Crepidula beklemishevi gen. et sp. n. and Dimeiospora palustris gen. et sp. n. (Microspora: Amblyosporidae)--new microsporidian genera and species from blood-sucking mosquitoes (Diptera: Culicidae) from the south of the western Siberia

Microsporidia parasitizing the adipose body of mosquito larvae of Anopheles beklemishevi and Aedes punctor has been studied. Two new genera of microsporidia are described based on lightmicroscopic and ultrastructural characteristics of spores and sporogony stages. The spore wall of Crepidula beklemishevi gen. n. et sp. n. is formed by two-membrane exospore, thick exospore, bilayer endospore and thin plasmolemma. Spores with single nucleus, polar filament anisofilar, with 6-7 coils (2+ 4-5), polaroplast consisting of three parts: macrochelicoidal, microhelicoidal and lamellar. Fixed spores 4.2 +/- 0.22 x 2 +/- 0.01 microns. The sporogony of Dimeiospora palustris gen. et. n. results in spore formation of two different types. Spores of the first type are oviform, with thick wall, single-nuclear, 6.1 x 4.9 microns. Spore wall with three layers, about 370 nm. Exospore electron-dense, subexospore moderately electrondense. Exospore and subexospore irregularly pleated on the almost spore surface and slightly thinner on anterior end only. Endospore electron-translucent. Polar filament anisofilar, with 9 coils (3 + 6). Polaroplas consists of three parts: lamellar, fine bubbled, and coarse bubbled. Spores of the second type broad-ovate, with apical pole narrower, distal pole concave, 4.6 x 3.7 microns. Spore wall with three layer, 355 nm. Exospore on the apical end irregularly pleated, consists of thin electrondense exospore, subexospore of variable electron density, endospore electron-translucent. Polar filament anisofilar, with 13 coils (3 + 10). Polaroplast has two parts: lamellar and vesicular.     

New species of Microsporidia in the genus Amblyospora from blood-sucking mosquitoes in the family Culicidae

Two microsporidians of the genus Amblyospora from mosquito larvae of Aedes and Culex were studied in Tomsk region (Siberia). Peculiarities of the sporogony and the meiospore ultrastructure were studied by light and electron microscopy. The meiospores of A. caspius sp. n. from Aedes caspius and Ae. communis measured on smears, were c. 4.8 x 3.6 microns in size. A polar filament was anisofilar with 5 wide (290 nm) and 9 narrow (190 nm) coils arranged in a single layer. A polaroplast of both species was composed of two parts: anterior lamellae and posterior fibrillae. Meiospores of A. burlaki sp. n. from Culex pipiens measured c. 4.8 x 3.5 microns. A polar filament was anisofilar with 3 wide (260 nm) and 6 narrow (110 nm) coils. The meiospore of this species had a 260 nm thick multilayered exospore.     

Six new species of microsporidia of the genus Amblyospora (Microspora: Amblyosporidae) from blood sucking mosquitoes (Diptera: Culicidae) from the west Siberia

Microsporidia of the genus Amblyospora parasiting the adipose body of mosquito larvae of the genus Aedes and Culex has been studied with both light and electron microscopy. Six new species of microsporidia are described based on ultrastructural characteristics of spores and sporogony stages. Amblyospora flavescens sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 165 nm. Exospore is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, large vesicular, lamellar. The anisofilar polar filament with 10--11 coils (3 1/2 + 2 1/2 + 4-5). Fixed spores are 6.3 +/- 0.1 x 4.24 +/- 0.1 microm. Amblyospora kolarovi sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 265-315 nm. Exospore shapes tucks on the surface of spore. It is two-membranous. Subexospore is quagge, structural. Endospore is electron-translucent. Polaroplast consists of two parts: lamellar and large vesicular. The anisofilar polar filament with 11-13 coils (3 + 8-10). Fixed spores are 5.4-5.6 x 3.5-4.2 microm. Amblyospora orbiculata sp. n. Mature spores are widely egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 155 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, vesicular, lamellar. Polar filament is anisofilar, with 11 1/2 coils (4 1/2 + 1 + 6). Fixed spores are 6.3 +/- 0.1 x x 4.0 +/- 0.1 microm. Amblyospora rugosa sp. n. Mature spores are egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 225 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 17 1/2 coils (3 1/2 + 1 + 13). Fixed spores are 5.3 +/- 0.1 x 3.7 +/- 0.1 microm. Amblyospora undata sp. n. Mature spores are egg-shaped. The spore wall is three-layered, about 220 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. The anisofilar polar filament with 8 coils (3 + 5). Fixed spores are 5.0 +/- 0.1 x 3.0 +/- 0.1 microm. Amblyospora urski sp. n. Mature spores have widely oval form. The back pole is concave. The spore wall with three layers, about 280 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 6 coils (2 + 4). Fixed spores are 4.4 +/- 0.1 x 2.9 +/- 0.1 microm.     

Ultrastructure of Tricornia muhezae N. G., n. sp. (Microspora, Thelohaniidae), a parasite of Mansonia africana (Diptera: Culicidae) from Tanzania.

