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Development of Amblyospora punctor sp.n. (Microspora: Amblyosporidae) in the Larval Mosquito Aedes punctor (Diptera: Culicidae)
Arch. Protistenkd. 140 (1991): 191-199 Gustav Fischer Verlag lena
Institute of Entomology, Dept. Insect Pathology, CS Academy of Sciences, Ceske Budejovice, Czechoslovakia; Institute of Microbiology, Dept. Electron Microscopy, CS Academy of Sciences, Praha, Czechoslovakia
Development of Amblyospora punctor sp.n. (Microspora: Amblyosporidae) in the Larval Mosquito Aedes punctor (Diptera: Culicidae) By JAROSLAv WEISER & ZDEN~K ZItKA With 4 Figures Key words: Microspora; Amblyospora; Aedes; Pathogen
Abstract A microsporidian infecting the fat body of last Aedes punctor larvae was found in Czechoslovakia. Only 2 % of larvae in the spring population were visibly infected. Three periods are identified in the development of the pathogen: The pregamogonial merogony ending with formation of minute spherical garnets which fuse to binucleate zygotes. The period of macronuclear stages with meiotic divisions resulting in two presporonts, closed in a splitt-off plasmatic membrane together with granular secretion masses. A third period is that of sporogony starting with modeling fingerlike protrusions of future sporoblasts. The covering pansporoblastic membrane is the sphorophorous vesicle which is finally filled with eight broad oval spores measuring 6 x 7 !-tm. Their polar filament is anisofilar, the manubrial part in three coils and the thin flagellar part in 9 coils. Secretion masses were formed during the sporogony and resorbed during spore maturation.
Introduction Microsporidia of the genus Amblyospora are common pathogens of mosquitoes, mainly of different species of the genus Aedes, of some species of Culex and only exceptionally of Anopheles. The taxonomy of different species is mostly based on the presumption of host specificity and some minute morphological features of their sporogony and spores, and on details of their development, mainly in the binucleate series appearing in adult female mosquitoes. Detailed evaluation of these differences is possible only with mosquitoes in laboratory colonies where positive transmission can be followed from the egg to the larvae and adults (HAZARD et al. 1985). Cross infections of other mosquitoes are possible to some extent (ANDREADIS 1989) and during these experimental transmissions details in morphology and development are changed making the species identifications more difficult. Under natural conditions of distribution in mosquito habitats in one region the relations to their host, their habitat and time of appearance are constant and the frequency of infections rather regular due to an equal frequency of infected eggs distributed in the region. In Central Europe all known cases of Amblyospora infections were recorded from the spring generation of "boreal mosquitoes" with Aedes cantans as the main recorded host, occurring in April and May in temporary snow pools in inundated forests (WEISER 1961). Only rare are the observations of infections during summer months in inundation waters when eggs of spring species are activated and the early spring population continues under new conditions. The present description is of an infection in Aedes punctor, a late spring mosquito appearing in temporary grassland pits with remains of plant materials. In this type of habitat an infection with Amblyospora was found for the first time. It differs in morphology and microstructures of the spore from the usual infection in forest Aedes mosquitoes.
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Fig. 1. A - Aedes punctor, larva infected with Amblyospora punctor. White cysts are in the fat body. B Gametocytes (g) and a zygote (z) together with stages of meiosis (m). C - Diplokaryon stage (d). D Diplokaryon (d) with quiescent nuclei between stages of meiosis (m). E - Meiotic divisions of nuclei (m) in macronuclear stages (m), on surface flat black secretions, resulting sporonts (s) with minute nuclei. F - Stages of sporogony with adherent black secretion masses (c) on macronuclear meiotic stages (c) and inside pansporoblasts (p).
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Material and methods The aquatic habitat of Aedes punctor was a roadside ditch near the Bilek railway station, 30 m long, 0.5 m broad, with depths varying from 20 to 30 cm in a distance of 300 m from the next forest and 1,500 m from a forest locality with population of Aedes cantans with a sporadic infection with Amblyospora barbata. In the habitat studied a population of A. punctor developed at the end of April, with adults hatching in mid May. After a rainy period, another population of larvae appeared at the end of June from eggs washed down by heavy rains. Larval density was first 10-15 larvae in 1 dm2 , of the second population 5-10 larvae. In both the frequency of visible infection was 2 %. Infected larvae with apparent yellow regions of the infected fat body were isolated from the remaining population and dissected. With parts of the infected fat body dry smears were prepared, other smears were fresh fixed in Bouin's fixative and some infected larvae were fixed in Bouin's for embedding into paraffin and were cut into sections 4 !lm thick. Parts of infected tissues were fixed in 4 % glutaraldehyde and osmic acid, and embedded in Vestopal W for preparation of ultrathin sections in the routine way. Smears were stained with Giemsa and Heidenhain's iron haematoxylin.
