The malaria parasite RhopH protein complex interacts with erythrocyte calmyrin identified from a comprehensive erythrocyte protein library.

Malaria merozoite apical organelles; microneme and rhoptry secreted proteins play functional roles during and following invasion of host erythrocytes. Among numerous proteins, the rhoptries discharge high molecular weight proteins known as RhopH complex. Recent reports suggest that the RhopH complex is essential for growth and survival of the malaria parasite within erythrocytes. However, an in-depth understanding of the host-parasite molecular interactions is indispensable. Here we utilized a comprehensive mouse erythrocyte protein library consisting of 443 proteins produced by a wheat germ cell-free system, combined with AlphaScreen technology to identify mouse erythrocyte calmyrin as an interacting molecule of the rodent malaria parasite Plasmodium yoelii RhopH complex (PyRhopH). The PyRhopH interaction was dependent on the calmyrin N-terminus and divalent cation capacity. The finding unveils a recommendable and invaluable usefulness of our comprehensive mouse erythrocyte protein library together with the AlphaScreen technology in investigating a wide-range of host-parasite molecular interactions.

Identification and prioritization of merozoite antigens as targets of protective human immunity to Plasmodium falciparum malaria for vaccine and biomarker development.

The development of effective malaria vaccines and immune biomarkers of malaria is a high priority for malaria control and elimination. Ags expressed by merozoites of Plasmodium falciparum are likely to be important targets of human immunity and are promising vaccine candidates, but very few Ags have been studied. We developed an approach to assess Ab responses to a comprehensive repertoire of merozoite proteins and investigate whether they are targets of protective Abs. We expressed 91 recombinant proteins, located on the merozoite surface or within invasion organelles, and screened them for quality and reactivity to human Abs. Subsequently, Abs to 46 proteins were studied in a longitudinal cohort of 206 Papua New Guinean children to define Ab acquisition and associations with protective immunity. Ab responses were higher among older children and those with active parasitemia. High-level Ab responses to rhoptry and microneme proteins that function in erythrocyte invasion were identified as being most strongly associated with protective immunity compared with other Ags. Additionally, Abs to new or understudied Ags were more strongly associated with protection than were Abs to current vaccine candidates that have progressed to phase 1 or 2 vaccine trials. Combinations of Ab responses were identified that were more strongly associated with protective immunity than responses to their single-Ag components. This study identifies Ags that are likely to be key targets of protective human immunity and facilitates the prioritization of Ags for further evaluation as vaccine candidates and/or for use as biomarkers of immunity in malaria surveillance and control.

Enhancement of dendritic cell activation via CD40 ligand-expressing γδ T cells is responsible for protective immunity to Plasmodium parasites.

Previous reports have shown that γδ T cells are important for the elimination of malaria parasites in humans and mice. However, how γδ T cells are involved in protective immunity against blood-stage malaria remains unknown. We infected γδ T-cell-deficient (TCRδ-KO) mice and control wild-type mice with Plasmodium berghei XAT, which is a nonlethal strain. Although infected red blood cells were eliminated within 30 d after infection, TCRδ-KO mice could not clear the infected red blood cells, showed high parasitemia, and eventually died. Therefore, γδ T cells are essential for clearance of the parasites. Here, we found that γδ T cells play a key role in dendritic cell activation after Plasmodium infection. On day 5 postinfection, γδ T cells produced IFN-γ and expressed CD40 ligand during dendritic cell activation. These results suggest that γδ T cells enhance dendritic cell activation via IFN-γ and CD40 ligand-CD40 signaling. This hypothesis is supported strongly by the fact that in vivo induction of CD40 signaling prevented the death of TCRδ-KO mice after infection with P. berghei XAT. This study improves our understanding of protective immunity against malaria and provides insights into γδ T-cell-mediated protective immunity against various infectious diseases.

Characteristic age distribution of Plasmodium vivax infections after malaria elimination on Aneityum Island, Vanuatu.

