Jiří Vávra

Chapter IV – Research methods for entomopathogenic Protozoa

Publisher Summary This chapter discusses research methods for entomopathogenic protozoa. Bioassays are used to test the presence, viability, or the quantity of infective forms of a protozoan. To conduct a quantitative bioassay, spores must be counted and a dilution series made so that a known concentration of spores can be fed to the host. To calculate a dosage or concentration for any bioassay, the spores, or other infectious stages must be accurately counted. Viability can be assessed without infecting host animals. Viable microsporidian spores must be capable of germination. Spores can be fed to the host on artificial diet or their natural food. Experimentation is necessary in order to find the optimum dosage for best spore production. Nosema and Endoreticulatus spp. in Lepidoptera develop more slowly than Vairimorpha necatrix, dictating that infections be initiated earlier in larval life than was recommended for V. necatrix. Purity facilitates counting and measuring of spores and is also essential for obtaining pure extracts for biochemical studies. It is found that freeing the spores of host tissues and microbial contaminants is often necessary for storage.

Microsporidia of the genus Trachipleistophora--causative agents of human microsporidiosis: description of Trachipleistophora anthropophthera n. sp. (Protozoa: Microsporidia).

Trachipleistophora anthropophthera n. sp., was found at autopsy in the brain of one and in the brain, kidneys, pancreas, thyroid, parathyroid, heart, liver, spleen, lymph nodes, and bone marrow of a second patient with AIDS. The parasite is similar to the recently described T. hominis Hollister, Canning, Weidner, Field. Kench and Marriott, 1996, in having isolated nuclei, meronts with a thick layer of electron dense material on the outer face of their plasmalemma and sporogony during which spores are formed inside a thick‐walled sporophorous vesicle. In contrast to T. hominis, this species is dimorphic as it forms two kinds of sporophorous vesicles and spores: Type I‐round to oval polysporous sporophorous vesicle. 7‐10 μm in size, usually with eight spores (3.7 × 2.0 μm), thick endospores, subterminal anchoring disc and anisofilar polar filaments forming seven thicker and two thinner terminal coils. This type of sporophorous vesicle is associated with 25‐30 nm filaments extending into the host cell cytoplasm. Type II—smaller, bisporous sporophorous vesicle (4‐5 times 2.2‐2.5 μm) with two, nearly round, thin‐walled spores, 2.2‐2.5 × 1.8‐2.0 μm in size, having 4‐5 isofilar coils. No outside filamentous elements are associated with the bisporous sporophorous vesicle. Both types of sporophorous vesicles were common in the infected brain tissue and could be found within the same cell. The newly described species, together with T. hominis and previously reported Pleistophora‐like parasites from human muscle, likely represent a group of closely related human microsporidia.

An Ultrastructural Comparison of the Attachment Sites Between Gregarina steini and Cryptosporidium muris

ABSTRACT. Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane‐like structure limiting the cortical zone of the epimerite, resembles the Y‐shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.

Redescriptjon of Pleistophora intestinalis Chatton, 1907, a microsporidian parasite of Daphnia magna and Daphnia puiex, with establishment of the new genus Glugoides (Microspora, glugeidae)

Summary The cytology of a microsporidium identified as Pleistophora intestinalis Chatton, 1907, is described with emphasis on the ultrastructure. The spores are somewhat asymmetrical, ovoid to lightly kidney-shaped, measuring 1.1–1.7 × 2.4–2.7 urn in fresh preparations. The spore wall is three-layered with an approximately 15 nm thick exospore, which is electron-dense with a more dense surface coat. The polar filament is isofilar with 5–8, mostly 6, coils in a single layer, and the polaroplast has two regions with lamellae, the anterior ones more densely packed. All life cycle stages have isolated nuclei and the spores are uninucleate. Merozoites and sporoblasts are produced by plasmotomy, and the sporogony is polysporoblastic. Merogonial stages are enclosed in parasitophorous vacuoles, sporogonial stages in double envelopes: the parasitophorous vacuole and a spor-ophorous vesicle, formed by duplication of the plasma membrane of the sporont. There is some doubt that Pleistophora intestinalis of Daphnia magna and of D. pulex is the same microsporidian species, but that was not proven by the cytological analysis. The identification has been verified by study of material originating from Chatton and of the microsporidium Otto Jirovec, the first reviser, identified as P. intestinalis . The microsporidium is not a Pleistophora species. The new genus Glugoides is established, and placed in the family Glugeidae.