Results Infected larvae of Aedes punctor are last instar larvae with yellow spots in the infected area of the fat body of the thorax (Fig. lA), later also in segmentary fat bodies of the abdomen. Infected larvae were sluggish in movement, and were usually hidden in the detritus on the bottom. Among collected pupae and hatched adults no microsporidian infection was found on smears and in watermounts of their internal organs. During the development of the mosquitoes there were no infected copepods present. The locality was dry from July to March. The infection in larvae is localized in the fat body only. In most infected larvae the main stage of development of the parasite were mature spores of one single type. In some larvae with visible signs of infection presporogonial and immature sporogonial stages were present. In infected lobes of the fat body the infection was spread over the whole lobe, the young, vegetative stages were in the central part, the mature spores on the periphery. The nuclei of invaded cells of the fat body disappear and the cell membranes are in the late stage of the infection dissolved and an uniform mass of spores fills the lobes. Some stages of the parasite released from broken cells are circulating in the hemolymph and adhere to the tracheal matrix. No phagocytosis or formation of nodules with granulocytes was observed.
Fig. 2. Sporogony of Amblyospora punctor. A - Sporoblasts inside the pansporoblastic sporophorous vesicle (p) separated by granular secretion deposits (c). B - Maturation of sporoblasts inside the pansporoblast. Besides granular secretions (c) also tubular secretions in its centre (t). C - Cementation of sporoblastic membranes in the sporophorous vesicle (p). Thin areas are open to the periphery, exposed to resorption of nutrients, thickened parts are close to secretion masses (c). 0 - Sporoblasts with formed exospore. Inside central nucleus with cross sections of chromosomes, ribosomes arranged along endoplasmic reticulum and cross sections of the forming polar filament. Fig. 3. Interior of the spore of Amblyospora punctor. A - Young spore with cross section of anchoring disc (a) of the polar filament which enters the lamellar (I) and foamy (f) part of the polaroplast. Nucleus is irregular lobated (n), laterally the posterosome (g). On surface of the polaroplast visible covering electron dense layer. Endospore (E) rather thick, exospore (e) dissolving. B - Longitudinal section of the spore with thick exospore (e) and thin endospore, on both poles attenuated. Polar filament with flat anchoring disc, polaroplast with the lamellar (I) and foamy (f) part following the sigmoidal bending of the filament (f,). Nucleus irregular (n), posterosome on the basis (g). C - Sporoblast of A. punctor with compact exospore, endospore not formed, network of endoplasmic reticulum and compact nucleus with sections of chromosomes (n). Anchoring part of the polar filament as spherical dark mass, polar filament (pf) coiled with 3 cross sections of the manubrial part and 6 sections of the flagellar part. 0 - Young spore of A. punctor with finished polar filament (pf) coiled in 3 coils of the thick manubrial and 9 coils of the thin flagellar part. Visible lamellar structure of exospore. First signs of formation of the endospore. 13 Arch. Protistenkd. Bd. 140, 2-3
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The development of the parasite has three distinct stages. Merogonial stages are minute spherical cells 3-4 f..l.m in diameter, with one rather large nucleus with dense chromatin and dense cytoplasm. In wet-fIxed material they are spherical, on dry smears often with nucleus in one end of an oval body. They divide their nuclei and form multinucleate plasmodia with 5-7 nuclei in fInger-like protrusions (Fig. 1B), and these break soon into uninucleate "gamets" remaining in groups (Fig. 4c, d). Here we fInd as a next stage binucleate individuals with nuclei adhering together in a broad flat area. All these stages have in wet smears nuclei with uniformly granular chromatin, not connected in chromosomes. This is not a fIxation artefact, next of them are other developmental stages with well differentiated meiotic chromosomes (Fig. 1C, D). The second series is characterized by macronucleate stages with well formed chromosomes (Fig. IE, F; Fig. 4g-I). They are broad oval to spherical, 10-15 f..l.m in diameter, with a nucleus 6-8 f..l.m in diameter and fInely granulated cytoplasm. The initial stage seems to be the diplokaryotic stage (Fig. IC, D; Fig. 4e, f) where both nuclei form chromosomes. Next seem to be stages with one large nucleus fIlled with a rather dense mass of forming chromosomes (Fig. 4h). From this stage a division of the nucleus is started which by some authors is characterized as meiosis due to synaptonemal complexes present. During meiotic fIgures more than 10 chromosomes were