Up to date, the tropical disease malaria is one of the most devastating infectious diseases in the world, causing 216 million new cases and approximately 655.000 casualties each year [1]. Disease treatment and control measures are undermined by the spread of drug resistance in malaria parasites, particularly in populations of Plasmodium falciparum, the agent responsible for malaria tropica (reviewed in [2]).

The transmission of malaria parasites from the human to the mosquito is mediated by specialized sexual precursor cells, the intraerythrocytic gametocytes. Maturation of P. falciparum gametocytes from stage I to stage V takes approximately 10 days, and during this period the gametocytes maintain a stable cell cycle arrest (reviewed in [3, 4]). The mature gametocytes circulate in the human’s blood stream, but remain dormant until they are taken up by a blood-feeding mosquito.

When entering the mosquito midgut together with the blood meal, the gametocytes become activated from the dormant stage by external stimuli, i.e. a drop in temperature and the contact with the mosquito-derived molecule xanthurenic acid (XA) (reviewed in [5, 6]). Gametocyte activation leads to rounding up of the cell, followed by parasite egress from the enveloping erythrocyte, which involves the rupture of two membranes, the parasitophorous vacuole membrane (PVM) and the erythrocyte membrane [7] (reviewed [8]). During gametogenesis the microgametocyte replicates its genome three times in order to produce eight motile microgametes. Following the fusion of micro- and macrogametes a zygote forms and develops into an infective ookinete within the following 24 hours. The motile ookinete possesses an apical complex which enables it to traverse the midgut epithelium before settling down and forming an oocyst between epithelium and basal lamina (reviewed in [5]).

During gametocytogenesis, P. falciparum expresses a new set of genes important for sexual development [9–14]. Ingestion by the blood-feeding mosquito again triggers molecular changes in the sexual stage parasites, which help adjusting the gametocytes to the insect and which on the one hand initiate sexual reproduction and further development of the parasite in the vector, on the other hand prepare the emerging gametes for the hostile environment of the mosquito midgut. Noteworthy, the midgut stages have to persevere outside a host cell for more than one day. During this time period, the cells are highly vulnerable to the aggressive factors of the gut, which among others include bacteria as well as human immune cells, antibodies and complement proteins present in the blood meal, and this exposure results in an approximate 1000-fold loss of parasite abundance [15] (reviewed in [5, 6]).

Gametocyte maturation and gametogenesis are particularly accompanied by the coordinated expression of numerous adhesive surface proteins, including the EGF domain-containing proteins Pfs25 and Pfs28, the 6-cys proteins Pfs230 and Pfs48/45, and the LCCL domain-containing PfCCp proteins. It is noteworthy that the majority of these proteins can be divided into two classes: One class of the adhesion proteins, including Pfs230, Pfs48/45 and the six PfCCp proteins, is expressed within the parasitophorous vacuole (PV) of the developing gametocyte, and the majority of these proteins assemble to multimeric protein complexes [16, 17] (reviewed in [18]). The adhesion proteins are subsequently present on the gamete surface, but expression of these proteins usually ceases during fertilization. The expression of the second class of surface proteins starts following parasite transmission to the mosquito, as was shown for Pfs25 and Pfs28, and expression often persists until the ookinete has formed (reviewed in [5]). One reason for this sudden onset of protein expression following gametocyte activation in the mosquito midgut is the translational repression of messenger RNA (mRNA) encoding for some of these proteins. This was inter alia shown in the rodent malaria parasite P. berghei for the repression of Pbs25 and Pbs28 by the RNA helicase DOZI (development of zygote inhibited) as part of a ribonucleoprotein complex [19]. DOZI and its subsequently identified interaction partner CITH (CAR-I and fly trailer hitch) are considered repressors of maternally supplied mRNA important for ookinete development [20].