Morphology, molecular phylogeny, and ecology of Binucleata daphniae n. g., n. sp. (Fungi: Microsporidia), a parasite of Daphnia magna Straus, 1820 (Crustacea: Branchiopoda)

ABSTRACT. We describe a new microsporidian species Binucleata daphniae, n. g., n. sp., that infects the integument cells lining the hemocoele cavity of the carapace and the postabdomen of the cladoceran Daphnia magna Straus. Infected cells filled with spores accumulate as large clusters in the carapace cavity and heavily infected hosts are detected by their opaque appearance. Despite the parasites presence, infected Daphnia grow and molt, but have a reduced fecundity. During the parasites life cycle, chain‐like meronts with isolated nuclei are formed, giving rise to binucleate presporonts, the most frequently observed, characteristic developmental stage. In sporogony, the nuclei of the presporont separate, divide, and eight spores enclosed in a thin‐walled sporophorous vesicle are formed. Spores are 4.9 × 2.5 μm in size (fresh) and have an anisofilar polar filament with eight coils. DNA sequence analysis places B. daphniae in a clade of microsporidians that parasitize crustaceans and mosquitoes and have assumed complex life cycles. Binucleata daphniae, however, has a simple and direct life cycle and can be transferred to naïve hosts and maintained as persistent infections in populations of its host D. magna. We propose that B. daphniae has simplified its life cycle by losing its secondary host, rendering it unique in this clade.

Opportunistic nature of the mammalian microsporidia: experimental transmission of Trachipleistophora extenrec (Fungi: Microsporidia) between mammalian and insect hosts

Spores of Trachipleistophora extenrec, originally isolated from the muscles of the Madagascan insectivore Hemicentetes semispinosus and maintained by serial passage in severe combined immunodeficiency (SCID) mice, were fed to larvae of the Egyptian cotton leafworm Spodoptera littoralis. Extensive infection of larval tissues ensued and caused larval and pupal mortality. The development of T. extenrec in the insect host, studied both by light and electron microscopy, followed generally the same life cycle as in the mammalian host. However, some differences in the fine structure of the parasite grown in both types of hosts were found. Spores isolated from the insect host caused infection of SCID mice when injected intramuscularly. Our results suggest that T. extenrec may be originally an insect microsporidian. This likelihood is corroborated by its structural similarity and phylogenetic relationship to two other microsporidia having insects either as unique hosts (Vavraia culicis) or being able to infect both mammalian and insect host (Trachipleistophora hominis).

Light and electron microscopic cytology of Trichotuzetia guttata gen. et. sp. n. (Microspora, Tuzetiidae), a microsporidian parasite of Cyclops vicinusUljanin, 1875 (Crustacea, Copepoda)

Summary The microsporidium Trichotuzetia guttata gen. et sp. n., a parasite of the copepod Cyclops vicinus in the Czech Republic, is described based on light microscopic and ultrastructural characteristics. All life cycle stages have isolated nuclei. In the merogonial reproduction multinucleate plasmodia divide by plasmotomy, yielding uninucleate merozoites. Sporonts develop into multinucleate plasmodia, which split in a rosette-like manner, initially into wide lobes with large nuclei, finally into narrow lobes with small nuclei. Sporoblasts are formed in individual sporophorous vesicles generated by the sporogonial plasmodium. Immature sporogonial stages are covered by fibrillar projections uniting the exospore with the envelope of the sporophorous vesicle. Mature spores, which are pyriform with pointed anterior end, normally lack projections. Unfixed spores measure 4.6-5.7x2.7-3.3 µm. The exospore is four-layered. The anterior lamellae of the polaroplast are wide. The posterior ones (present in a short zone) are narrow close to the filament, wider at the periphery. The polar filament is isofilar with 9-10, 102-131 nm wide, coils in a single layer close to the spore wall in posterior half of the spore. The angle of tilt is 60-65°. Isometric, 21-25 nm wide virus-like particles were observed in the nuclei of mature spores. Experimental transmission of the microsporidium per os has failed. The small subunit rRNA gene has been sequenced and the data have been used in a phylogenetic analysis (PAUP). The species is compared to previously described microsporida of copepods, and the taxonomy is discussed.

