025.07.21.665974; doi: https://doi.org/10.1101/2025.07.21.665974 Share This Article: Copy Citation Tools Spatiotemporal analysis of sporozoite maturation and infectivity Lisa H Verzier , Jean-Michel Thiberge , Eduardo Aliprandini , Vanessa Lagal , Pauline Formaglio , Olivier Silvie , Rogerio Amino bioRxiv 2025.07.21.665974; doi: https://doi.org/10.1101/2025.07.21.665974 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)