While astonishing advances have been made in previous years in unveiling the molecular basis of parasite transmission from the mammalian host to the mosquito, the currently available data are fragmented and major key players for gametogenesis and adaptation have not yet been identified. We thus aimed at investigating the changes in transcript levels during gametocyte activation in order to gain in-depth knowledge on the molecular switch-over that takes place in the parasite during transmission. We selected the suppression subtractive hybridization (SSH) technique to experimentally screen for genes differentially expressed during gametocyte activation, because this PCR-based method amplifies induced genes while simultaneously suppressing house-keeping genes, and is thus particularly suited to discover new or unexpected genes [21]. Our data provide insights into the regulated gene expression of malaria parasites during transmission to the mosquito and enable the identification of novel proteins with essential functions for the mosquito midgut stages, which might be suitable targets for transmission blocking interventions.

To determine changes in the transcriptome of malaria parasites during the initial phase of transmission to the mosquito, we conducted a SSH on mRNA isolated from mature non-activated gametocytes and from gametocytes at 30 min after in vitro activation, which have completed gametogenesis [7]. A subtracted cDNA library was constructed and the resulting cDNA was assigned to the respective plasmodial genes.

We identified a total number of 126 genes, for which expression levels changed in the gametocytes during activation. The majority of genes can be assigned to six major ontology groups (Figure 1, Table 1): 22 genes (17.5%) have putative functions in signaling, 18 genes (14.3%) are assigned to processes linked to cell cycle and gene expression, 11 genes (8.7%) can be linked to the cytoskeleton or the inner membrane complex (IMC), 10 genes (7.9%) have putative functions in protein trafficking, stabilization and degradation (proteostasis), eight genes (6.4%) are linked to general metabolic functions, and 16 genes (12.7%) are proteins of the cell surface or PVM. Furthermore, 15 genes (11.9%) can be assigned to other functions, including protein synthesis and processing. A total of 26 genes (20.6%) encode for proteins with unknown function, eight of which have sequences for transmembrane domains and five of which encode for a signal peptide.

We used real-time RT-PCR analyses to confirm the transcript changes in gametocytes during activation for a subset of the identified genes and to determine, if these genes become up- or down-regulated in their expression during gametocyte activation. Total RNA was isolated from immature (stages III and IV) and mature (stage V) gametocytes of P. falciparum strain NF54 and from activated gametocytes at 30 min post-activation (p.a.). Furthermore, we isolated RNA from mixed asexual blood stages of the gametocyte-less P. falciparum strain F12. Initially, the synthesized cDNA of each sample was tested for its stage-specificity by diagnostic RT-PCR, using primers for the asexual blood stage gene ama-1[24, 25] and for the gametocyte-specific gene pfccp2[26]. The tests confirmed that no pfccp2 transcript was present in the F12 sample, while no ama-1 expression was detected in the purified gametocyte samples of strain NF54 (Figure 2A, left). The absence of ama-1 signals in the gametocyte samples and the absence of pfccp2 in the F12 asexual blood stage sample further showed that these were devoid of any contamination by genomic DNA (gDNA). An additional test for gDNA contamination was performed by using primers specific for the gene hdac1 (histone deacetylase 1; PF3D7_0925700). In all parasite samples, i.e. in samples of F12 asexual blood stages as well as in NF54 immature, mature and activated gametocytes, hdac1 transcript was present (Figure 2A, right), as shown by diagnostic RT-PCR. In sample preparations lacking reverse transcriptase, on the other hand, no hdac1-specific PCR bands were detected.

We chose 27 genes from the SSH analysis for comparison of transcript abundance, with representatives from all ontology groups. The gene pfs16 was selected as an internal control, because it is known to be highly expressed in gametocytes throughout development, while it is absent in the gametes [7, 27, 28]. Furthermore two sexual stage-specific genes, actin II and pfs25, served as external controls. Pfs25 is expressed in vesicular structures during gametocyte maturation and relocates to the surface of macrogametes following activation. Pfs25 is subsequently present on the parasite surface until the ookinete stage [16, 29–32]. Actin II is a sexual stage-specific actin isomer, and in P. berghei actin II was reported to play a role during microgametogenesis [33, 34]. In addition, the gene su α5, encoding for subunit (SU) α5 of the α-ring of the proteasome core particle [35], was included in the investigations.