Resurgence is a major concern after malaria elimination. After the initiation of the elimination program on Aneityum Island in 1991, microscopy showed that Plasmodium falciparum disappeared immediately, whereas P. vivax disappeared from 1996 onward, until P. vivax cases were reported in January 2002. By conducting malariometric surveys of the entire population of Aneityum, we investigated the age distribution of individuals with parasites during this epidemic in the context of antimalarial antibody levels and parasite antigen diversity. In July 2002, P. vivax infections were detected by microscopy in 22/759 individuals: 20/298 born after the beginning of the elimination program in 1991, 2/126 born between 1982 and 1991, and none of 335 born before 1982. PCR increased the number of infections detected to 77, distributed among all age groups. Prevalences were 12.1%, 16.7%, and 6.0%, respectively (P < 0.001). In November, a similar age pattern was found, but with fewer infections: 6/746 and 39/741 individuals were found to be infected by microscopy and PCR, respectively. The frequencies of antibody responses to P. vivax were significantly higher in individuals born before 1991 than in younger age groups and were similar to those on Malakula Island, an area of endemicity. Remarkably low antigen diversity (h, 0.15) of P. vivax infections was observed on Aneityum compared with the other islands (h, 0.89 to 1.0). A P. vivax resurgence was observed among children and teenagers on Aneityum, an age distribution similar to those before elimination and on islands where P. vivax is endemic, suggesting that in the absence of significant exposure, immunity may persist, limiting infection levels in adults. The limited parasite gene pool on islands may contribute to this protection.

RALP1 is a rhoptry neck erythrocyte-binding protein of Plasmodium falciparum merozoites and a potential blood-stage vaccine candidate antigen.

Erythrocyte invasion by merozoites is an obligatory stage of Plasmodium infection and is essential to disease progression. Proteins in the apical organelles of merozoites mediate the invasion of erythrocytes and are potential malaria vaccine candidates. Rhoptry-associated, leucine zipper-like protein 1 (RALP1) of Plasmodium falciparum was previously found to be specifically expressed in schizont stages and localized to the rhoptries of merozoites by immunofluorescence assay (IFA). Also, RALP1 has been refractory to gene knockout attempts, suggesting that it is essential for blood-stage parasite survival. These characteristics suggest that RALP1 can be a potential blood-stage vaccine candidate antigen, and here we assessed its potential in this regard. Antibodies were raised against recombinant RALP1 proteins synthesized by using the wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that RALP1 is a rhoptry neck protein of merozoites. Moreover, our IFA data showed that RALP1 translocates from the rhoptry neck to the moving junction during merozoite invasion. Growth and invasion inhibition assays revealed that anti-RALP1 antibodies inhibit the invasion of erythrocytes by merozoites. The findings that RALP1 possesses an erythrocyte-binding epitope in the C-terminal region and that anti-RALP1 antibodies disrupt tight-junction formation, are evidence that RALP1 plays an important role during merozoite invasion of erythrocytes. In addition, human sera collected from areas in Thailand and Mali where malaria is endemic recognized this protein. Overall, our findings indicate that RALP1 is a rhoptry neck erythrocyte-binding protein and that it qualifies as a potential blood-stage vaccine candidate.

Single amino acid substitution in Plasmodium yoelii erythrocyte ligand determines its localization and controls parasite virulence.

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host-pathogen interactions and malaria virulence.

Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen.

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.

Plasmodium vivax: comparison of immunogenicity among proteins expressed in the cell-free systems of Escherichia coli and wheat germ by suspension array assays.

BACKGROUND: In vitro cell-free systems for protein expression with extracts from prokaryotic (Escherichia coli) or eukaryotic (wheat germ) cells coupled to solid matrices have offered a valid approach for antigen discovery in malaria research. However, no comparative analysis of both systems is presently available nor the usage of suspension array technologies, which offer nearly solution phase kinetics. METHODS: Five Plasmodium vivax antigens representing leading vaccine candidates were expressed in the E. coli and wheat germ cell-free systems at a 50 µl scale. Products were affinity purified in a single-step and coupled to luminex beads to measure antibody reactivity of human immune sera. RESULTS: Both systems readily produced detectable proteins; proteins produced in wheat germ, however, were mostly soluble and intact as opposed to proteins produced in E. coli, which remained mostly insoluble and highly degraded. Noticeably, wheat germ proteins were recognized in significantly higher numbers by sera of P. vivax patients than identical proteins produced in E. coli. CONCLUSIONS: The wheat germ cell-free system offers the possibility of expressing soluble P. vivax proteins in a small-scale for antigen discovery and immuno-epidemiological studies using suspension array technology.

