Ronny Larsson

Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites

Significance Intracellular obligate parasitism results in extreme adaptations, whose evolutionary history is difficult to understand, because intermediate forms are hardly ever found. Microsporidia are highly derived intracellular parasites that are related to fungi. We describe the evolutionary history of a new microsporidian parasite found in the hindgut epithelium of the crustacean Daphnia and conclude that the new species has retained ancestral features that were lost in other microsporidia, whose hallmarks are the evolution of a unique infection apparatus, extreme genome reduction, and loss of mitochondrial respiration. The first evolutionary steps leading to the extreme metabolic and genomic simplification of microsporidia involved the adoption of a parasitic lifestyle, the development of a specialized infection apparatus, and the loss of diverse regulatory proteins. Intracellular parasitism results in extreme adaptations, whose evolutionary history is difficult to understand, because the parasites and their known free-living relatives are so divergent from one another. Microsporidia are intracellular parasites of humans and other animals, which evolved highly specialized morphological structures, but also extreme physiologic and genomic simplification. They are suggested to be an early-diverging branch on the fungal tree, but comparisons to other species are difficult because their rates of molecular evolution are exceptionally high. Mitochondria in microsporidia have degenerated into organelles called mitosomes, which have lost a genome and the ability to produce ATP. Here we describe a gut parasite of the crustacean Daphnia that despite having remarkable morphological similarity to the microsporidia, has retained genomic features of its fungal ancestors. This parasite, which we name Mitosporidium daphniae gen. et sp. nov., possesses a mitochondrial genome including genes for oxidative phosphorylation, yet a spore stage with a highly specialized infection apparatus—the polar tube—uniquely known only from microsporidia. Phylogenomics places M. daphniae at the root of the microsporidia. A comparative genomic analysis suggests that the reduction in energy metabolism, a prominent feature of microsporidian evolution, was preceded by a reduction in the machinery controlling cell cycle, DNA recombination, repair, and gene expression. These data show that the morphological features unique to M. daphniae and other microsporidia were already present before the lineage evolved the extreme host metabolic dependence and loss of mitochondrial respiration for which microsporidia are well known.

A new microsporidium Berwaldia singularis gen. et sp.nov. from Daphnia pulex and a survey of microsporidia described from Cladocera

A new microsporidian parasite of a freshwater cladoceran from southern Sweden is described using light and electron microscopical methods. Development comprises 2 merogonial sequences, the first resulting in a cluster of 8 merozoites, the second in a chain of 4 merozoites. Each secondary merozoite develops into a sporont which divides into 2 sporoblasts, each of which develops into a spore. The spores are broadly oval and in fresh smears measure about 6 μm in length, with a single nucleus and a posterosome. The polar filament is about 40 μm long, of even thickness throughout, and appears as 15—18 coils in a single layer. The anchoring disc is small and the polaroplast is composed of 2 lamellar parts. Outside the plasma membrane of the sporont a 5-layered, electron-dense substance is produced, which further differentiates into endo- and exospore, an electron-dense substance occurring patchily on the exospore and a pansporoblast membrane. During development the sporoblasts and the young spores are connected by a dense substance. Mature spores appear single or paired. The pansporoblast membrane is composed of 2 structurally different layers, namely a thin outer, single membrane and an inner layer composed of tubular structures. It is connected to the spore coat by patches of the dense substance. The new microsporidium is considered to belong to a new genus of the family Telomyxidae, and its systematic relationship with this and the related family Tuzetiidae is discussed. A survey of microsporidia from Cladocera is included.

A rickettsial pathogen of the amphipod Rivulogammarus pulex

Abstract A microorganism attributed to the genus Rickettsiella was found as a pathogen of the amphipod Rivulogammarus pulex collected in the south of Sweden. The rickettsiae were studied using light and electron microscopical methods, and different stainings were tested. The polychromatic staining by J. M. Vetterling and D.E. Thompson (1972, Stain Technol. , 47 , 164–165) appeared most suitable. Several tissues were infected, most heavily in the fat cells. Infection was restricted to the cytoplasm and infected cells were hypertrophied. The rickettsiae developed inside membrane-lined vacuoles and three morphological types were observed. Type 1 was irregular rods with a length of 0.6–1.4 μm; type 2 electron-dense, slightly bent rods of regular shape, 0.5–0.6 μm long; type 3 rounded cells with a diameter of 1.1–2.8 μm containing irregular crystal-like bodies.