Transcript expression levels were measured via real-time RT-PCR and calculated by the 2^-ΔCt method [36, 37] in which the threshold cycle number (Ct), was normalized to the Ct of the endogenous control gene encoding for P. falciparum seryl tRNA synthetase (PF3D7_0717700) as reference gene [38, 39]. Transcript levels with 2^-ΔCt values below 0.5 were considered negligible. Real-time RT-PCR revealed transcription in mature gametocytes for 22 out of the 27 genes. Out of these, 15 genes showed increased transcript expression in gametocytes compared to asexual blood stage parasites (Figure 2B).

Seven genes were identified, i.e. PF3D7_0827800 (set3), Pf3D7_1246200 (actin I), PF3D7_1218800 (psop17), PF3D7_1239400, PF3D7_0704100, PF3D7_0417400, and PF3D7_1321000, for which transcript levels were increased in activated gametocytes as compared to asexual blood stages, immature gametocytes and non-activated gametocytes (Figure 2B), indicating that these genes may play an important role downstream of gametocyte activation in the mosquito midgut. SET3 was previously described to accumulate in male gametocytes, where it contributes to a prompt entry and execution of S/M phases during microgametogenesis [40]. SET domains are assigned to chromatin dynamics and are often found in histone methyltransferases, thus they play a role in the epigenetic control of gene regulation. The P. falciparum genome encodes for at least nine SET-domain-containing proteins which exhibit five different types of substrate specificities [41].

Actin I was described as part of the plasmodial motor complex (reviewed in [42]) and was recently also reported to be present in gametocytes [33, 43]. Furthermore, PSOP17 (putative secreted ookinete protein 17) was previously reported to be expressed in mature gametocytes of P. falciparum[9, 12] and in ookinetes of P. berghei[44]. In addition, transcript expression of the two control genes, PF3D7_1031000 (pfs25) and PF3D7_1412500 (actin II) was strong in gametocytes compared to asexual blood stage parasites and increased during gametocyte activation (Figure 2B).

Eight genes had an increased transcript expression in gametocytes compared to asexual blood stage parasites, and transcript levels either remained constant or decreased following activation. These include PF3D7_0302100 (clk4), PF3D7_1115200 (set7), PF3D7_0422300 (α-tubulin II), PF3D7_1103500 (encoding for a CPW-WPC protein), PF3D7_406200 (pfs16), PF3D7_1457000 (spp), PF3D7_0817600 (encoding for a MAC/PF domain, here termed PPLP6) and PF3D7_1225600 (Figure 2B). The mRNA splicing kinase CLK4 (also termed SRPK1) is expressed in the asexual blood stages and gametocytes of P. falciparum[45, 46] and a gene knock-out of the orthologous protein in P. berghei failed to exflagellate upon gametocyte activation [47]. The gene set7 encodes for one of the nine SET-domain-containing proteins (see above). Transcript of set7 is destabilized in the absence of DOZI and CITH (compare with Table 1), pointing at a translational repression of set7 in gametocytes and a role in epigenetic control mechanisms of the ookinete.

Pfs16 is a transmembrane protein of the gametocyte PVM [12, 27, 48, 49]. After the PVM has ruptured during the egress of the activated gametocyte from the host cell, Pfs16 is not detectable any longer in the sexual stage parasites [7] (see below), explaining the decrease of transcript following activation. Furthermore, α-tubulin II represents a tubulin isoform that is expressed in asexual blood stage parasites, in gametocytes and in microgametes [50–52]. Targeted gene modification studies in P. berghei indicated that α-tubulin II plays an important role for microgametogenesis [53]. The fact that α-tubulin II transcript levels decrease during gametogenesis lets us conclude that the α-tubulin II filaments needed for the formation of the microgametes are already present in the mature gametocytes prior to activation.