present, but the number could not be defIned exactly due to minute size. During telophase two new nuclei are formed (Fig. 4j, k, m) with a reduced mass of chromosomal material and the fInal stage seem to be two sporonts with nuclei fIlled with chromatin organized in a coil of chromosomes, less dense than in the beginning of the macronucleate stages (Fig. 4n). This stage is spherical, 8-9 f..l.m in diameter, with a nucleus 4 f..l.m in diameter. During this series of divisions granular secretions are formed under the covering membrane of this series of stages and a second membrane includes the newly formed macronucleate stage (Fig. IE, F, Fig. 4j, k). The third series are sporogonial stages. They are formed inside the original membrane of the macronucleated stage and it forms a sporophorous vesicle (pansporoblastic membrane). Sporogonial stages are characterized by individual membranes with thickened areas where the secretion of granular electron dense material is concentrated. Later these areas coalesce and a complete cover of sporogonial individual membrane is formed (Fig. 2A, B). With three nuclear divisions eight nuclei are formed and the nuclei enter spherical protrusions which arise from a central connecting part. At the stage of the octonucleate cluster, the bridge is lysed and the stages of prosporoblasts are maturating individually. Secretion granules form large masses which fIll the space between individual protrusions. During maturation of the sporoblasts the secretions are dissolved (Fig. 2B) and there remain only some tubular structures in the mature pansporoblast. The sporophorous vesicle fIxes the maturing spores together till complete maturation, the pansporoblast grows to a fInal diameter of 20 f..l.m. Spores are broad oval to subspherical, 6x7 f..l.m when fresh, 6x7.2 f..l.m in dry smears and 5.4 x 6 f..l.m in wet fIxed smears. In Giemsa stained dry smears a bright red granule in the posterior end is the posterosome. On ultrathin sections (Fig. 3A-D) the spores have a rather thick, irregularly contracted electron dense exospore with a thin zone on both poles. The single nucleus is oval, in HCI-hydrolysed spores obliquely located in the posterior end. The anterior pole is occupied by a polaroplast with surrounds the polar fIlament from its flat anchoring disc most of the length of the spore up to the coiled part. The vacuolated part is larger than the lamellar part. The polar fIlament is anisofIlar, with three coils of the thicker, manubrial part and nine coils of the thinner part (flagellum). The spores of Amblyospora barbata in Aedes cantans differ in shape and have three coils of the thicker part and four of the thin part of the polar fIlament, the maximum of thin coils in young spores is going up to 6. Based on indicated differences we consider the microsporidian in Aedes punctor as a new species and propose for it the new name Amblyospora punctor sp.n.
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Discussion The described life cycle of the new microsporidian gives an opportunity to discuss a sequence which is subject of very different classifications of the authors. The reason is that it is difficult to identify in static material of sections or smears the sequences of stages which appear all together in the same smear. A second source of difficulties is that the scheme of life cycle, especially the appearance of sexual stages and the macronuclear mitotic/meiotic series is unique in protozoa and is interpreted in a different way due to absence of easily recognized markers in normal morphology and ultrastructures. The situation seems to be cleared up in studies of HAZARD et al. (1985); BECNEL (1986); BECNEL et al. (1986) and ANDREADIS (1983). They identified the series of stages arising from released spore germ plasms, (sporoplasmas, planonts) as the gamete development, ending with a still mysterious zygote. The lack of morphological characteristics of this stage makes its identification difficult. Among species and genera of the Amblyosporidae WEISER (1977), the development proceeds with the same morphological series, and facts disclosed in one species can be confirmed in another. In our material we could confirm that minute uninucleate gametes are formed in quantity in the early phase of invasion of A. punctor, but we could find them only in the fat body and in rare cases in the salivary glands, near the centres with macronuclear stages. In our material it was evident that they end as typical diplokarya in the stage of a zygote. We could
a
b
Fig. 4. Development of Amblyospora punctor, a - planont; b, c - development of gametes; d - zygote; e, f - diplokarya; g-m - macronuclear series of meiotic stages in nuclear division; n - sporonts, o-p - division of the sporont inside the pansporoblastic sporophorous vesicle; r - octosporous pansporoblast with dissolved secretions; s - mature spore.