Ultracytology of a Tetrasporoblastic Microsporidium of the Caddis flyHolocentropus picicornis(Trichoptera, Polycentropodidae), with Description ofEpiseptuni inversum gen.et sp. nov. (Microspora, Gurleyidae)

Summary The microsporidium Episeptum inversum gen. et sp. nov. (fam. Gurleyidae) is a parasite of the caddisfly Holocentropus picicornis (Polycentropodidae) in southern Sweden. Infection is restricted to the adipose tissue. Infected cells and their nuclei are hypertrophied, and in the final phase disintegrated. In the merogonial development multinucleate plasmodia with isolated nuclei give rise to numerous uninucleate merozoites, which mature to uninucleate sporonts. The tetranucleate sporogonial plasmodium is divided into four symmetrical lobes, and finally four rounded uninucleate sporoblasts are budded off. Sporogonial plasmodium and sporoblasts are enclosed in a sporophorous vesicle, produced by the sporont, with a thin electron-dense envelope and a fibrillar content. The vesicle is subpersistent and disappears at spore maturation. Mature spores are always free and ungrouped, even in situ in the host. Spores are pyriform, c. 2.1 X 2.5-3.2 Itm unfixed, 1.2-1.6 X 2.1-2.6,am fixed and stained. The membrane-lined posterior vacuole is visible also in unfixed spores, in an oblique position close to the posterior pole. The uninucleate spores have a polaroplast with two lamellar parts. The anterior lamellae are wide, the posterior ones narrow. The polar filament is anisofilar, with 3 wide (130-140 nm) anterior coils and 2 to 3 narrow (108-113 nm) posterior coils in a single layer close to the spore wall. The angle of tilt is acute, 30-35 degrees. The spore wall is 187-194 nm thick. The internal plasmalemma is c. 10 nm thick and the electron-translucent endospore - 100 nm. The plurilayered exospore has two c. 16 nm thick internal layers of somewhat different electron densities, and a 54 nm thick reticulate or chambered coat, with approximately 10 nm wide compartments and a 6 nm thick cover resembling a unit membrane. The ultracytology of the different developmental stages and the morphogenesis of the characteristic organelles are described. The new microsporidium is compared to the microsporidia of Trichoptera, to the three tetrasporoblastic microsporidian genera, and to the microsporidia with a specialized exospore.

Insect pathological investigations on Swedish thysanura: Observations on Malamoeba locustae (Protozoa, Amoebidae) from Lepisma saccharina (Thysanura, Lepismatidae)

Abstract A case of spontaneous infection of Malamoeba locustae from a free-living indoor population of Lepisma saccharina is reported. The source of infection was probably a culture of Schistocerca gregaria reared in the same room. Measurements of fixed cysts from L. saccharina are compared with measurements of fixed and unfixed cysts from S. gregaria and with published data from other species of grasshoppers and locusts.

Thelohania capillata n. sp. (microspora, Thelohaniidae) — An ultrastructural study with remarks on the taxonomy of the genus ThelohaniaHenneguy, 1892

Summary Thelohania capillata sp. nov. is described from larvae of the blackfly Odagmia omata ( Meigen , 1818), collected in a small stream in the south of Sweden, Infection was restricted to the adipose tissue, which was transformed into a syncytial xenorna. The earliest stage observed was diplokaryotic merozoites, which mature directly into diplokaryotic sporonts. The first division of the sporont is a meiosis, identified by the occurrence of synaptonemal complexes, and it is usually followed by a mitotical division, resulting in a plasmodium with 8 single nuclei. The cytoplasm is further divided into 8 lobes which finally give rise to 8 rounded sporoblasts. Each sporoblast matures into a barrel-shaped spore, with a spore-wall composed of three layers, 280-350 nm thick. Most spores are octosporous microspores, dimensions 2.0-3.1 x 2.0-2.7 μm in fixed preparations, but a small number of macrospores, 3.5-4.5 x 2.8 - 3.0 μm, are produced in pansporoblasts with four spores. Both spore types with a single nucleus and a nearly identical fine structure. The anchoring disc is small and the straight part of the polar filament reaches the posterior pole of the spore. It is surrounded by a polaroplast composed of two structurally different parts. The anterior part is about 1/3 of the spore length, the posterior about 2/3. The polar filament is of approximately uniform thickness all over its length. In microspores the diameter is 120-140 nm, in macrospores it is slightly bigger, 125-150 nm. The polar filament of microspores is posteriorly arranged as 4-5 coils in a single layer close to the spore wall. The angle of tilt of the first filament coil is approximately 45 degrees. In macrospores the posterior part of the filament is arranged as 8 irregularly stacked coils. Sporogony occurs in a pansporoblast transversed by a fibrous material continuous from the spore wall to the pansporoblast membrane, which has a hair-like fibrous appearance. The relationship of the new species to other species of the genus Thelohania and to Thelohania-like microsporidia from blackflies is discussed.

