A central role for P48/45 in malaria parasite male gamete fertility

Fertilization and zygote development are obligate features of the malaria parasite life cycle and occur during parasite transmission to mosquitoes. The surface protein PFS48/45 is expressed by male and female gametes of Plasmodium falciparum and PFS48/45 antibodies prevent zygote development and transmission. Here, gene disruption was used to show that Pfs48/45 and the ortholog Pbs48/45 from a rodent malaria parasite P. berghei play a conserved and important role in fertilization. p48/45- parasites had a reduced capacity to produce oocysts in mosquitoes due to greatly reduced zygote formation. Unexpectedly, only male gamete fertility of p48/45- parasites was affected, failing to penetrate otherwise fertile female gametes. P48/45 is shown to be a surface protein of malaria parasites with a demonstrable role in fertilization.     

Target antigens in malaria transmission blocking immunity

Malaria transmission blocking immunity has been found to operate against two distinct phases of development of malaria parasites in the mosquito midgut: (i) against the extracellular gametes and newly fertilized zygotes shortly after ingestion by a mosquito of parasitized blood and (ii) against the zygotes during their subsequent development into ookinetes. Immunity is antibody-mediated and stage-specific. A set of three proteins, synthesized in the gametocytes, expressed on the surface of the gametes and newly fertilized zygotes and subsequently shed during later transformation of the zygotes, has been identified as the target antigens of anti-gamete fertilization blocking antibodies. A single protein, synthesized and expressed on the zygote surface during its development to ookinetes, has been identified as the target of antibodies which block the development of the fertilized parasites in the mosquito. Immunization of human populations against gamete or zygote antigens, while not directly protecting an immunized individual from inflection, would reduce the transfer of malaria within the population. Such immunity, in addition to reducing the overall rate of malaria transmission, would, if combined with a vaccine against the asexual (disease-causing) stages, reduce the chance of selection of parasites that are resistant to the asexual vaccine by preventing their entry into the mosquito population.     

Targeting and sequestration of truncated Pfs230 in an intraerythrocytic compartment during Plasmodium falciparum gametocytogenesis

For malaria to be transmitted, the Plasmodium falciparum parasite must invade an erythrocyte and undergo gametocytogenesis. When mature intraerythrocytic gametocytes are taken up in a blood meal by a mosquito they emerge as gametes and, once fertilized, continue to differentiate into infectious sporozoites. One of the major proteins associated with the surface of the parasite during gamete differentiation is Pfs230, a 360 kDa member of a family of P. falciparum proteins that contains a repeated cysteine motif domain. To characterize the role of different regions of Pfs230, the gene was disrupted by targeted integration and clones isolated that expressed distinct sections of Pfs230. Independent clones D1.356 a and b express the first 452 amino acids (aa) of Pfs230 and do not contain a cysteine motif domain, whereas clones D2.850 a and b express the first 950 aa, including the first cysteine motif domain. Although both sets of clones undergo gametogenesis and produce morphologically normal gametes, neither truncated Pfs230 is located on the surface of the gamete. In clones D1.356 a and b, the 452 aa Pfs230 is secreted into the parasitophorous vacuole and released as a soluble protein when the parasite emerges from the erythrocyte as a gamete. In marked contrast, the 950 aa form of Pfs230 expressed by clones D2.850 a and b is sequestered in a novel tubular compartment in the erythrocyte cytoplasm. This sexual-stage tubular intraerythrocytic compartment (STIC) is not recognized by antibodies specific for proteins associated with the parasitophorous vacuole membrane (Pfs16 or Exp-1) or Maurer's clefts (Pfsbp 1 or mAb LWL1) or intraerythrocytic asexual parasite proteins (PfEMP2 or HRP II).     

Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement

Gametocytes are the intraerythrocytic stages of malaria parasites that infect mosquitoes. When gametocytes of the chicken malaria parasite Plasmodium gallinaceum are ingested by a mosquito they become extracellular in the mosquito midgut, form gametes, and fertilize within 10 to 15 min after the insect has taken a blood meal. Gametocytes of P. gallinaceum were infectious when fed to Aedes aegypti mosquitoes in blood meals containing native serum from chickens or from the non-host species, man or sheep. Gametocytes stimulated to undergo gametogenesis and to fertilize in vitro were also infectious when fed to mosquitoes in native chicken serum. However, native serum from most non-host species, including sheep and man, suppressed the infectivity of newly fertilized zygotes to mosquitoes and lysed the zygotes in vitro. These effects were shown to be due to the activity of the alternative pathway of complement (APC) in the serum of the non-host species. After mild trypsin treatment, the zygotes of P. gallinaceum no longer infected mosquitoes in the presence of native chicken serum, although in heat-inactivated chicken serum their infectivity was normal. We conclude that trypsin-sensitive components on the zygotes surface protect them from destruction by the APC of their native host. The ability of gametocytes of P. gallinaceum to infect mosquitoes in the presence of native human serum is probably due to proteases that inactivate the APC of human serum before the gametes and zygotes emerge as extracellular parasites in the blood meal.     

Hybridization and pre-zygotic reproductive barriers in Plasmodium

Sexual reproduction is an obligate step in the life cycle of many parasites, including the causative agents of malaria (Plasmodium). Mixed-species infections are common in nature and consequently, interactions between heterospecific gametes occur. Given the importance of managing gene flow across parasite populations, remarkably little is understood about how reproductive isolation between species is maintained. We use the rodent malaria parasites P. berghei and P. yoelii to investigate the ecology of mixed-species mating groups, identify proteins involved in pre-zygotic barriers, and examine their evolution. Specifically, we show that (i) hybridization occurs, but at low frequency; (ii) hybridization reaches high levels when female gametes lack the surface proteins P230 or P48/45, demonstrating that these proteins are key for pre-zygotic reproductive isolation; (iii) asymmetric reproductive interference occurs, where the fertility of P. berghei gametes is reduced in the presence of P. yoelii and (iv) as expected for gamete recognition proteins, strong positive selection acts on a region of P230 and P47 (P48/45 paralogue). P230 and P48/45 are leading candidates for interventions to block malaria transmission. Our results suggest that depending on the viability of hybrids, applying such interventions to populations where mixed-species infections occur could either facilitate or hinder malaria control.     

A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei

Differentiation of malaria parasites into sexual forms (gametocytes) in the vertebrate host and their subsequent development into gametes in the mosquito vector are crucial steps in the completion of the parasite's life cycle and transmission of the disease. The molecular mechanisms that regulate the sexual cycle are poorly understood. Although several signal transduction pathways have been implicated, a clear understanding of the pathways involved has yet to emerge. Here, we show that a Plasmodium berghei homologue of Plasmodium falciparum mitogen-activated kinase-2 (Pfmap-2), a gametocyte-specific mitogen-activated protein kinase (MAPK), is required for male gamete formation. Parasites lacking Pbmap-2 are competent for gametocytogenesis, but exflagellation of male gametocytes, the process that leads to male gamete formation, is almost entirely abolished in mutant parasites. Consistent with this result, transmission of mutant parasites to mosquitoes is grossly impaired. This finding identifies a crucial role for a MAPK pathway in malaria transmission.     

The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a monoclonal antibody to ookinetes

We studied the point at which a monoclonal antibody (mAb C5) to a surface protein (Pgs25) on Plasmodium gallinaceum ookinetes blocked the infection of Aedes aegypti mosquitoes. The antibody did not block the development of zygotes to ookinetes in vitro. Development of ookinetes to oocysts in the mosquito was blocked to the same extent whether zygotes grew to ookinetes in the presence of mAb C5 or the antibody was added after the ookinetes had reached full development. When ookinetes developed in vitro in the presence of mAb C5, antibody remained on the surface of the parasite for the next 50 hr and did not block attachment to the peritrophic membrane. When ookinetes were fed to mosquitoes, two subpopulations of mosquitoes were observed (high numbers of oocysts per midgut and low numbers of oocysts per midgut). mAb C5 reduced the number of oocysts per midgut in the subpopulation that had low numbers of oocysts. The subpopulation that had high numbers of oocysts was unaffected by antibody, indicating that the antibody did not block invasion of the midgut epithelium. When mAb C5 was fed with gametocytes, the parasites invaded the epithelium at the same time (between 30 and 35 hr after the blood meal) as in controls, although at a markedly reduced rate. The ultrastructural observations were consistent with a block of parasites within the peritrophic membrane and not with a block at the epithelium, as parasites were not seen to accumulate within the space between the peritrophic membrane and the epithelium. The mechanism by which mAb C5 to Pgs25 of P. gallinaceum blocks the penetration of the peritrophic membrane remains undefined. We present evidence that the parasite modifies the peritrophic membrane during penetration, an observation first made for Babesia microti during penetration of the peritrophic membrane in Ixodes ticks. Ookinetes in the absence of antibodies appeared to disrupt the layers of the peritrophic membrane, suggesting an enzymatic mechanism for penetration.     