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not find any nipple on the gametes but could identify repeatedly the zygotes as quiescent diplokaryotic stages in the centre of micronuclear and macronuclear stages with typical activated nuclei with chromosomes. This observation may help, if confmned in other species, to the characterization of the gametal series of the microsporidian (Fig. 4). Of further interest is the process of activation of the nuclei of the zygote, fusion of the nuclei with an evidently denser mass of formed chromosomes and the subsequent nuclear division, resulting in two prosporonts. The question if this is a real meiosis or an aberrant mitosis is still open. Ultrastructures in electron microscopy do not help to see the procedure in toto and the wet-fixed smears stained with iron haematoxylin are at the moment the only efficient method. In theoretical considerations the formation of the sporophorous vesicle is indicated as the initial point of the sporogony. Secretions of electron dense material indicate the stage when the outer membrane of the stage is separated from the forming sporont in its interior. This procedure starts early in the macronuclear series and therefore this series must be actually included in the sporogonial sequence. After the macronuclear sequence the development of sporoblasts is connected with a distinct morphological differentiation when secretion granules fill the space between the forming morula-like sporoblasts. The cleaved-off membrane of the sporophorous vesicle remains inpermeable for the secretion deposits and retains afterwards its character during the whole sporogony. During further divisions in sporogony plasmatic membranes are no more split off. Deposits of secretion granules are not just the feature of Amblyospora. In the octosporous sequence of Vairimorpha there are similar deposits, but usually of the granular and tubular type. In Amblyospora a few tubules are formed in the centre of the rosette and disappear soon. The deposits are re-used by the microsporidian during its sporulation. This period is generally characterized by formation of empty spaces around individual sporoblasts and young spores when they are in the host tissue. The generic characteristic of Amblyospora includes the characteristics of the exospore and the anisofllar polar fllament. Specific differences include differences in the size and shape of the gametes, in the duration and, eventually, the division of diplokaryotic zygotes, and in a persistence of the pansporoblastic membrane during the sporulation, finally in the size, shape and ultrastructural details of their spores. The differences are evident also in relations of development of thickwalled and thinwalled spores to the host stage (development in larvae of both types in mixture) and sex (apparent infection in male hosts ending with mortality before pupation, and inapparent infections in female larvae, pupae and adults ending with transovarial transmission). The whole group needs a comparative study of all known species with the same methods of investigation.
Acknowledgement This research was party supported by a grant for the Collaborating Centre WHO for vector pathology from the UNDPlWorld BanklWHO Special Programme for Research and Training in Tropical Diseases. The authors wish to thank to Dr. J. VAVRA, Parasitology Dept., Charles University, Prague, for discussion and constructive criticism of the draft of this manuscript and Mrs. JImNA MARTtNKoVA for technical assistance.
References ANDREADIS, T. G. (1983): Life cycle and epizootiology of Amblyospora sp. (Microspora, Amblyosporidae) in the mosquito Aedes cantator. J. Protozool. 30: 509-518. BECNEL, J. J. (1986): Microsporidian sexuality in culicine mosquitoes. In: SAMSON, R. A., VLAK, J. M., & PETERSON, D. (eds.), Fundamental and Applied Aspects of Invertebrate Pathology. ICIP, Wageningen, pp. 331-334. HAZARD, E. I., & FUKUDA, T. (1986): Fine structure and development of Pilosporella chaprrumi (Microspora, Thelohaniidae) in the mosquito Aedes triseriatus (SAY.). J. Protozool. 33: 60-66.
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HAZARD, E. I., FUKUDA, T., & BECNEL, J. J., (1985): Gametogenesis and plasmogamy in certain species of Microspora. J. Invertebr. Pathol. 46: 63-69. WEISER, J., (1961): Mikrosporidien a1s Parasiten der Insekten. Monographien der Zeit. Angew. Entomol. 17, Hamburg, 149 pp. - (1977): Contribution to the classification of microsporidia. Vest. Cs. Spol. Zool. 41: 308-320. Address of the authors: JAROSLAV WEISER, Department of Insect Pathology, Institute of Entomology, Acad. Sci., Flemingovo n. 2, Praha 6, Czechoslovakia.