Berwaldia schaefernai (Jírovec, 1937) comb. n. (protozoa, microsporida), fine structure, life cycle, and relationship to Berwaldia singularis Larsson, 1981

Summary Fine structure studies of the microsporidian Plistophora schaefernai Jirovec, 1937 (syn. Pleistophora schaefernai and Microsporidium schaefernai ) revealed that each spore is surrounded by an individual sporophorous vesicle, composed of a thin outer membrane-like sheath and an inner layer of tubular structures. This type of sporophorous vesicle is structurally identical to that of the genus Berwaldia Larsson, 1981. Berwaldia schaefernai (Jirovec, 1937) comb. n. is proposed and its relationship to Berwaldia singularis is established. Although both species are structurally very similar, they differ in spore width, arrangement of polar filament coils and probably in host specificity. Obviously they are sibling species.

Life cycle of Amblyospora weiseri n.sp.: (Microsporidia) in Aedes cantans (Diptera, Culicidae)

Amblyospora weiseri n.sp., parasite of the mosquito Aedes cantans in Czechoslovakia has two sporulation sequences: the octosporous sporulation sequence occurs in oenocytes and fat body tissue of larvae, involves meiosis and results in the formation of eight uninucleate spores (octospores) enclosed in a sporophorous vesicle and is fatal for the host. Octospores are not infectious to other larvae. The oenocytic sequence localized in oenocytes of larvae and adults of both sexes probably effects transovarial transmission to the next mosquito generation and involves the formation of isolated binucleate spores. The oenocytic development differs from the same sequence found in other mosquito Amblyospora, as it involves the development of uninucleate meronts and large plasmodia with single nuclei as well as binucleate stages. A. weiseri n.sp. is another member of the genus Amblyospora in which sporulation is not dependent upon the host taking a blood meal. Preliminary attemps to infect Cyclops strenuus and Megacyclops gigas with octospores failed.

Trachipleistophora extenrec n. sp. a New Microsporidian (Fungi: Microsporidia) Infecting Mammals

ABSTRACT. A new microsporidian Trachipleistophora extenrec n. sp. was isolated from a muscle lesion of the streaked tenrec Hemicentetes semispinosus Cuvier, 1798 (Mammalia, Tenrecidae), an insectivore endemic to Madagascar. The spores isolated from the tenrec were infectious to severe combined immunodeficient (SCID) mice by intramuscular injection. Material obtained from muscular lesions in mice was used for the parasite description. All developmental stages of the microsporidian were covered by a dense coat, which during sporogony changed into the sporophorous vesicle wall. Eight, 16, 32, or more spores were formed inside the sporophorous vesicle as the result of the division by plasmotomy and sequential fission of a multinucleate sporogonial plasmodium. Spores were ovoid, 4.7 × 2.8 μm in size, had a large posterior vacuole, and had an isofilar polar tube with 15–16 coils. Although the fine structure and the developmental pattern of the organism were in some respects similar to the genus Vavraia, molecular phylogeny based on the gene sequences of the small subunit rRNA and RNA polymerase subunit II indicated that the organism belongs to the genus Trachipleistophora. The diagnostic characters of the genera Trachipleistophora and Vavraia are discussed as well as the discrepancies between the phylogenies of these two microsporidian genera based on morphology and molecules.