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ABSTRACTMosquito saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis and immune responses. Here, we generated two knockout (KO) mosquito lines by CRISPR/Cas9 to functionally characterize D7L1 and D7L2, two abundantly expressed salivary proteins from the yellow fever mosquito vector Aedes aegypti. The D7s bind and scavenge biogenic amines and eicosanoids involved in hemostasis at the bite site. The absence of D7 proteins in the salivary glands of KO mosquitoes was confirmed by mass spectrometry, enzyme-linked immunosorbent assay, and fluorescence microscopy of the salivary glands with specific antibodies. D7-KO mosquitoes had longer probing times than parental wildtypes. The differences in probing time were abolished when mutant mice resistant to inflammatory insults were used. These results confirmed the role of D7 proteins as leukotriene scavengers in vivo. We also investigated the role of D7 salivary proteins in Plasmodium gallinaceum infection and transmission. Both KO lines had significantly fewer oocysts per midgut. We hypothesize that the absence of D7 proteins in the midgut of KO mosquitoes might be responsible for creating a harsh environment for the parasite. The information generated by this work highlights the biological functionality of salivary gene products in blood feeding and pathogen infection.IMPORTANCEDuring blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti, the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission.

ABSTRACT The mosquito immune system limits Plasmodium infection and malaria transmission. Plasmodium falciparum evades the mosquito defense response by expressing the surface protein P. falciparum P47 (Pfs47) that inhibits midgut epithelial nitration by interacting with the mosquito midgut P47 receptor (P47Rec). However, the mechanism by which P47Rec suppresses caspase-mediated nitration is unknown. Here, we show that epithelial invasion by Pfs47 knockout parasites is followed by an extrusion of cells undergoing caspase-mediated apoptosis, which triggers the release of hemocyte-derived microvesicles (HdMv’s), known to promote complement activation and ookinete lysis. In contrast, invasion by P. falciparum parasites that express Pfs47 accelerates the extrusion of damaged cells and disrupts caspase activation and HdMv release. The Anopheles gambiae heat shock protein 70 cognate 3 (Hsc70-3), the ortholog of Drosophila binding immunoglobulin protein, was identified as a molecular partner of P47Rec. Silencing of Hsc70-3 promotes lysis of Pfs47 wild-type parasites through a caspase S2-dependent mechanism. The interaction of P47Rec activates a Hsc70-3-mediated anti-apoptotic response that prevents caspase activation. The interaction of P47Rec with Hsc70-3 is necessary for P. falciparum to evade the mosquito early immune responses that target the ookinete stage. IMPORTANCE Malaria transmission by Anopheles gambiae mosquitoes is very effective, in part because the parasite expresses a surface protein called Pfs47 that allows it to evade the mosquito immune system. Here we investigate how this protein changes the response of mosquito midgut epithelial cells to invasion by the parasite. Pfs47 is known to interact with P47Rec, a mosquito midgut receptor. We found that Pf47Rec inhibits caspase-mediated apoptosis by interacting with the Hsc70-3. This disrupts nitration of midgut epithelial cells invaded by the parasite and the release of hemocyte-derived microvesicles, which are critical for effective activation of the mosquito complement system that eliminates the parasite.

Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. Results We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. Conclusions It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