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Institute of Entomology, Dept. Insect Pathology, CS Academy of Sciences, Ceske Budejovice, Czechoslovakia; Institute of Microbiology, Dept. Electron Microscopy, CS Academy of Sciences, Praha, Czechoslovakia
Development of Amblyospora punctor sp.n. (Microspora: Amblyosporidae) in the Larval Mosquito Aedes punctor (Diptera: Culicidae) By JAROSLAv WEISER & ZDEN~K ZItKA With 4 Figures Key words: Microspora; Amblyospora; Aedes; Pathogen
Abstract A microsporidian infecting the fat body of last Aedes punctor larvae was found in Czechoslovakia. Only 2 % of larvae in the spring population were visibly infected. Three periods are identified in the development of the pathogen: The pregamogonial merogony ending with formation of minute spherical garnets which fuse to binucleate zygotes. The period of macronuclear stages with meiotic divisions resulting in two presporonts, closed in a splitt-off plasmatic membrane together with granular secretion masses. A third period is that of sporogony starting with modeling fingerlike protrusions of future sporoblasts. The covering pansporoblastic membrane is the sphorophorous vesicle which is finally filled with eight broad oval spores measuring 6 x 7 !-tm. Their polar filament is anisofilar, the manubrial part in three coils and the thin flagellar part in 9 coils. Secretion masses were formed during the sporogony and resorbed during spore maturation.
Introduction Microsporidia of the genus Amblyospora are common pathogens of mosquitoes, mainly of different species of the genus Aedes, of some species of Culex and only exceptionally of Anopheles. The taxonomy of different species is mostly based on the presumption of host specificity and some minute morphological features of their sporogony and spores, and on details of their development, mainly in the binucleate series appearing in adult female mosquitoes. Detailed evaluation of these differences is possible only with mosquitoes in laboratory colonies where positive transmission can be followed from the egg to the larvae and adults (HAZARD et al. 1985). Cross infections of other mosquitoes are possible to some extent (ANDREADIS 1989) and during these experimental transmissions details in morphology and development are changed making the species identifications more difficult. Under natural conditions of distribution in mosquito habitats in one region the relations to their host, their habitat and time of appearance are constant and the frequency of infections rather regular due to an equal frequency of infected eggs distributed in the region. In Central Europe all known cases of Amblyospora infections were recorded from the spring generation of "boreal mosquitoes" with Aedes cantans as the main recorded host, occurring in April and May in temporary snow pools in inundated forests (WEISER 1961). Only rare are the observations of infections during summer months in inundation waters when eggs of spring species are activated and the early spring population continues under new conditions. The present description is of an infection in Aedes punctor, a late spring mosquito appearing in temporary grassland pits with remains of plant materials. In this type of habitat an infection with Amblyospora was found for the first time. It differs in morphology and microstructures of the spore from the usual infection in forest Aedes mosquitoes.
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Fig. 1. A - Aedes punctor, larva infected with Amblyospora punctor. White cysts are in the fat body. B Gametocytes (g) and a zygote (z) together with stages of meiosis (m). C - Diplokaryon stage (d). D Diplokaryon (d) with quiescent nuclei between stages of meiosis (m). E - Meiotic divisions of nuclei (m) in macronuclear stages (m), on surface flat black secretions, resulting sporonts (s) with minute nuclei. F - Stages of sporogony with adherent black secretion masses (c) on macronuclear meiotic stages (c) and inside pansporoblasts (p).
Development of Amblyospora punctor sp.n.
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Material and methods The aquatic habitat of Aedes punctor was a roadside ditch near the Bilek railway station, 30 m long, 0.5 m broad, with depths varying from 20 to 30 cm in a distance of 300 m from the next forest and 1,500 m from a forest locality with population of Aedes cantans with a sporadic infection with Amblyospora barbata. In the habitat studied a population of A. punctor developed at the end of April, with adults hatching in mid May. After a rainy period, another population of larvae appeared at the end of June from eggs washed down by heavy rains. Larval density was first 10-15 larvae in 1 dm2 , of the second population 5-10 larvae. In both the frequency of visible infection was 2 %. Infected larvae with apparent yellow regions of the infected fat body were isolated from the remaining population and dissected. With parts of the infected fat body dry smears were prepared, other smears were fresh fixed in Bouin's fixative and some infected larvae were fixed in Bouin's for embedding into paraffin and were cut into sections 4 !lm thick. Parts of infected tissues were fixed in 4 % glutaraldehyde and osmic acid, and embedded in Vestopal W for preparation of ultrathin sections in the routine way. Smears were stained with Giemsa and Heidenhain's iron haematoxylin.
