Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts.

The circumsporozoite protein, CSP, is the major surface protein of Plasmodium sporozoites, the form of malaria parasites transmitted by mosquitoes. CSP is involved in sporozoite formation within and egress from oocysts, entry into mosquito salivary glands and mammalian liver as well as migration in the skin. Yet, how CSP facilitates sporozoite formation, oocyst egress and hepatocyte specific invasion is still not fully understood. Here, we aimed at generating a series of parasites expressing full-length versions of CSP with internally inserted green fluorescent protein between known domains at the endogenous csp locus. This enabled the investigation of sporozoite formation in living oocysts. GFP insertion after the signal peptide leads to cleavage of GFP before the fusion protein reached the plasma membrane while insertion of GFP before or after the TSR domain prevented sporozoite egress and liver invasion. These data suggest different strategies for obtaining mature salivary gland sporozoites that express GFP-CSP fusions.

Vancomycin improves Plasmodium yoelii malaria parasite in vitro liver stage cultures by controlling Elizabethkingia anophelis, a bacterium in the microbiome of lab-reared Anopheles mosquitoes.

Studies of Plasmodium sporozoites and liver stages require dissection of Anopheles mosquitoes to obtain sporozoites for experiments. Sporozoites from the rodent parasite P. yoelii are routinely used to infect hepatocytes for liver stage culture, but sometimes these cultures become contaminated. Using standard microbiological techniques, a single colony type of Gram-negative rod-shaped bacteria was isolated from contaminated cultures. Mass spectrometry and sequencing of the bacterial 16S ribosomal RNA gene identified the contaminant as Elizabethkingia spp. Based on sequence comparison and published studies of the Anopheles microbiome, the best match was E. anophelis. Culture contamination was not ameliorated by density gradient purification of sporozoites. However, the addition of vancomycin to the culture media consistently reduced contamination and improved culture outcomes as measured by liver stage parasite size. Thus, mosquito salivary gland-derived E. anophelis is identified a potential contaminant of Plasmodium liver stage cultures that can be mitigated by the addition of antibiotics.

Deciphering host lysosome-mediated elimination of Plasmodium berghei liver stage parasites.

Liver stage Plasmodium parasites reside in a parasitophorous vacuole (PV) that associates with lysosomes. It has previously been shown that these organelles can have beneficial as well as harmful effects on the parasite. Yet it is not clear how the association of lysosomes with the parasite is controlled and how interactions with these organelles lead to the antagonistic outcomes. In this study we used advanced imaging techniques to characterize lysosomal interactions with the PV. In host cells harboring successfully developing parasites we observed that these interaction events reach an equilibrium at the PV membrane (PVM). In a population of arrested parasites, this equilibrium appeared to shift towards a strongly increased lysosomal fusion with the PVM witnessed by strong PVM labeling with the lysosomal marker protein LAMP1. This was followed by acidification of the PV and elimination of the parasite. To systematically investigate elimination of arrested parasites, we generated transgenic parasites that express the photosensitizer KillerRed, which leads to parasite killing after activation. Our work provides insights in cellular details of intracellular killing and lysosomal elimination of Plasmodium parasites independent of cells of the immune system.

Detection of Plasmodium berghei infected Anopheles stephensi using near-infrared spectroscopy.

BACKGROUND: The proportion of mosquitoes infected with malaria is an important entomological metric used to assess the intensity of transmission and the impact of vector control interventions. Currently, the prevalence of mosquitoes with salivary gland sporozoites is estimated by dissecting mosquitoes under a microscope or using molecular methods. These techniques are laborious, subjective, and require either expensive equipment or training. This study evaluates the potential of near-infrared spectroscopy (NIRS) to identify laboratory reared mosquitoes infected with rodent malaria. METHODS: Anopheles stephensi mosquitoes were reared in the laboratory and fed on Plasmodium berghei infected blood. After 12 and 21 days post-feeding mosquitoes were killed, scanned and analysed using NIRS and immediately dissected by microscopy to determine the number of oocysts on the midgut wall or sporozoites in the salivary glands. A predictive classification model was used to determine parasite prevalence and intensity status from spectra. RESULTS: The predictive model correctly classifies infectious and uninfectious mosquitoes with an overall accuracy of 72%. The false negative and false positive rates were 30 and 26%, respectively. While NIRS was able to differentiate between uninfectious and highly infectious mosquitoes, differentiating between mid-range infectious groups was less accurate. Multiple scans of the same specimen, with repositioning the mosquito between scans, is shown to improve accuracy. On a smaller dataset NIRS was unable to predict whether mosquitoes harboured oocysts. CONCLUSIONS: To our knowledge, we provide the first evidence that NIRS can differentiate between infectious and uninfectious mosquitoes. Currently, distinguishing between different intensities of infection is challenging. The classification model provides a flexible framework and allows for different error rates to be optimised, enabling the sensitivity and specificity of the technique to be varied according to requirements.