Abstract Background Functional screens based on dsRNA-mediated gene silencing identified several Anopheles gambiae genes that limit Plasmodium berghei infection. However, some of the genes identified in these screens have no effect on the human malaria parasite Plasmodium falciparum; raising the question of whether different mosquito effector genes mediate anti-parasitic responses to different Plasmodium species. Results Four new An. gambiae (G3) genes were identified that, when silenced, have a different effect on P. berghei (Anka 2.34) and P. falciparum (3D7) infections. Orthologs of these genes, as well as LRIM1 and CTL4, were also silenced in An. stephensi (Nijmegen Sda500) females infected with P. yoelii (17XNL). For five of the six genes tested, silencing had the same effect on infection in the P. falciparum-An. gambiae and P. yoelii-An. stephensi parasite-vector combinations. Although silencing LRIM1 or CTL4 has no effect in An. stephensi females infected with P. yoelii, when An. gambiae is infected with the same parasite, silencing these genes has a dramatic effect. In An. gambiae (G3), TEP1, LRIM1 or LRIM2 silencing reverts lysis and melanization of P. yoelii, while CTL4 silencing enhances melanization. Conclusion There is a broad spectrum of compatibility, the extent to which the mosquito immune system limits infection, between different Plasmodium strains and particular mosquito strains that is mediated by TEP1/LRIM1 activation. The interactions between highly compatible animal models of malaria, such as P. yoelii (17XNL)-An. stephensi (Nijmegen Sda500), is more similar to that of P. falciparum (3D7)-An. gambiae (G3).

Background and Objective Folate and vitamin B12 deficiencies can impair proper growth and brain development in children. Data on the folate and vitamin B12 status of children aged 6-59 months in Guatemala are scarce. Identification of factors associated with higher prevalence of these micronutrient deficiencies within the population is needed for national and regional policymakers. Objective To describe national and regional post-fortification folate and vitamin B12 status of children aged 6-59 months in Guatemala. Methods A multistage, cluster probability study was carried out with national and regional representation of children aged 6-59 months. Demographic and health information was collected for 1246 preschool children, but blood samples for red blood cell (RBC) folate and vitamin B12 were collected and analyzed for 1,245 and 1143 preschool children, respectively. We used the following deficiency criteria as cutoff points for the analyses: < 305 nmol/L for RBC folate, < 148 pmol/L for vitamin B12 deficiency, and 148-221 pmol/L for marginal vitamin B12 deficiency. Prevalence of RBC folate deficiency and vitamin B12 deficiency and marginal deficiency were estimated. Prevalence risk ratios of RBC folate and vitamin B12 deficiency were estimated comparing subpopulations of interest. Results The national prevalence estimates of RBC folate deficiency among children was 33.5% [95% CI 29.1, 38.3]. The prevalence of RBC folate deficiency showed wide variation by age (20.3-46.6%) and was significantly higher among children 6-11 months and 12-23 months (46.6 and 37.0%, respectively), compared to older children aged 48-59 months (20.3%). RBC folate deficiency also varied widely by household wealth index (22.6-42.0%) and geographic region (27.2-46.7%) though the differences were not statistically significant. The national geometric mean for RBC folate concentrations was 354.2 nmol/L. The national prevalences of vitamin B12 deficiency and marginal deficiency among children were 22.5% [95% CI 18.2, 27.5] and 27.5% [95% CI 23.7, 31.7], respectively. The prevalence of vitamin B12 deficiency was significantly higher among indigenous children than among non-indigenous children (34.5% vs. 13.1%, aPRR 2.1 95% CI 1.4, 3.0). The prevalence of vitamin B12 deficiency also significantly varied between the highest and lowest household wealth index (34.3 and 6.0%, respectively). The national geometric mean for vitamin B12 concentrations was 235.1 pmol/L. The geometric means of folate and B12 concentrations were significantly lower among children who were younger, had a lower household wealth index, and were indigenous (for vitamin B12 only). Folate and vitamin B12 concentrations showed wide variation by region (not statistically significant), and the Petén and Norte regions showed the lowest RBC folate and vitamin B12 concentrations, respectively. Conclusions In this study, a third of all children had RBC folate deficiency and half were vitamin B12 deficient. Folate deficiency was more common in younger children and vitamin B12 deficiency was more common in indigenous children and those from the poorest families. These findings suggest gaps in the coverage of fortification and the need for additional implementation strategies to address these gaps in coverage to help safeguard the health of Guatemalan children., Author(s): E. Wong [sup.1] , R. Molina-Cruz [sup.2] , C. Rose [sup.3] , L. Bailey [sup.4] , G. P. A. Kauwell [sup.5] , J. Rosenthal [sup.3] Author Affiliations: (1) grid.410711.2, [...]