Plasmodium sex determination and transmission to mosquitoes

In order to be transmitted by their mosquito vector, malaria parasites undergo sexual reproduction, which occurs between specialized male and female parasites (gametes) within the blood meal in the mosquito. Nothing was known about how Plasmodium determines the sex of its gametocytes (gamete precursors), which are produced in the vertebrate host. Recently, erythropoietin, the vertebrate hormone controlling erythropoiesis in response to anaemia, was implicated in Plasmodium sex determination in animal models of malaria. This review examines the available information and addresses the relevance of such a sex determining mechanism for Plasmodium falciparum transmission to mosquitoes, with special reference to low gametocytaemias.     

Human T clones reactive to the sexual stages of Plasmodium falciparum malaria. High frequency of gamete-reactive T cells in peripheral blood from nonexposed donors

Malarial gametocytes, which are taken up by mosquitoes during a blood meal, develop in the gut of the mosquito into gametes. Gametes and gametocytes contain the target antigens of transmission-blocking immunity. Here, we show that the peripheral blood of nonexposed donors contains Plasmodium falciparum gamete-reactive T cells at frequencies ranging from 1/300 to 1/4000. Studies on long-term clones demonstrated that these cells often recognized antigens shared between gametes and asexual stage parasites or even between heterologous gametes, although it has been possible to derive a P. falciparum gamete-specific T clone. The T clones examined were T3+, T4+, T8-, and either HLA-DR- or HLA-DQ-restricted. They responded to gametes by both proliferation and the secretion of gamma-interferon. The gamete-specific clone and other asexual cross-reactive clones examined could be stimulated in vitro by a preparation of mature gametocytes within RBC, but not by RBC alone, suggesting that gametocytes are immunogenic or can become immunogenic for T cells in vivo. The significance of these observations to mosquito transmission of malaria and development and application of a gamete vaccine are discussed.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.     

Target antigens of transmission blocking immunity of Plasmodium vivax malaria. Characterization and polymorphism in natural parasite isolates.

A panel of 20 anti-Plasmodium vivax female gamete mAb has been established and was characterized with respect to their transmission-blocking properties in membrane-feeding experiments and their target Ag identified. Seven mAb suppressed the infectivity of P. vivax parasites to Anopheles tesselatus mosquitoes. The m.w. of the Ag recognized by these mAb were ascertained by SDS-PAGE and Western blots. Three sets of polypeptides of low Mr--20, 24, and a doublet of 37/42 kDa--have been defined as target Ag of transmission-blocking antibodies of P. vivax. All epitopes of these target Ag were found to be dependent on the tertiary conformational structure of the Ag. Polymorphism of target Ag of transmission-blocking immunity was investigated in over 30 natural isolates of P. vivax in Sri Lanka based on the reactivity of a mAb with an isolate as assessed by the indirect immunofluorescent test with the use of live extracellular female gametes, and in Western blots with the use of extracted gametes. The functional consequences of antigenic polymorphism on immunity was investigated in transmission-blocking assays by using membrane-feeding experiments. A majority of target Ag of transmission-blocking immunity were found to be polymorphic, exhibiting size as well as epitope polymorphism. Results indicate that failure of a mAb to affect the infectivity of a parasite isolate of P. vivax to mosquitoes can be caused by polymorphism of the target Ag among isolates.     

Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut

Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.     

Plasmodium falciparum: IgG subclass antibody response to merozoite surface protein-1 among Amazonian gold miners, in relation to infection status and disease expression

The merozoite surface protein-1 (MSP-1) of Plasmodium falciparum comprises two major targets of antibody-mediated immunity: the polymorphic block 2 and the 19-kDa C-terminal domain MSP-1(19). Here, we measured antibodies to three block 2 variants and MSP-1(19) among Amazonian gold miners and examined the repertoire of block 2 variants in local parasites. Main findings were as follows: (1) Only seven different block 2 variants were found in 18 DNA sequences analyzed. (2) No major difference was observed in IgG subclass distribution of antibodies from symptomatic P. falciparum-infected patients, asymptomatic parasite carriers, and non-infected subjects. (3) Antibodies to all block 2 antigens, but not to MSP-1(19), were biased towards IgG3 across different strata of cumulative malaria exposure. (4) Similar proportions of symptomatic and asymptomatic subjects failed to recognize the block 2 variant expressed by infecting parasites. These negative results underscore the limits of conventional antibody assays to evaluate clinical immunity to malaria.     

