Molecular phylogenetical identification of bear roundworms (Baylisascaris transfuga) derived from wild Japanese black bears (Ursus thibetanus japonicus) around Lake Towada.

The bear roundworm Baylisascaris transfuga has been identified in several host bears (Ursinae). However, limited genetic information is available on the bear roundworm in Japanese populations. This study evaluated the genetic composition of bear roundworms isolated from wild Japanese black bears indigenous to Lake Towada, Japan. First, we conducted genetic and/or molecular phylogenetic analyses based on cytochrome c oxidase subunit II and internal transcribed spacer 2 among Baylisascaris species. These analyses revealed that the identified roundworms were genetically B. transfuga. In addition, the average body size of the obtained roundworms in this study was almost the same as that previously reported for B. transfuga. This study represents an important step in genetic research on the roundworm B. transfuga in Ursinae bears not only from Japan but also from the rest of the world.

The C-terminal region of the Plasmodium berghei gamete surface 184-kDa protein Pb184 contributes to fertilization and male gamete binding to the residual body.

BACKGROUND: Malaria, a global health concern, is caused by parasites of the Plasmodium genus, which undergo gametogenesis in the midgut of mosquitoes after ingestion of an infected blood meal. The resulting male and female gametes fuse to form a zygote, which differentiates into a motile ookinete. After traversing the midgut epithelium, the ookinete differentiates into an oocyst on the basal side of the epithelium. METHODS: Membrane proteins with increased gene expression levels from the gamete to oocyst stages in P. berghei were investigated utilizing PlasmoDB, the functional genomic database for Plasmodium spp. Based on this analysis, we selected the 184-kDa membrane protein, Pb184, for further study. The expression of Pb184 was further confirmed through immunofluorescence staining, following which we examined whether Pb184 is involved in fertilization using antibodies targeting the C-terminal region of Pb184 and biotin-labeled C-terminal region peptides of Pb184. RESULTS: Pb184 is expressed on the surface of male and female gametes. The antibody inhibited zygote and ookinete formation in vitro. When mosquitoes were fed on parasite-infected blood containing the antibody, oocyst formation decreased on the second day after feeding. Synthesized biotin-labeled peptides matching the C-terminal region of Pb184 bound to the female gamete and the residual body of male gametes, and inhibited differentiation into ookinetes in the in vitro culture system. CONCLUSIONS: These results may be useful for the further studying the fertilization mechanism of Plasmodium protozoa. There is also the potential for their application as future tools to prevent malaria transmission.

Searching for new molecules involved in Anopheles mosquitoes' response to Plasmodium infection.

Plasmodium parasites within mosquitoes are exposed to various physiological processes, such as blood meal digestion activity, the gonotrophic cycle, and host responses preventing the entry of parasites into the midgut wall. However, when in vitro-cultured ookinetes are injected into the hemocoel of mosquitoes, Plasmodium parasites are not affected by the vertebrate host's blood contents and do not pass through the midgut epithelial cells. This infection method might aid in identifying mosquito-derived factors affecting Plasmodium development within mosquitoes. This study investigated novel mosquito-derived molecules related to parasite development in Anopheles mosquitoes. We injected in vitro-cultured Plasmodium berghei (ANKA strain) ookinetes into female and male Anopheles stephensi (STE2 strain) mosquitoes and found that the oocyst number was significantly higher in males than in females, suggesting that male mosquitoes better support the development of parasites. Next, RNA-seq analysis was performed on the injected female and male mosquitoes to identify genes exhibiting changes in expression. Five genes with different expression patterns between sexes and greatest expression changes were identified as being potentially associated with Plasmodium infection. Two of the five genes also showed expression changes with infection by blood-feeding, indicating that these genes could affect the development of Plasmodium parasites in mosquitoes.

Detection and molecular analysis of Philopinna higai (Digenea: Didymozoidae) from domestically introduced Sarcocheilichthys fishes in the Tohoku region, Japan.

Philopinna higai is a species of Didymozoidae (Digenea: Hemiuroidea). The definitive hosts of this parasite only belong to the fish genus Sarcocheilichthys. Sarcocheilichthys fishes are endemic to Lake Biwa and southwestern Japan and were introduced into the northeastern (Tohoku) region. However, P. higai parasitism has not been investigated in the Tohoku region. In this study, we surveyed the distribution of P. higai in the Tohoku region and sequenced 28S rDNA (994 bp) and cytochrome oxidase subunit 1 (CO1) gene (721 bp) of P. higai. We also sequenced mitochondrial cytochrome b (581 bp) of Sarcocheilichthys fishes from the Tohoku region and Lake Biwa. Our findings confirmed the distribution of P. higai in all seven surveyed river systems in the four prefectures of the Tohoku region. The 28S rDNA sequence of P. higai did not differ among regions, whereas 10 haplotypes of CO1 were identified and clustered into two major clades. The haplotypes of Sarcocheilichthys fishes introduced in the Tohoku region were identical to the dominant haplotypes in Lake Biwa. Thus, P. higai from Lake Biwa and the Tohoku region were genetically the same species, although genetically differentiated populations formed in the Tohoku region.

Formation of free oocysts in Anopheles mosquitoes injected with Plasmodium ookinetes.