Re-evaluation of the life cycle of Eimeria maxima Tyzzer, 1929 in chickens (Gallus domesticus).

A time-course study was conducted to resolve discrepancies in the literature and better define aspects of the Eimeria maxima life cycle such, as sites of development and both morphology and number of asexual stages. Broiler chickens were inoculated orally with five million E. maxima oocysts (APU1), and were necropsied at regular intervals from 12 to 120 h p.i. Small intestine tissue sections and smears were examined for developmental stages. The jejunum contained the highest numbers of developmental stages. At 12 h p.i., sporozoites were observed inside a parasitophorous vacuole (PV) in the epithelial villi and the lamina propria. By 24 h, sporozoites enclosed by a PV were observed in enterocytes of the glands of Lieberkühn. At 48 h p.i., sporozoites, elongated immature and mature schizonts, were all seen in the glands with merozoites budding off from a residual body. By 60 h, second-generation, sausage-shaped schizonts containing up to 12 merozoites were observed around a residual body in the villar tip of invaded enterocytes. At 72 and 96 h, profuse schizogony associated with third- and fourth-generation schizonts was observed throughout the villus. At 120 h, another generation (fifth) of schizonts were seen in villar tips as well as in subepithelium where gamonts and oocysts were also present; a few gamonts were in epithelium. Our finding of maximum parasitization of E. maxima in jejunum is important because this region is critical for nutrient absorption and weight gain.

Plasmodium Sporozoite Biology.

Plasmodium sporozoite transmission is a critical population bottleneck in parasite life-cycle progression and, hence, a target for prophylactic drugs and vaccines. The recent progress of a candidate antisporozoite subunit vaccine formulation to licensure highlights the importance of sporozoite transmission intervention in the malaria control portfolio. Sporozoites colonize mosquito salivary glands, migrate through the skin, penetrate blood vessels, breach the liver sinusoid, and invade hepatocytes. Understanding the molecular and cellular mechanisms that mediate the remarkable sporozoite journey in the invertebrate vector and the vertebrate host can inform evidence-based next-generation drug development programs and immune intervention strategies.

Isospora lunaris n. sp. (Apicomplexa: Eimeriidae) from the domestic Java sparrow in Japan.

Five individuals of the domestic Java sparrows, Lonchura oryzivora (Aves: Estrildidae), were examined for coccidian parasites. Sporulated oocysts had two sporocysts containing four sporozoites each. Sporulated oocysts (n=30) were spherical, with a two splinter-like polar granules. Oocyst size was 22.1×20.7 (20.0-25.0×20.0-22.5)µm. They had a thick wall that consisted of a pale yellow outer layer and a dark yellow inner layer, and lacked micropyle and residuum. Sporocysts (n=60) were elongated ovoid 14.1×9.8 (12.5-15.0×7.5-10.0)µm, smooth walled, and colorless, with crescent-shaped Stieda and indistinct substieda bodies. Sporocyst residuum was interspersed between sporozoites. Sporozoites were oriented transverse to the sporocyst longitudinal axis. On the basis of morphological data, the species isolated in the present study is a new species of Isospora and propose the name Isospora lunaris n. sp.

Progress in imaging methods: insights gained into Plasmodium biology.

Over the past decade, major advances in imaging techniques have enhanced our understanding of Plasmodium spp. parasites and their interplay with mammalian hosts and mosquito vectors. Cryoelectron tomography, cryo-X-ray tomography and super-resolution microscopy have shifted paradigms of sporozoite and gametocyte structure, the process of erythrocyte invasion by merozoites, and the architecture of Maurer's clefts. Intravital time-lapse imaging has been revolutionary for our understanding of pre-erythrocytic stages of rodent Plasmodium parasites. Furthermore, high-speed imaging has revealed the link between sporozoite structure and motility, and improvements in time-lapse microscopy have enabled imaging of the entire Plasmodium falciparum erythrocytic cycle and the complete Plasmodium berghei pre-erythrocytic stages for the first time. In this Review, we discuss the contribution of key imaging tools to these and other discoveries in the malaria field over the past 10 years.

Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalisation.