Results Infected larvae of Aedes punctor are last instar larvae with yellow spots in the infected area of the fat body of the thorax (Fig. lA), later also in segmentary fat bodies of the abdomen. Infected larvae were sluggish in movement, and were usually hidden in the detritus on the bottom. Among collected pupae and hatched adults no microsporidian infection was found on smears and in watermounts of their internal organs. During the development of the mosquitoes there were no infected copepods present. The locality was dry from July to March. The infection in larvae is localized in the fat body only. In most infected larvae the main stage of development of the parasite were mature spores of one single type. In some larvae with visible signs of infection presporogonial and immature sporogonial stages were present. In infected lobes of the fat body the infection was spread over the whole lobe, the young, vegetative stages were in the central part, the mature spores on the periphery. The nuclei of invaded cells of the fat body disappear and the cell membranes are in the late stage of the infection dissolved and an uniform mass of spores fills the lobes. Some stages of the parasite released from broken cells are circulating in the hemolymph and adhere to the tracheal matrix. No phagocytosis or formation of nodules with granulocytes was observed.
Fig. 2. Sporogony of Amblyospora punctor. A - Sporoblasts inside the pansporoblastic sporophorous vesicle (p) separated by granular secretion deposits (c). B - Maturation of sporoblasts inside the pansporoblast. Besides granular secretions (c) also tubular secretions in its centre (t). C - Cementation of sporoblastic membranes in the sporophorous vesicle (p). Thin areas are open to the periphery, exposed to resorption of nutrients, thickened parts are close to secretion masses (c). 0 - Sporoblasts with formed exospore. Inside central nucleus with cross sections of chromosomes, ribosomes arranged along endoplasmic reticulum and cross sections of the forming polar filament. Fig. 3. Interior of the spore of Amblyospora punctor. A - Young spore with cross section of anchoring disc (a) of the polar filament which enters the lamellar (I) and foamy (f) part of the polaroplast. Nucleus is irregular lobated (n), laterally the posterosome (g). On surface of the polaroplast visible covering electron dense layer. Endospore (E) rather thick, exospore (e) dissolving. B - Longitudinal section of the spore with thick exospore (e) and thin endospore, on both poles attenuated. Polar filament with flat anchoring disc, polaroplast with the lamellar (I) and foamy (f) part following the sigmoidal bending of the filament (f,). Nucleus irregular (n), posterosome on the basis (g). C - Sporoblast of A. punctor with compact exospore, endospore not formed, network of endoplasmic reticulum and compact nucleus with sections of chromosomes (n). Anchoring part of the polar filament as spherical dark mass, polar filament (pf) coiled with 3 cross sections of the manubrial part and 6 sections of the flagellar part. 0 - Young spore of A. punctor with finished polar filament (pf) coiled in 3 coils of the thick manubrial and 9 coils of the thin flagellar part. Visible lamellar structure of exospore. First signs of formation of the endospore. 13 Arch. Protistenkd. Bd. 140, 2-3
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The development of the parasite has three distinct stages. Merogonial stages are minute spherical cells 3-4 f..l.m in diameter, with one rather large nucleus with dense chromatin and dense cytoplasm. In wet-fIxed material they are spherical, on dry smears often with nucleus in one end of an oval body. They divide their nuclei and form multinucleate plasmodia with 5-7 nuclei in fInger-like protrusions (Fig. 1B), and these break soon into uninucleate "gamets" remaining in groups (Fig. 4c, d). Here we fInd as a next stage binucleate individuals with nuclei adhering together in a broad flat area. All these stages have in wet smears nuclei with uniformly granular chromatin, not connected in chromosomes. This is not a fIxation artefact, next of them are other developmental stages with well differentiated meiotic chromosomes (Fig. 1C, D). The second series is characterized by macronucleate stages with well formed chromosomes (Fig. IE, F; Fig. 4g-I). They are broad oval to spherical, 10-15 f..l.m in diameter, with a nucleus 6-8 f..l.m in diameter and fInely granulated cytoplasm. The initial stage seems to be the diplokaryotic stage (Fig. IC, D; Fig. 4e, f) where both nuclei form chromosomes. Next seem to be stages with one large nucleus fIlled with a rather dense mass of forming chromosomes (Fig. 4h). From this stage a division of the nucleus is started which by some authors is characterized as meiosis due to synaptonemal complexes present. During meiotic fIgures more than 10 chromosomes were