Measurement of naturally acquired humoral immune responses against the C-terminal region of the Plasmodium vivax MSP1 protein using protein arrays.

Protein arrays are powerful tools for antibody profiling and vaccine development against infectious agents. In the previous report, we successfully applied an antibody-based protein array for immunoprofiling of Plasmodium vivax infection. Herein, we developed a Ni-NTA surface based protein array to detect immune responses against the recombinant C-terminal region (19 and 42 kDa) of the P. vivax merozoite surface protein 1 (PvMSP1-19 and -42) from sera of vivax malaria patients. The PvMSP1-19 arrays detected P. vivax in 112 of 130 (86.2%; 95% CI, 83.2-89.2%) microscopically positive samples and 2 false positives were obtained among 100 sera samples from healthy subjects (2.0%; 95% CI, 0.6-3.4%). These results were in concordance with results of enzyme-linked immunosorbent assays (ELISA). Kappa values represented excellent agreement for the recombinant PvMSP1-19 protein against sera samples as measured by protein arrays and ELISA (Kappa=0.904, 95% CI: 0.849-0.960). The PvMSP1-42 protein arrays detected antibody response in 100 of 130 microscopically positive samples (76.9%; 95% CI, 72.4-86.8%) and 8 false positives were obtained in 100 healthy subjects (8.0%; 95% CI, 2.7-13.3%). There is no significant difference between the fluorescent intensity of antibody response to PvMSP1-19 and PvMSP1-42 in the positive sera samples (P>0.05). The novel protein array platform may be used for profiling naturally acquired humoral immune responses to P. vivax infection.

Immunoproteomics profiling of blood stage Plasmodium vivax infection by high-throughput screening assays.

Completed genome sequences and stage-specific transcriptomes of the intraerythrocytic developmental cycle of Plasmodium vivax offers the opportunity to profile immune responses against P. vivax infection using innovative screening approaches. To detect the immune responses to blood stage-specific proteins, we applied a protein array technology to screen the sera of vivax malaria patients. Herein, a set of genes from the P. vivax blood stage was cloned using the In-Fusion cloning method and expressed by a wheat germ cell-free system. A total of 94 open reading frames (ORFs) were cloned and 89 (95%, 89/94) proteins were expressed, which were screened with sera from P. vivax-infected patients and healthy individuals using protein arrays. A total of 18 (19.1%, 18/94) highly immunoreactive proteins were identified, including 7 well-characterized vivax vaccine candidates. The remaining 11 ORFs have not been previously described as immunologically reactive. These novel immunoproteomes of the vivax malaria blood stage will be further studied as potential vaccine candidates. In this first report, high-throughput screening assays have been applied to investigate blood stage-specific immunoproteomes from vivax malaria. These methods may be used to determine immunodominant candidate antigens from the P. vivax genome.

Evidence for the transmission of Plasmodium vivax in the Republic of the Congo, West Central Africa.

Plasmodium vivax is not thought to be transmitted in western and central Africa, because of the very high prevalence of the red blood cell Duffy-negative phenotype in local populations, a condition which is thought to confer complete resistance against blood infection with P. vivax. There are, however, persistent reports of travelers returning from this region with P. vivax infections. To investigate whether transmission occurs in this region, the presence of antibodies specific to P. vivax preerythrocytic-stage antigens was assessed in individuals from the Republic of the Congo. A total of 55 (13%) of 409 samples tested by enzyme-linked immunosorbent assay had antibodies to P. vivax-specific antigens.

Identification of a Novel RAMA/RON3 Rhoptry Protein Complex in Plasmodium falciparum Merozoites.