The presenilin-like signal peptide peptidase (SPP) was previously reported to be involved in merozoite invasion and cleavage of the erythrocyte cytoskeletal protein band 3 [54]. A recent study, however, disagreed with these findings, and reported that SPP is an ER-resident protease required for growth of the erythrocytic stages [55]. SPP is considered a potential drug target of liver and blood stage parasites [55–57]. The combined data indicate that SPP is present in the liver, blood and gametocyte stages, pointing to a role of SPP in multiple life-cycle stages of Plasmodium.

The gene product of PF3D7_0817600 comprises a MAC/PF domain, similar to the MAC/PF domains of the previously described plasmodial perforin-like proteins PPLP1-5 [58] (reviewed in [8]). We therefore termed the protein PPLP6 in this study. Furthermore, the gene product of PF3D7_1103500 is a member of a family of nine secreted proteins with cysteine-rich CPW-WPC domains [59] (reviewed in [60]). The CPW-WPC domain is a conserved domain of about 61 residues in length that exhibits six well-conserved cysteine residues and six well-conserved aromatic sites. The functions and life cycle expression patterns of the plasmodial CPW-WPC proteins are hitherto not known.

Three genes were predominantly expressed in the asexual blood stages and show a resurgence in transcript expression in gametocytes during maturation, i.e. PF3D7_1238900 (pk2), PF3D7_1136400 (encoding for a tetratrico peptide repeat region; TPR), and PF3D7_1021700. The calcium/calmodulin-dependent protein kinase PK2 was hitherto only investigated in the asexual blood stages of P. falciparum[61, 62] and protein expression in gametocytes has not yet been investigated. The gene product of PF3D7_1136400 comprises a TPR domain, which is known to mediate protein-protein interactions and the assembly of multiprotein complexes (reviewed in [63]), but the function of the plasmodial TPR domain protein is not yet known. While transcript levels of PF3D7_1136400 increase during gametocyte development, a drop following activation was observed.

For four genes, PF3D7_0818900 (hsp70-1), PF3D7_080700 (su α6), PF3D7_0807500 (gap50), and PF3D7_1444800 (encoding for fructose 1,6 bisphosphate aldolase, FBPA) expression was high in asexual blood stages and decreased during gametocyte development and following activation (Figure 2B). SU α6 is a component of the plasmodial proteasome, a proteolytic complex composed of more than 33 SUs that is responsible for the degradation and recycling of ubiquitinated proteins. As an external control we thus investigated the transcript levels of another α-ring component, su α5, and revealed similar changes in the transcript levels of su α5 in asexual blood stage parasites and gametocytes before and after activation (Figure 2B).

The genome of P. falciparum encodes for a variety of chaperones, including heat shock proteins (HSPs) of the HSP70, HSP90 and DnaJ/HSP40 families (reviewed in [64]). HSP70-1 was previously described to be located in the cytoplasm and nucleus of the parasite blood stages [65] as well as in the PV, pointing to a role in protein transport to the erythrocyte [66]. Another proposed function of HSP70-1 includes the protein trafficking to the apicoplast [67, 68], indicating that the chaperone has several essential functions and is important for multiple life-cycle stages. It remains to be elucidated, if other of the identified HSPs have more specific functions during parasite transmission from human to mosquito. Interestingly in this context, HSP90 belongs to a family of evolutionarily conserved chaperones which has been suggested as a capacitor for morphological evolution because reduction of its function results in phenotypic variation in Drosophila[69]. In addition, HSP90 displays multiple roles in stress adaptation and development, including spermatogenesis, oogenesis and embryogenesis in insects [70].

The transmembrane protein GAP50 is part of the IMC of the parasite invasive stages, including the ookinete [42, 71]. Here, it links myosin with the outer membrane of the IMC and thus contributes to gliding motility of the parasite. Recently, GAP50 was also described as a component of the gametocyte IMC [43, 72, 73], which appears to be important for the stability of the crescent-shaped cell. Noteworthy, transcript of the P. berghei orthologous protein was shown to co-precipitate with DOZI and CITH (G.R. Mair, unpublished observations), indicating that gap50 is translationally repressed in gametocytes. Thus, the release of the translational repression and onset of protein synthesis during gametocyte activation might cause the detected decrease in the gap50 transcript level.