Protective antigens of rodent and human bloodstage malaria

Bloodstage malaria parasites are antigenically complex, but individual antigens can be identified and analysed using monoclonal antibodies. Two monoclonal antibodies that recognize a 235 000 molecular mass Plasmodium yoelii rhoptry protein provide some protection when injected into mice against a challenge infection. The purified rhoptry protein also provides protective immunity against P. yoelii YM when used to vaccinate mice and fulminating infections are converted into self-limiting, reticulocyte-restricted infections. Another monoclonal antibody immunoprecipitates a 230 000 molecular mass protein and a series of proteolytic processing fragments. At least one of these processing fragments, probably a 90 000 molecular mass species, is located on the merozoite surface. Mice immunized with the purified protein were protected against challenge infection with P. yoelii YM. This antigen may provide protection by inducing a cell-mediated immune response. A monoclonal antibody raised against P. falciparum schizonts reacts with a 195 000 molecular mass protein which is synthesized in schizonts and subsequently cleaved. Fragments of the 195 000 molecular mass protein are expressed as major antigens on the merozoite surface. The 195 000 molecular mass P. falciparum protein and the 230 000 molecular mass P. yoelii protein belong to a class of malaria parasite antigens which probably is important in the induction of a protective immune response in the host.     

PfCCp proteins of Plasmodium falciparum: gametocyte-specific expression and role in complement-mediated inhibition of exflagellation

The sexual phase of the malaria parasite Plasmodium falciparum is essential for transmission of the disease and is accompanied by the co-ordinated expression of sexual stage proteins. Six of these proteins belong to a highly conserved apicomplexan family of multi-domain adhesion proteins, termed PfCCps. PfCCp1, PfCCp2 and PfCCp3 are co-dependently expressed in the parasitophorous vacuole associated with the gametocyte plasma membrane. PfCCp2 and PfCCp3 also play an essential role for parasite development in the mosquito. We show that the six PfCCp proteins are expressed in stages II-V of gametocytogenesis as well as during early gamete formation. The proteins are expressed in association with the surface of both male and female gametocytes and macrogametes, but are not present in exflagellating microgametes. Further, the newly described protein PfCCp4 co-localizes with the transmission blocking candidate Pfs230, with which it forms a protein complex. In contrast to the phenotypes that are observed following targeted gene disruption of PfCCp2, PfCCp3 or Pfs230, the lack of PfCCp4 expression does not inhibit parasite development in the mosquito vector. This indicates a non-essential role for this protein during parasite transmission. Exflagellation assays revealed that antibodies directed against distinct domains of PfCCp1 through PfCCp4 and PfFNPA support a complement-mediated decrease in gametocyte emergence. We conclude that the six PfCCp proteins are specifically expressed during gametocytogenesis and gamete formation, and that select members may represent prospective candidates for transmission blocking vaccines.     

Biology and biochemistry of sexual and sporogenic stages of Plasmodium falciparum: a review

Gametocyte development has been studied in synchronous cultures of P. falciparum. These studies have confirmed that maturation of this stage occurs over 7 days and doubling of the DNA content takes place early in the development stage. There appears to be no direct relationship between the number of gametocytes ingested by a mosquito and the oocyst load, nor between the latter and the number of salivary gland sporozoites. The variability in oocyst load could in part be explained by the individual differences in digestion speed of mosquitoes. The number of salivary gland sporozoites and thus also that of sporozoites inoculated per bite of a mosquito is influenced by the number of blood meals which the mosquito has ingested after the infectious blood meal. Moreover, the longer the mosquito survives the more infective it becomes due to increased density of sporozoites in the glands. Anti-sporozoite antibodies present in the blood meal of an already infected mosquito do not hinder migration of sporozoites to the salivary glands. It has been demonstrated that monoclonal antibodies directed against epitopes of gamete surface proteins of molecular weight of 230, 45/48 and 25 kDa could block sporogony. Transmission blocking antibodies are also present in the sera of individuals exposed to natural transmission. These epitopes of the target proteins are non-repetitive and conformational and thus rendered non-reactive by reduction. Moreover, some of these surface proteins are glycosylated and/or acylated, and may depend on the presence of the native disulphide bonds for the preservation of immune reactivity. These post-translational modifications have made screening of gene libraries with immunoproteins difficult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular and humoral immune responses to Plasmodium falciparum gametocyte antigens in malaria-immune individuals. Limited response to the 48/45-kilodalton surface antigen does not appear to be due to MHC restriction