Malaria causes a half a million deaths annually. The parasite intraerythrocytic lifecycle in the human bloodstream is the major cause of morbidity and mortality. Apical organelles of merozoite stage parasites are involved in the invasion of erythrocytes. A limited number of apical organellar proteins have been identified and characterized for their roles during erythrocyte invasion or subsequent intraerythrocytic parasite development. To expand the repertoire of identified apical organellar proteins we generated a panel of monoclonal antibodies against Plasmodium falciparum schizont-rich parasites and screened the antibodies using immunofluorescence assays. Out of 164 hybridoma lines, 12 clones produced monoclonal antibodies yielding punctate immunofluorescence staining patterns in individual merozoites in late schizonts, suggesting recognition of merozoite apical organelles. Five of the monoclonal antibodies were used to immuno-affinity purify their target antigens and these antigens were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two known apical organelle protein complexes were identified, the high-molecular mass rhoptry protein complex (PfRhopH1/Clags, PfRhopH2, and PfRhopH3) and the low-molecular mass rhoptry protein complex (rhoptry-associated proteins complex, PfRAP1, and PfRAP2). A novel complex was additionally identified by immunoprecipitation, composed of rhoptry-associated membrane antigen (PfRAMA) and rhoptry neck protein 3 (PfRON3) of P. falciparum. We further identified a region spanning amino acids Q221-E481 within the PfRAMA that may associate with PfRON3 in immature schizonts. Further investigation will be required as to whether PfRAMA and PfRON3 interact directly or indirectly.

Serodiagnostic antigens of Clonorchis sinensis identified and evaluated by high-throughput proteogenomics.

Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis.

Pyruvate kinase type-II isozyme in Plasmodium falciparum localizes to the apicoplast.

Bioinformatics research on Plasmodium falciparum revealed two isoforms of pyruvate kinase: type-I and type-II enzymes. The type-I enzyme shows typical glycolytic properties, while type-II enzyme is involved in fatty acid type-II biosynthesis and has been predicted to localize to the apicoplast with the targeting signal in its N-terminus. The type-I and type-II isoforms have the same evolutionary origin as Toxoplasma gondii isozymes, TgPyKI and TgPyKII, respectively; however, TgPyKII localizes to both the mitochondrion and the apicoplast. Accordingly, we made a recombinant full length of P. falciparum pyruvate kinase type-II protein using a wheat germ cell-free expression system and obtained a specific antibody against the type-II protein. Fluorescent microscopic analysis revealed that P. falciparum type-II enzyme was localized only to the apicoplast, not to the mitochondrion. The data suggest differences in localization and metabolic pathways between P. falciparum and T. gondii pyruvate kinase isoforms.

Anti-MSP11 IgG inhibits Plasmodium falciparum merozoite invasion into erythrocytes in vitro.

Merozoite surface proteins (MSPs) are considered as promising blood-stage malaria vaccine candidates. MSP3 has long been evaluated for its vaccine candidacy, however, the candidacy of other members of MSP3 family is insufficiently characterized. Here, we investigated Plasmodium falciparum MSP11 (PF3D7_1036000), a member of the MSP3 family, for its potential as a blood-stage vaccine candidate. The full-length protein (MSP11-FL) as well as the N-terminal half-MSP11 (MSP11-N), known to be unique among the MSP3 family members, were expressed by wheat germ cell-free system, and used to raise antibodies in rabbit. Immunoblot analysis of schizont lysates probed with anti-MSP11-N antibodies detected double bands at approximately 40 and 60 kDa, consistent with the previous report thus confirming antibodies specificity. However, inconsistent with previously reported merozoite's surface localization, immunofluorescence assay (IFA) revealed that MSP11 likely localizes to rhoptry neck of merozoites in mature schizonts. After invasion, MSP11 localized to parasitophorous vacuole and thereafter in Maurer's clefts in trophozoites. Anti-MSP11-FL antibody levels were significantly higher in asymptomatic than symptomatic P. falciparum cases in malaria low endemic Thailand. This reconfirmed that anti-MSP11 antibodies play an important role in protection against clinical malaria, as previously reported. Furthermore, in vitro growth inhibition assay revealed that anti-MSP11-FL rabbit antibodies biologically function by inhibiting merozoite invasion of erythrocytes. These findings further support the vaccine candidacy of MSP11.

Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates.

One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 mug/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.

Wavefront analysis of acrylic spherical and aspherical intraocular lenses.