Toxoplasma gondii is a common parasite of humans and animals, which is transmitted via oocysts in cat faeces or tissue cysts in contaminated meat. The robust oocyst and sporocyst walls protect the infective sporozoites from deleterious external attacks including disinfectants. Upon oocyst acquisition, these walls lose their integrity to let the sporozoites excyst and invade host cells following a process that remains poorly understood. Given the resistance of the oocyst wall to digestive enzymes and the ability of oocysts to cause parenteral infections, the present study investigated the possible contribution of macrophages in supporting sporozoite excystation following oocyst internalisation. By using single cell micromanipulations, real-time and time-point imaging techniques, we demonstrated that RAW macrophages could interact rapidly with oocysts and engulfed them by remodelling of their actin cytoskeleton. Internalised oocysts were associated to macrophage acidic compartments and showed evidences of wall disruption. Sporozoites were observed in macrophages containing oocyst remnants or in new macrophages, giving rise to dividing tachyzoites. All together, these results highlight an unexpected role of phagocytic cells in processing T. gondii oocysts, in line with non-classical routes of infection, and open new perspectives to identify chemical factors that lead to oocyst wall disruption under physiological conditions.

Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites.

BACKGROUND: Malaria is transmitted when an infected mosquito delivers Plasmodium sporozoites into a vertebrate host. There are many species of Plasmodium and, in general, the infection is host-specific. For example, Plasmodium gallinaceum is an avian parasite, while Plasmodium berghei infects mice. These two parasites have been extensively used as experimental models of malaria transmission. Plasmodium falciparum and Plasmodium vivax are the most important agents of human malaria, a life-threatening disease of global importance. To complete their life cycle, Plasmodium parasites must traverse the mosquito midgut and form an oocyst that will divide continuously. Mature oocysts release thousands of sporozoites into the mosquito haemolymph that must reach the salivary gland to infect a new vertebrate host. The current understanding of the biology of oocyst formation and sporozoite release is mostly based on experimental infections with P. berghei, and the conclusions are generalized to other Plasmodium species that infect humans without further morphological analyses. RESULTS: Here, it is described the microanatomy of sporozoite escape from oocysts of four Plasmodium species: the two laboratory models, P. gallinaceum and P. berghei, and the two main species that cause malaria in humans, P. vivax and P. falciparum. It was found that sporozoites have species-specific mechanisms of escape from the oocyst. The two model species of Plasmodium had a common mechanism, in which the oocyst wall breaks down before sporozoites emerge. In contrast, P. vivax and P. falciparum sporozoites show a dynamic escape mechanism from the oocyst via polarized propulsion. CONCLUSIONS: This study demonstrated that Plasmodium species do not share a common mechanism of sporozoite escape, as previously thought, but show complex and species-specific mechanisms. In addition, the knowledge of this phenomenon in human Plasmodium can facilitate transmission-blocking studies and not those ones only based on the murine and avian models.

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites.

Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes.

A new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from Alectoris barbara (Aves: Phasianidae) from the Canary Islands (Spain).

The present study was conducted with the objective of identifying the species of Eimeria present in a cynegetic farm. A new coccidian (Apicomplexa: Eimeriidae) species is described from Barbary partridge, Alectoris barbara, from the Canary Islands. Experimental infections were carried out in order to determine the prepatent period, sporulation time, site of infection, and morphology of endogenous stages. One species is described as new. Eimeria barbarae n. sp. has ellipsoidal oocysts, 20.0 × 14.4 (16-23 × 13-16) µm, with a shape-index (SI) of 1.39. Sporocysts are almond-shaped, 9.0 × 5.4 (6.5-11 × 4.5-6) µm, SI = 1.56. The endogenous development takes place along the intestine. The present study showed that E. barbarae causes severe pathologies in A. barbara chickens, with impact on their health condition. Control strategies needs to be implemented to reduce the loss due to coccidiosis at studied farm.

Reclassification of Eimeria pogonae Walden (2009) as Choleoeimeria pogonae comb. nov. (Apicomplexa: Eimeriidae).

The presented paper provides a reclassification of Eimeria pogonae from Pogona vitticeps into the correct genus Choleoeimeria. A description of exogenous and endogenous stages of biliary coccidium is given. Sporulation of the oocysts was endogenous. The mature oocysts contained four sporocysts each with two sporozoites. Oocysts were ellipsoidal in shape, with average length/width ratio 1.7 and measured 28.4 (SD1.5) × 16.8 (SD 1.5). The micropyle, residuum, and polar granules were absent from the sporulated oocysts. Ovoidal in shape, sporosysts without Steida bodies contained residuum and two elongated and boat-shaped sporozoites. The endogenous stages of the coccidia were located mainly in the epithelium of bile ducts; however, single-epithelium cells of the gallbladder were also infected.

Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms.

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.

Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent.

Eimeria bovis is an important coccidian parasite that causes high economic losses in the cattle industry. We recently showed that polymorphonuclear neutrophils (PMN) react upon E. bovis sporozoite exposure by neutrophil extracellular trap (NET) formation. We focused here on the molecular mechanisms that are involved in this process. The sporozoite encounter led to an enhanced surface expression of neutrophil CD11b suggesting a potential role of this receptor in E. bovis-mediated NETosis. Antibody-mediated blockage of CD11b confirmed this assumption and led to a significantly decreased sporozoite-triggered NET. In addition, E. bovis-induced NETosis was found to be Ca(2+)-dependent since the inhibition of store-operated calcium entry (SOCE) significantly diminished NET. Furthermore, NADPH oxidase, neutrophil elastase (NE) and myeloperoxidase (MPO) were confirmed as key molecules in sporozoite-triggered NETosis, as inhibition thereof blocked parasite-triggered NET. PMN degranulation analyses revealed a significant release of matrix metalloprotease-9 containing granules upon sporozoite exposure. We further show a significantly enhanced phosphorylation of ERK1/2 and p38 MAPK in sporozoite-exposed PMN indicating a key role of this signaling pathway in E. bovis-mediated NETosis. Accordingly, ERK 1/2 and p38 MAPK inhibition led to a significant decrease in NET formation. Finally, we demonstrate that sporozoite-induced NETosis is neither a stage-, species-, nor host-specific process.

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages.

The repeat region of the circumsporozoite protein is critical for sporozoite formation and maturation in Plasmodium.

The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH2-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH2-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective.

An in vitro model of infection of chicken embryos by Cryptosporidium baileyi.

Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged and wild birds. Cryptosporidium baileyi is the most common species reported in a wide range of avian hosts. Although this parasite is well investigated, there is no adequate in vitro model for its endogenous development, and therefore, knowledge of each life cycle phase is scarce. In the present study, an in vitro model for C. baileyi in chicken embryos was developed and the complete life cycle investigated by light and electron microscopy, including both the sexual and asexual reproduction stages. The complete life cycle of C. baileyi was observed during 1-96 h post inoculation (PI), and the average reproduction number of C. baileyi oocysts in allantoic fluid of each chicken embryo was greatest at 168 h PI. These results suggest that chicken embryos could adequately represent the natural host cells and support the development of all the endogenous life cycle stages of C. baileyi, and also provide a new and effective in vitro cultivation system for further studies on antigens, virulence, infectivity, metabolites, and sensitivity of drugs against parasites.

Three new species of coccidia (Apicomplexa: Eimeriidae) from skinks, Lipinia spp. (Sauria: Scincidae), from Oceania.

Between September 1991 and March 1993, 25 moth skinks (Lipinia noctua) were collected from various localities on the Cook Islands, Fiji, Papua New Guinea (PNG), and Vanuatu and examined for coccidians. In addition, a single Roux's lipinia skink (Lipinia rouxi) was collected from PNG and examined for coccidia. Sixteen (64%) L. noctua were found to harbor 2 new eimerians, and L. rouxi harbored another new Eimeria sp. Oocysts of Eimeria lipinia n. sp. from 9 (36%) L. noctua from the Cook Islands, Fiji, and PNG were subspherical with a bilayered wall and measured (L × W) 18.6 × 16.9 µm, with a L/W ratio of 1.1. Both micropyle and oocyst residuum were absent, but a polar granule was present. Oocysts of Eimeria melanesia n. sp. from 6 (24%) L. noctua from Fiji and Vanuatu and a single L. rouxi from PNG were subspherical to ellipsoidal with a bilayered wall and measured 19.8 × 17.5 µm, and L/W was 1.1. Both micropyle and oocyst residuum were absent, but a single or fragmented polar granule was present. Oocysts of Eimeria lessoni n. sp. from 1 (4%) L. noctua from PNG were cylindroidal with a bilayered wall and measured 28.1 × 15.7 µm, and L/W was 1.8. Both micropyle and oocyst residuum were absent, but a single polar granule was present. These represent the third report of Eimeria spp. reported from any host on PNG and the only coccidians, to our knowledge, ever described from L. noctua and L. rouxi and from the Cook Islands and Vanuatu.