present, but the number could not be defIned exactly due to minute size. During telophase two new nuclei are formed (Fig. 4j, k, m) with a reduced mass of chromosomal material and the fInal stage seem to be two sporonts with nuclei fIlled with chromatin organized in a coil of chromosomes, less dense than in the beginning of the macronucleate stages (Fig. 4n). This stage is spherical, 8-9 f..l.m in diameter, with a nucleus 4 f..l.m in diameter. During this series of divisions granular secretions are formed under the covering membrane of this series of stages and a second membrane includes the newly formed macronucleate stage (Fig. IE, F, Fig. 4j, k). The third series are sporogonial stages. They are formed inside the original membrane of the macronucleated stage and it forms a sporophorous vesicle (pansporoblastic membrane). Sporogonial stages are characterized by individual membranes with thickened areas where the secretion of granular electron dense material is concentrated. Later these areas coalesce and a complete cover of sporogonial individual membrane is formed (Fig. 2A, B). With three nuclear divisions eight nuclei are formed and the nuclei enter spherical protrusions which arise from a central connecting part. At the stage of the octonucleate cluster, the bridge is lysed and the stages of prosporoblasts are maturating individually. Secretion granules form large masses which fIll the space between individual protrusions. During maturation of the sporoblasts the secretions are dissolved (Fig. 2B) and there remain only some tubular structures in the mature pansporoblast. The sporophorous vesicle fIxes the maturing spores together till complete maturation, the pansporoblast grows to a fInal diameter of 20 f..l.m. Spores are broad oval to subspherical, 6x7 f..l.m when fresh, 6x7.2 f..l.m in dry smears and 5.4 x 6 f..l.m in wet fIxed smears. In Giemsa stained dry smears a bright red granule in the posterior end is the posterosome. On ultrathin sections (Fig. 3A-D) the spores have a rather thick, irregularly contracted electron dense exospore with a thin zone on both poles. The single nucleus is oval, in HCI-hydrolysed spores obliquely located in the posterior end. The anterior pole is occupied by a polaroplast with surrounds the polar fIlament from its flat anchoring disc most of the length of the spore up to the coiled part. The vacuolated part is larger than the lamellar part. The polar fIlament is anisofIlar, with three coils of the thicker, manubrial part and nine coils of the thinner part (flagellum). The spores of Amblyospora barbata in Aedes cantans differ in shape and have three coils of the thicker part and four of the thin part of the polar fIlament, the maximum of thin coils in young spores is going up to 6. Based on indicated differences we consider the microsporidian in Aedes punctor as a new species and propose for it the new name Amblyospora punctor sp.n.
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Discussion The described life cycle of the new microsporidian gives an opportunity to discuss a sequence which is subject of very different classifications of the authors. The reason is that it is difficult to identify in static material of sections or smears the sequences of stages which appear all together in the same smear. A second source of difficulties is that the scheme of life cycle, especially the appearance of sexual stages and the macronuclear mitotic/meiotic series is unique in protozoa and is interpreted in a different way due to absence of easily recognized markers in normal morphology and ultrastructures. The situation seems to be cleared up in studies of HAZARD et al. (1985); BECNEL (1986); BECNEL et al. (1986) and ANDREADIS (1983). They identified the series of stages arising from released spore germ plasms, (sporoplasmas, planonts) as the gamete development, ending with a still mysterious zygote. The lack of morphological characteristics of this stage makes its identification difficult. Among species and genera of the Amblyosporidae WEISER (1977), the development proceeds with the same morphological series, and facts disclosed in one species can be confirmed in another. In our material we could confirm that minute uninucleate gametes are formed in quantity in the early phase of invasion of A. punctor, but we could find them only in the fat body and in rare cases in the salivary glands, near the centres with macronuclear stages. In our material it was evident that they end as typical diplokarya in the stage of a zygote. We could
a
b
Fig. 4. Development of Amblyospora punctor, a - planont; b, c - development of gametes; d - zygote; e, f - diplokarya; g-m - macronuclear series of meiotic stages in nuclear division; n - sporonts, o-p - division of the sporont inside the pansporoblastic sporophorous vesicle; r - octosporous pansporoblast with dissolved secretions; s - mature spore.