PURPOSE: To compare higher order wavefront aberrations in eyes implanted with acrylic aspherical intraocular lenses (IOLs) with a modified prolate anterior surface with conventional acrylic spherical IOLs by using the optical path difference method. METHODS: In a nonrandomized parallel cohort investigation, 36 eyes of 31 patients implanted with aspherical IOLs (Tecnis ZA9003) and 37 eyes of 23 age-matched patients implanted with spherical IOLs (SENSOR AR40e) were evaluated with a wavefront analyzer (OPD-Scan II) preoperatively and 1 month after surgery. The higher order aberrations for a 4.0-mm pupil diameter were expanded into Zernike's polynomial expression. Coma aberration, spherical aberration, and total higher order aberrations were evaluated as root mean square values. RESULTS: Postoperatively, coma and total higher order aberrations of spherical and aspherical IOLs significantly improved in both eyes. Spherical aberration improved in eyes with aspherical IOLs only (P < 0.01). CONCLUSION: After implantation of acrylic aspherical IOLs, postoperative higher order aberrations were not necessarily lower than after implantation of acrylic spherical IOLs, but compared with levels following implantation of acrylic spherical IOLs, a significant reduction in spherical aberration was achieved.

Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission.

The target molecules of antibodies against falciparum malaria remain largely unknown. Recently we have identified multiple proteins as targets of immunity against Plasmodium falciparum using African serum samples. To investigate whether potential targets of clinical immunity differ with transmission intensity, we assessed immune responses in residents of low malaria transmission region in Thailand. Malaria asymptomatic volunteers (Asy: n=19) and symptomatic patients (Sym: n=21) were enrolled into the study. Serum immunoreactivity to 186 wheat germ cell-free system (WGCFS)-synthesized recombinant P. falciparum asexual-blood stage proteins were determined by AlphaScreen, and subsequently compared between the study groups. Forty proteins were determined as immunoreactive with antibody responses to 35 proteins being higher in Asy group than in Sym group. Among the 35 proteins, antibodies to MSP3, MSPDBL1, RH2b, and MSP7 were significantly higher in Asy than Sym (unadjusted p<0.005) suggesting these antigens may have a protective role in clinical malaria. MSP3 reactivity remained significantly different between Asy and Sym groups even after multiple comparison adjustments (adjusted p=0.033). Interestingly, while our two preceding studies using African sera were conducted differently (e.g., cross-sectional vs. longitudinal design, observed clinical manifestation vs. functional activity), those studies similarly identified MSP3 and MSPDBL1 as potential targets of protective immunity. This study further provides a strong rationale for the application of WGCFS-based immunoprofiling to malaria vaccine candidate and biomarker discovery even in low or reduced malaria transmission settings.

The Plasmodium vivax homolog of the ookinete adhesive micronemal protein, CTRP.

The Plasmodium circumsporozoite protein/thrombospondin-related anonymous protein-related protein (CTRP) is expressed at the mosquito midgut ookinete stage and is considered to be a transmission-blocking vaccine candidate. CTRP is composed of multiple von Willebrand factor A (vWA) and thrombospondin type 1 domains in the extracellular portion of the molecule, and a short acidic cytoplasmic domain that interacts with the actomyosin machinery. As a means to predict functionally relevant domains within CTRP we determined the nucleotide sequences of CTRP from the Plasmodium vivax Sall and the Plasmodium yoelii 17XL strains and characterized the conservation of domain architectures and motifs across Plasmodium genera. Sequence alignments indicate that the CTRP 1st to 4th vWA domains exhibit greater conservation, and thereby are perhaps functionally more important than the 5th and 6th domains. This point should be considered for the development of a transmission-blocking vaccine that includes CTRP recombinant subunit. To complement previous cellular studies on CTRP, we further determined the expression and cellular localization of CTRP protein in P. vivax and P. yoelii.

Role of complex II in anaerobic respiration of the parasite mitochondria from Ascaris suum and Plasmodium falciparum.

Parasites have developed a variety of physiological functions necessary for existence within the specialized environment of the host. Regarding energy metabolism, which is an essential factor for survival, parasites adapt to low oxygen tension in host mammals using metabolic systems that are very different from that of the host. The majority of parasites do not use the oxygen available within the host, but employ systems other than oxidative phosphorylation for ATP synthesis. In addition, all parasites have a life cycle. In many cases, the parasite employs aerobic metabolism during their free-living stage outside the host. In such systems, parasite mitochondria play diverse roles. In particular, marked changes in the morphology and components of the mitochondria during the life cycle are very interesting elements of biological processes such as developmental control and environmental adaptation. Recent research has shown that the mitochondrial complex II plays an important role in the anaerobic energy metabolism of parasites inhabiting hosts, by acting as quinol-fumarate reductase.