FBPA is an enzyme of glycolysis, and in Plasmodium is also reported to be part of the motor complex, here linking TRAP to actin I [71, 74]. Noteworthy in this context, during gametocytogenesis, malaria parasites shift from glycolysis towards mitochondrial respiration [75], which might explain the decrease in FBPA expression in gametocytes compared to asexual blood stage parasites.

Transcript expression for five genes, PF3D7_0825800 (vsp9), PF3D7_0215400 (encoding for a WD40 motif), PF3D7_1351700 (alv6), PF3D7_1033200 (etramp10.2), and PF3D7_0207700 (sera4), is negligible in gametocytes. The gene PF3D7_0815800 encodes for a homolog to the yeast vacuolar sorting protein VSP9 with a predicted function in protein-protein interactions, while PF3D7_0215400 encodes for a protein with a WD40 motif. WD-repeat proteins are a large family found in all eukaryotes, implicated in a variety of functions ranging from signal transduction and transcription regulation to cell cycle control and apoptosis. The WD40 motifs act as a site for protein-protein interaction, and proteins containing WD40 repeats are known to serve as platforms for the assembly of protein complexes or mediators of transient interplay among other proteins (reviewed in [76]). WD-repeat proteins of P. falciparum have hitherto been described as receptors for protein kinase C, and as components of the myosin-driven motor complex [77, 78]. Furthermore, ALV6 is a member of the alveolin family, which comprises seven proteins in P. falciparum, associated with the membranous sacs of the IMC [79]. The fact that ALV6 expression decreases during gametocytogenesis is fairly surprising, considering that gametocytes possess an IMC (see above). It has to be elucidated, if the expression of any other alveolin is up-regulated in gametocytes.

The early transcribed membrane proteins (ETRAMPs) are proteins of the plasmodial PVM [80, 81]. In P. falciparum the proteins were shown to form complexes with the PVM protein exported protein 1 (EXP-1) [82]. To date, ETRAMP10.2 transcripts were found in P. falciparum trophozoites and the mixed asexual stages and the liver stages of P. yoelii[80, 81]. Because EXP-1 is also present in the gametocyte PVM [7], the expression of some of the ETRAMPs in these stages can be expected. It thus remains to be elucidated, if the other two SSH-identified ETRAMPs, ETRAMP2 and ETRAMP4, might play a specific role for gametocytes. Expression of serine repeat antigen SERA4 in the asexual blood stages has previously been described [83]. The SERA family comprises nine proteins with functions in asexual blood stage growth and host cell egress (reviewed in [8, 84]).