We have examined immune responses to a cultured Plasmodium falciparum gametocyte lysate and to an affinity-purified preparation of the 48/45-kDa gamete surface Ag in a group of 30 malaria immune individuals and in 24 Europeans with no previous exposure to malaria. Cellular responses were assessed in vitro by lymphoproliferation and production of IFN-gamma; antigamete antibodies were detected by immunofluorescence, Western blotting, and competitive ELISA. Cells from all the malaria immune donors responded to the gametocyte lysate in both assays while cells from nonimmune donors gave only weak proliferative responses. Antigamete antibodies were detected in the serum of all the immune donors but not in serum from nonimmunes. Nonimmune donors were completely unresponsive to the purified 48/45-kDa surface Ag while cells from 40% of immune donors responded by either proliferation or IFN-gamma production. Only 3 of 30 immune donors had detectable antibodies to the 48/45-kDa Ag. Class II HLA type was determined for 27 of the immune donors but no relationship between HLA-DR or -DQ and responsiveness to the 48/45-kDa Ag was discerned. The possible reasons for limited recognition of this gamete surface Ag are discussed.     

A NIMA-related protein kinase is essential for completion of the sexual cycle of malaria parasites

The molecular mechanisms regulating the sexual development of malaria parasites from gametocytes to oocysts in their mosquito vector are still largely unexplored. In other eukaryotes, NIMA-related kinases (Neks) regulate cell cycle progression and have been implicated in the regulation of meiosis. Here, we demonstrate that Nek-4, a new Plasmodium member of the Nek family, is essential for completion of the sexual cycle of the parasite. Recombinant Plasmodium falciparum Nek-4 possesses protein kinase activity and displays substrate preferences similar to those of other Neks. Nek-4 is highly expressed in gametocytes, yet disruption of the nek-4 gene in the rodent malaria parasite P. berghei has no effect on gamete formation and subsequent fertilization. However, further differentiation of zygotes into ookinetes is abolished. Measurements of nuclear DNA content indicate that zygotes lacking Nek-4 fail to undergo the genome replication to the tetraploid level that precedes meiosis. Cell cycle progression in the zygote is identified as a likely precondition for its morphological transition to the ookinete and for the successful establishment of a malaria infection in the mosquito.     

Three Members of the 6-cys Protein Family of Plasmodium Play a Role in Gamete Fertility

The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates.     

An Electron Microscopical Study of the Interaction of Monoclonal Antibodies with Gametes of the Malarial Parasite Plasmodium gallinaceum1

Anti-malarial gamete antibodies prevent the fertilization of gametes in the mosquito midgut and prevent transmission of malaria. Recently, hybridoma cell lines secreting monoclonal antibodies (10G3 and 11C7) against gametes of the malarial parasite have been developed. These antibodies act synergistically to mediate 80–90% suppression of the infectivity of gametocytes, although neither monoclonal antibody alone has a significant effect on gametocyte infectivity. We performed immuno-electron microscopy to characterize the interactions of these monoclonal antibodies with gametes of Plasmodium gallinaceum. Male gametes exposed to either 10G3 or 11C7 agglutinated into loose clusters, while those exposed to a mixture of 10G3 and 11C7 agglutinated into long, rope-like bundles. This difference appears to be related to the distribution of the antibodies on the surface of the gametes. When 10G3 or 11C7 labeled with a ferritin-conjugated anti-mouse Ig were used singly, the ferritin particles were distributed in focal areas over the surface of the parasites. By contrast, when the male gametes were exposed to a mixture of 10G3 and 11C7, the ferritin particles were distributed over their entire surface. Female gametes reacted similarly to these antibodies. These observations indicate that combinations of antibody specificities that reduce fertilization efficiency coat the entire surface of the gametes. On the other hand, focal interactions resulting from a single antibody are unable to block fertilization.