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not find any nipple on the gametes but could identify repeatedly the zygotes as quiescent diplokaryotic stages in the centre of micronuclear and macronuclear stages with typical activated nuclei with chromosomes. This observation may help, if confmned in other species, to the characterization of the gametal series of the microsporidian (Fig. 4). Of further interest is the process of activation of the nuclei of the zygote, fusion of the nuclei with an evidently denser mass of formed chromosomes and the subsequent nuclear division, resulting in two prosporonts. The question if this is a real meiosis or an aberrant mitosis is still open. Ultrastructures in electron microscopy do not help to see the procedure in toto and the wet-fixed smears stained with iron haematoxylin are at the moment the only efficient method. In theoretical considerations the formation of the sporophorous vesicle is indicated as the initial point of the sporogony. Secretions of electron dense material indicate the stage when the outer membrane of the stage is separated from the forming sporont in its interior. This procedure starts early in the macronuclear series and therefore this series must be actually included in the sporogonial sequence. After the macronuclear sequence the development of sporoblasts is connected with a distinct morphological differentiation when secretion granules fill the space between the forming morula-like sporoblasts. The cleaved-off membrane of the sporophorous vesicle remains inpermeable for the secretion deposits and retains afterwards its character during the whole sporogony. During further divisions in sporogony plasmatic membranes are no more split off. Deposits of secretion granules are not just the feature of Amblyospora. In the octosporous sequence of Vairimorpha there are similar deposits, but usually of the granular and tubular type. In Amblyospora a few tubules are formed in the centre of the rosette and disappear soon. The deposits are re-used by the microsporidian during its sporulation. This period is generally characterized by formation of empty spaces around individual sporoblasts and young spores when they are in the host tissue. The generic characteristic of Amblyospora includes the characteristics of the exospore and the anisofllar polar fllament. Specific differences include differences in the size and shape of the gametes, in the duration and, eventually, the division of diplokaryotic zygotes, and in a persistence of the pansporoblastic membrane during the sporulation, finally in the size, shape and ultrastructural details of their spores. The differences are evident also in relations of development of thickwalled and thinwalled spores to the host stage (development in larvae of both types in mixture) and sex (apparent infection in male hosts ending with mortality before pupation, and inapparent infections in female larvae, pupae and adults ending with transovarial transmission). The whole group needs a comparative study of all known species with the same methods of investigation.
Acknowledgement This research was party supported by a grant for the Collaborating Centre WHO for vector pathology from the UNDPlWorld BanklWHO Special Programme for Research and Training in Tropical Diseases. The authors wish to thank to Dr. J. VAVRA, Parasitology Dept., Charles University, Prague, for discussion and constructive criticism of the draft of this manuscript and Mrs. JImNA MARTtNKoVA for technical assistance.
References ANDREADIS, T. G. (1983): Life cycle and epizootiology of Amblyospora sp. (Microspora, Amblyosporidae) in the mosquito Aedes cantator. J. Protozool. 30: 509-518. BECNEL, J. J. (1986): Microsporidian sexuality in culicine mosquitoes. In: SAMSON, R. A., VLAK, J. M., & PETERSON, D. (eds.), Fundamental and Applied Aspects of Invertebrate Pathology. ICIP, Wageningen, pp. 331-334. HAZARD, E. I., & FUKUDA, T. (1986): Fine structure and development of Pilosporella chaprrumi (Microspora, Thelohaniidae) in the mosquito Aedes triseriatus (SAY.). J. Protozool. 33: 60-66.
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HAZARD, E. I., FUKUDA, T., & BECNEL, J. J., (1985): Gametogenesis and plasmogamy in certain species of Microspora. J. Invertebr. Pathol. 46: 63-69. WEISER, J., (1961): Mikrosporidien a1s Parasiten der Insekten. Monographien der Zeit. Angew. Entomol. 17, Hamburg, 149 pp. - (1977): Contribution to the classification of microsporidia. Vest. Cs. Spol. Zool. 41: 308-320. Address of the authors: JAROSLAV WEISER, Department of Insect Pathology, Institute of Entomology, Acad. Sci., Flemingovo n. 2, Praha 6, Czechoslovakia.
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