For selected genes we subsequently investigated the expression changes on the protein level. Antibodies against the proteins PK2, CLK-4, actin I and actin II, GAP50, proteasome SU α5, Pfs16, Pfs25 and PPLP6 were used to immunolabel the respective proteins in samples of P. falciparum F12 asexual blood stages, non-activated NF54 gametocytes and gametocytes at 30 min p.a. via indirect immunofluorescence assay (Figure 3A). Asexual blood stage parasites were highlighted by MSP-1 labeling; gametocytes were highlighted by labeling of Pfs25 or Pfs230, respectively. The abundance of the respective proteins in gametocytes before and after activation was quantified by measuring the average fluorescence signal intensity of a total number of 20 plotted cells per setting (Figure 3B). A significant up-regulation in the expression of PK2, actin II and Pfs25 following gametocyte activation was confirmed (Figure 3A, B). While PK2 is also expressed in asexual blood stage parasites, actin II and Pfs25 cannot be detected in these stages. On the other hand, CLK4, proteasome SU α5, and PPLP6 were detected in asexual blood stage parasites and gametocytes during maturation, but the proteins were down-regulated in gametocytes at 30 min p.a. (Figure 3B). While labeling of CLK4 and SU α5 revealed a homogenous expression of these proteins in the parasite cytoplasm and nucleus, PPLP6-labeling exhibited a punctuated expression (Figure 3A). Noteworthy, the PPLP6 labeling disappeared after the gametes have fully egressed from the enveloping erythrocyte. A role of the perforin PPLP1 was assigned to rupturing the PVM during host cell egress by the Apicomplexan parasite Toxoplasma gondii[85], and a recent study reported the involvement of PPLP2 in the lysis of the erythrocyte membrane during the exflagellation of male P. berghei gametocytes [86]. We thus hypothesize that the plasmodial PPLP6 might also play a role in the rupture of the enveloping membranes of the activated gametocytes, explaining its disappearance during gametogenesis. Similarly, the PVM-based protein Pfs16 was not present in gametocytes at 30 min p.a., since at this time point the parasites have egressed from the enveloping erythrocyte and the PVM was destroyed [7]. Furthermore, in agreement with the real-time RT-PCR results, actin I is present in both, asexual blood stages and gametocytes, and a minor up-regulation in the activated gametocytes was detected (Figure 3A, B).

Interestingly, we demonstrated a slight up-regulation in the protein expression of GAP50 at 30 min p.a., although a decrease in transcript levels was described during this process. This phenomenon can be explained by the fact that translationally repressed gap50 transcript is present in the non-activated gametocytes (see above), and that the release of repression during activation leads to rapid translation and in consequence to a loss in transcript abundance and an increase in protein abundance. It is worth mentioning in this context, that we recently showed a relocation of GAP50 from the gametocyte IMC to the plasma membrane during gametogenesis. Once on the gamete surface, GAP50 binds the human complement regulator protein factor H from the blood meal, which enables the extracellular parasite to prevent lysis by the human complement [43].

In summary we demonstrate that the majority of genes, which are regulated in their expression levels during gametocyte activation, have functions assigned to signaling, cell cycle and gene regulation, and proteostasis, or that they are cytoskeletal or cell surface proteins. The regulated expression of proteins involved in signaling and cell cycle control is on the one hand important for the signaling pathways that result in the induction of gametogenesis, once temperature drop and XA are perceived by the parasite [47] (reviewed in [6, 87]; on the other hand gametocyte activation releases the parasites from cell cycle arrest, subsequently leading to three rounds of rapid DNA replication during microgametogenesis [88].

Furthermore, induction of gametogenesis following gametocyte activation results on the one hand in the destruction of the gametocyte IMC and relocation of cytoskeletal proteins [7, 33, 34, 72, 73], on the other hand in a drastic turn-over of surface proteins (reviewed in [6]). The molecular changes in the composition of cytoskeletal and surface proteins are important for stage conversion during gametogenesis and for adaptation of the parasite to the insect host and also promote the development of the parasite midgut stages, like the ookinete. The changes in protein expression during parasite transmission from human to insect further explain the importance of chaperones and assembly proteins. The proteasome on the other hand appears to play only a minor role during gametogenesis. We hypothesize that proteins which lost their function, once the parasite has entered the mosquito vector, are only degraded after the gametes have developed and after fertilization has occurred, thus after the phase of rapid stage conversions has finished.

Noteworthy, the SSH analysis identified a number of unknown proteins with transmembrane domains and/or signal peptides, and for six of them, high transcript levels were shown in mature and activated gametocytes. For three of the genes, expression in the gametocytes was higher as compared to asexual blood stage parasites, and these genes might encode for proteins important for gametocyte development or gametogenesis.

The here presented data on the changes in the P. falciparum transcriptome during gametogenesis provide first insights into the regulated gene expression occurring while the malaria parasite passes the initial phase of transmission to the mosquito. Our findings form the basis for further studies on genes important for malaria transmission and support the identification of proteins involved in the development of the mosquito midgut stages, which might lead to the discovery of new transmission blocking targets.