Malaria needs new strategies for its control. Plasmodium spp., the causative agent of malaria, is transmitted by mosquitoes. These parasites develop into oocysts and sporozoites in the body of the mosquitoes. A deeper understanding of oocysts that produce the infectious form of the parasite, sporozoites, can facilitate the development of novel countermeasures. However, the isolation of Plasmodium oocysts is challenging as these are formed between midgut epithelial cells and basal lamina after gametocytes enter the mosquito's body through blood feeding. Further research on oocysts has been impeded by issues related to oocyst isolation. Therefore, in this study, we injected Plasmodium into mosquitoes-an artificial and unique method-and aimed to clarify how oocysts were formed in mosquitoes after Plasmodium injection and whether free oocysts were formed from the mosquito tissue. Plasmodium berghei (ANKA strain) ookinetes cultured in vitro were injected into the thoracic body cavity (hemocoel) of female and male Anopheles stephensi mosquitoes. Oocysts were formed in the body of female and male mosquitoes at 14 days post injection. In addition, oocysts formed as a result of injection developed into sporozoites, which were infectious to mice. These findings suggest that P. berghei can complete its developmental stage in mosquitoes by injection. Some of the oocysts formed were free from mosquito tissue, and it was possible to collect oocysts with minimal contamination of mosquito tissue. These free oocysts can be used for investigating oocyst proteins and metabolism.

Plasmodium berghei Brca2 is required for normal development and differentiation in mice and mosquitoes.

BACKGROUND: Malaria is a major global parasitic disease caused by species of the genus Plasmodium. Zygotes of Plasmodium spp. undergo meiosis and develop into tetraploid ookinetes, which differentiate into oocysts that undergo sporogony. Homologous recombination (HR) occurs during meiosis and introduces genetic variation. However, the mechanisms of HR in Plasmodium are unclear. In humans, the recombinases DNA repair protein Rad51 homolog 1 (Rad51) and DNA meiotic recombinase 1 (Dmc1) are required for HR and are regulated by breast cancer susceptibility protein 2 (BRCA2). Most eukaryotes harbor BRCA2 homologs. Nevertheless, these have not been reported for Plasmodium. METHODS: A Brca2 candidate was salvaged from a database to identify Brca2 homologs in Plasmodium. To confirm that the candidate protein was Brca2, interaction activity between Plasmodium berghei (Pb) Brca2 (PbBrca2) and Rad51 (PbRad51) was investigated using a mammalian two-hybrid assay. To elucidate the functions of PbBrca2, PbBrca2 was knocked out and parasite proliferation and differentiation were assessed in mice and mosquitoes. Transmission electron microscopy was used to identify sporogony. RESULTS: The candidate protein was conserved among Plasmodium species, and it was indicated that it harbors critical BRCA2 domains including BRC repeats, tower, and oligonucleotide/oligosaccharide-binding-fold domains. The P. berghei BRC repeats interacted with PbRad51. Hence, the candidate was considered a Brca2 homolog. PbBrca2 knockout parasites were associated with reduced parasitemia with increased ring stage and decreased trophozoite stage counts, gametocytemia, female gametocyte ratio, oocyst number, and ookinete development in both mice and mosquitoes. Nevertheless, the morphology of the blood stages in mice and the ookinete stage was comparable to those of the wild type parasites. Transmission electron microscopy results showed that sporogony never progressed in Brca2-knockout parasites. CONCLUSIONS: Brca2 is implicated in nearly all Plasmodium life cycle stages, and especially in sporogony. PbBrca2 contributes to HR during meiosis.

Morphological and molecular phylogenetical identification of Tricodectes pinguis from Japanese black bears (Ursus thibetanus japonicus) in Aomori Prefecture, Japan.

Trichodectes pinguis, referred to commonly as the bear-biting louse, has been reported in several bear species. However, graphical (blurred or coarse) and genetic information on the louse is limited. In this study, we identified T. pinguis collected from Japanese black bears in the Aomori Prefecture, Japan. We confirmed 12S rDNA sequences derived from the collected T. pinguis and performed molecular phylogenetic analysis based on 12S rDNA. The analysis revealed the parasitic louse to be T. pinguis. Interestingly, the body size of T. pinguis found in this study was smaller than the previous recorded body size of them in Japan and Turkey. To better understand the biting louse infesting bears, morphometric and genetic information from other bear hosts needs to be accumulated.

Role of Plasmodium berghei ookinete surface and oocyst capsule protein, a novel oocyst capsule-associated protein, in ookinete motility.

BACKGROUND: Plasmodium sp., which causes malaria, must first develop in mosquitoes before being transmitted. Upon ingesting infected blood, gametes form in the mosquito lumen, followed by fertilization and differentiation of the resulting zygotes into motile ookinetes. Within 24 h of blood ingestion, these ookinetes traverse mosquito epithelial cells and lodge below the midgut basal lamina, where they differentiate into sessile oocysts that are protected by a capsule. METHODS: We identified an ookinete surface and oocyst capsule protein (OSCP) that is involved in ookinete motility as well as oocyst capsule formation. RESULTS: We found that knockout of OSCP in parasite decreases ookinete gliding motility and gradually reduces the number of oocysts. On day 15 after blood ingestion, the oocyst wall was significantly thinner. Moreover, adding anti-OSCP antibodies decreased the gliding speed of wild-type ookinetes in vitro. Adding anti-OSCP antibodies to an infected blood meal also resulted in decreased oocyst formation. CONCLUSION: These findings may be useful for the development of a transmission-blocking tool for malaria.

Raccoon dogs (Nyctereutes procyonoides) are the new natural definitive hosts of Metagonimus hakubaensis.

The definitive hosts of Metagonimus hakubaensis are reported to be hamsters, rats, mice, dogs, cats, chickens, and quails in experimental infection and Japanese water shrews in natural infection. Here we report that raccoon dogs are new natural definitive hosts of M. hakubaensis, based on morphological and molecular analyses of Metagonimus flukes collected from the host species from Aomori Prefecture, Japan. Moreover, M. hakubaensis recovered from raccoon dogs showed higher fecundity than those recovered from Japanese water shrews. Therefore, raccoon dogs were considered as a more suitable natural definitive host of M. hakubaensis than Japanese water shrews.