Molina-Cruz et al. analyzed 4,971 sequences of the Plasmodium falciparum Pfs47 gene and identified a combination of two SNP haplotypes highly correlated with geographic origin and transmission potential by mosquito vectors. A simple High-Resolution Melting RT-PCR assay was developed to genotype these SNPs and establish the likely continent of origin and transmission risk of P. falciparum isolates.

Abstract Background The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. Methods RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. Results Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. Conclusion SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.

Combinations of monoclonal antibodies (mAbs) against different epitopes on the same antigen synergistically neutralize many viruses. However, there are limited studies assessing whether combining human mAbs against distinct regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) enhances in vivo protection against malaria compared to each mAb alone or whether passive transfer of PfCSP mAbs would improve protection following vaccination against PfCSP. Here, we isolated a panel of human mAbs against the subdominant C-terminal domain of PfCSP (C-CSP) from a volunteer immunized with radiation-attenuated Pf sporozoites. These C-CSP-specific mAbs had limited binding to sporozoites in vitro that was increased by combination with neutralizing human "repeat" mAbs against the NPDP/NVDP/NANP tetrapeptides in the central repeat region of PfCSP. Nevertheless, passive transfer of repeat- and C-CSP-specific mAb combinations did not provide enhanced protection against in vivo sporozoite challenge compared to repeat mAbs alone. Furthermore, combining potent repeat-specific mAbs (CIS43, L9, and 317) that respectively target the three tetrapeptides (NPDP/NVDP/NANP) did not provide additional protection against in vivo sporozoite challenge. However, administration of either CIS43, L9, or 317 (but not C-CSP-specific mAbs) to mice that had been immunized with R21, a PfCSP-based virus-like particle vaccine that induces polyclonal antibodies against the repeat region and C-CSP, provided enhanced protection against sporozoite challenge when compared to vaccine or mAbs alone. Collectively, this study shows that while combining mAbs against the repeat and C-terminal regions of PfCSP provide no additional protection in vivo, repeat mAbs do provide increased protection when combined with vaccine-induced polyclonal antibodies. These data should inform the implementation of PfCSP human mAbs alone or following vaccination to prevent malaria infection.

ABSTRACT Drug development efforts have focused mostly on the asexual blood stages of the malaria parasite Plasmodium falciparum. Except for primaquine, which has its own limitations, there are no available drugs that target the transmission of the parasite to mosquitoes. Therefore, there is a need to validate new parasite proteins that can be targeted for blocking transmission. P. falciparum calcium-dependent protein kinases (PfCDPKs) play critical roles at various stages of the parasite life cycle and, importantly, are absent in the human host. These features mark them as attractive drug targets. In this study, using CRISPR/Cas9 we successfully knocked out PfCDPK2 from blood-stage parasites, which was previously thought to be an indispensable protein. The growth rate of the PfCDPK2 knockout (KO) parasites was similar to that of wild-type parasites, confirming that PfCDPK2 function is not essential for the asexual proliferation of the parasite in vitro. The mature male and female gametocytes of PfCDPK2 KO parasites become round after induction. However, they fail to infect female Anopheles stephensi mosquitoes due to a defect(s) in male gametocyte exflagellation and possibly in female gametes. IMPORTANCE Despite reductions in the number of deaths it causes, malaria continues to be a leading infectious disease of the developing world. For effective control and elimination of malaria, multiple stages of the parasite need to be targeted. One such stage includes the transmission of the parasite to mosquitoes. Here, we demonstrate the successful knockout of PfCDPK2, which was previously thought to be indispensable for parasite growth in red blood cells. The PfCDPK2 KO parasites are incapable of establishing an infection in mosquitoes. Therefore, our study suggests that targeting PfCDPK2 may be a good strategy to control malaria transmission in countries with high transmission. Moreover, molecular understanding of the signaling pathway of PfCDPK2 may provide additional targets for malaria control.

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.