Insect pathological investigations on Swedish thysanura: A nuclear polyhedrosis virus of the bristletail Dilta hibernica

Abstract A singly enveloped NPV with fragile, spindle-shaped inclusion bodies is described from the free-living bristletail, Dilta hibernica , collected on an island on the southeastern coast of Sweden. The infection, which induces nuclear hypertrophy, is restricted to the midgut epithelium, and polyhedra are shed into the gut lumen, where virions are released, enabling reinfection to occur. The virus could not be transmitted per os to Lepisma saccharina .

Baculovirus-like particles in the midgut epithelium of the phantom midge, Chaoborus crystallinus (Diptera, Chaoboridae)

Abstract A rod-shaped microorganism of presumed baculoviral nature is described from larvae of Chaoborus crystallinus collected in Sweden. The infection was restricted to the midgut epithelium, causing strong nuclear hypertrophy. Sectioned rods measured 75–106 × 260–300 nm. Their envelope, similar to a unit membrane, was approximately 10 nm thick. The electron-dense center, interpreted as the nucleocapsid, measured 38–43 × 210–226 nm. The rods had a longitudinal unilateral evagination. Individual rods were connected to chains and arranged as paracrystalline structures, most prominent at the nuclear periphery. Infected midguts had a yellow discoloration, but showed no iridescence. The particles were similar in size and shape to nonoccluded baculoviruses, and the microorganism of C. crystallinus is compared to other nonoccluded microorganisms of verified or suspected baculoviral nature.

Description of Hyalinocysta expilatoria n. sp., a microsporidian parasite of the blackfly Odagmia ornata

Abstract Hyalinocysta expilatoria n. sp. is described from a larva of Odagmia ornata collected in Sweden. Infection was restricted to the adipose tissue which was transformed into a syncytium. The earliest stage observed was diplokaryotic merozoites, which mature directly into diplokaryotic sporonts. Each sporont produces a sporophorous vesicle (pansporoblast), which persists, also enclosing mature spores. Usually nuclear divisions result in a plasmodium with 8 nuclei, which fragments into 8 sporoblasts, each of which develops into a spore without further division. Occasionally an aberrant number of spores (2, 4, 6) is formed. The spores are pyriform with a flattened area at the posterior pole. Spores in sporophorous vesicles with 8 spores are 4.0–6.0 μm long, in vesicles with 4 spores 4.0–5.0 μm, and in vesicles with 2 spores 7.0–8.0 μm. In some vesicles the spores develop asynchronously, and 2, 4, or 6 mature spores are found together with 6, 4, or 2 immature. There was also a small number of vesicles with supernumerary spores, less than 8 normally developed. The 325–350 nm thick spore wall is composed of three layers. The polar filament is anisofilar with 7 coils in a single layer. The anterior 5–6 coils are wide, the posterior 2-1 thin. The angle of tilt of the anterior filament coil is approximately 50°. The spore has a single nucleus. The sporophorous vesicle is delimited by a thin membrane, also visible in haematoxylin stained preparations. Vesicles with mature spores are void of metabolic inclusions.

Development, ultracytology and taxonomical position of Thelohania corethrae Schuberg and Rodriguez, 1915 (Microspora, Thelohaniidae)

Summary The development and ultracytology of a microsporidium identified as Thelohania corethrae are briefly described. The development conforms with the characteristics of Thelohaniidae: 8 diplokaryotic merozoites are formed by rosette-like budding, the diplokaryotic sporont divides meiotically and gives rise to a sporogonial plasmodium with 8 isolated nuclei, and 8 sporoblasts are formed. The uninucleate spores are nearly oval in fresh preparations, pyriform in stained smears. The envelope of the sporophorous vesicle is subpersistent and mature spores are rarely seen in groups of 8. The exospore has the layered construction typical for the Thelohania -like microsporidia, the polaroplast is lamellar with narrow anterior and wide posterior lamellae, and the polar filament is anisofilar. The species is variable, especially concerning the persistence of the sporophorous vesicle, the occurrence of macrospores, and the ratio wide to narrow coils of the polar filament. In the characteristic lesions formed by the microsporidium the external part is devoid of microsporidia, and mitochondria are prominently aggregated externally to the microsporidia-filled zone. The characteristics used to discriminate between T. corethrae and Plistophora chaobori are found to lack diagnostic value and P. chaobori is considered to be a younger synonym of T. corethrae . The ultrastructural findings exclude T. corethrae from the genus Thelohania , and the new combination Systenostrema corethrae is proposed.