Collections of Mansonia africana mosquito larvae were made at one site in N.E. Tanzania in 1985 and 1987 and from two additional sites, both within about 2 mi of the original one in 1987. An octosporous microsporidian, present at all three sites, was found in both years infecting from 7 to 22% of larvae. Spores (stained in Giemsa) measured 3.0 microns +/- 0.25 microns x 2.25 microns +/- 0.26 microns. Ultrastructurally, spores were seen to have an anterior rim surrounding a depressed area where the endospore was at its thinnest. In transmission electron microscopy section, the rim appeared as two processes into which all layers of the wall extended. At the posterior end all layers of the wall extended into a simple knob-like structure which could be interpreted as a section through a crest running longitudinally around the spore. The polar filament was anisofilar, with two anterior coils of greater diameter than the three posterior coils. Although most closely resembling the genera Amblyspora and Parathelohania in the family Thelohaniidae, the species in M. africana differs from the former, which has oval spores, broadly rounded at the ends, and from the latter, which has a prominent, ridged posterior extension to the spores. The new species has been placed in a new genus and the name Tricornia muhezae proposed.     

Ecology and epizootology of Microsporidia in malarial mosquitoes (diptera: culicidae) from the south of Western Siberia

The ecology and epizootology of Microsporidia were studied in the natural population of malarial mosquitoes from Western Siberia over a 30-year period. Symptoms of the disease, host specificity and character of parasite localization in host tissues were investigated. Microsporidia of 9 species from 4 genera, namely Amblyospora, Crepidulospora, Senoma, and Parathelohania, were found in the malarial mosquito larvae from the territory examined. The mosquito species Anopheles messeae was infested by larger number of the microsporidian species, than A. beklemishevi. Spores and active stages of the microsporidian Senoma sp. from mosquito larvae are localized in epithelial cells of the host's intestine. There are no external signs of the infestation. The infested larvae do not die, and the infection proceeds to the pupal stage. Microsporidians of the genus Parathelohania infest larvae of both male and female mosquitoes. The parasites localized in the fat body cells. Body of the infested host gets an opaque white color in the period of spore maturation. The infested larvae perish at 4th stage. Microsporidian spores are formed in the mosquito larvae of both sexes. However, infection rate is much higher in the male larvae (77.8%), than that in the female larvae (22.2%). Symptoms of the disease under the infestation by the genera Crepidulospora and Amblyospora are similar to those under the infestation by Parathelohania sp. There are no external signs of the infestation in the adult mosquito females. In these hosts microsporidians form large two-nuclear spores with a thin capsule accounted for transovarial transmission. An about two times decrease of fertility in the Anopheles messeae females infested by the Parathelohania microsporidians is revealed. Mean number of eggs laid by the infested females was 121, while that of the noninfested ones was 232. Thus, the cause of the decrease in the mosquito abundance is both the elimination of male larvae and the decrease of fertility in females. In Siberia malarial mosquitoes yield 2 or 3 generations per year. Infested larvae are rarely occurred in the beginning of summer. Maximal extensiveness of the invasion may be observed in the end of summer, because of the accumulation of the infection in water bodies. Number of the peaks of infestation coincides with the peaks of abundance of mosquito larvae of 4th stage. Peaks of infestation are delayed as compared with the peaks of mosquito abundance. It is an evidence of the development delay in the infested larvae. Long-term dynamics of the infestation by microsporidians is studied. The epizooty caused by Parathelohania was observed in the malarial mosquitoes in the eighties (with the infestation rate up to 62 %). In the last ten years the infestation rate was low (from 0.1 to 2.6%).     

A new genus of microsporidans Cristulospora gen. n. (Amblyospiridae) with 3 new species from blood-sucking mosquitoes in Uzbekistan

Three new species of microsporidans of the new genus Cristulospora are described from mosquitoes of the genera Culex and Aedes (floodlands of the Syr Darya, Syrdarya Province, Bukharski? oasis). In mosquitoes microsporidans have sporogony of two types that is characteristic of the family Amblyosporidae. The development in larvae ends in the formation of 8 uninucleate spores in pansporoblasts. In females single binucleate spores are formed. The new genus differs from other amblyosporids in the presence of appendages in shape of magnificent plumes on both poles of octospores, which are distinct without staining. C. sherbani sp. n. from Culex modestus forms spores measuring 5.0-8.1 X 4.3-5.6 microns in larvae. During the fixation the poles become flattened, plumes are equal, most of them having wide bases. In genital ducts of females oval cylindrical thin-walled spores of 6.3-8.7 X 2.5-3.7 microns are formed. C. cadyrovi sp. n. from Culex pipiens forms spores of 5.6-6.8 X 3.7-5.0 microns in larvae. After the fixation the poles do not become flattened, plumes are equal, with narrow base. The second type of spores has not been discovered. C. aedis sp. n. from Aedes caspius has spores of 6.2-7.5 X 4.3-5.6 microns in larvae. Plumes with narrow base, the anterior and posterior ones are different in shape. In genital ducts of females thin-walled spores of 8.7-11.8 X 3.7-5.0 microns are formed.     

Horizontal transmission of Amblyospora albifasciati García and Becnel, 1994 (Microsporidia: amblyosporidae), to a copepod intermediate host and the neotropical mosquito, Aedes albifasciatus (Macquart, 1837)

The life cycle of Amblyospora albifasciati is characterized by three sporulation sequences involving the definitive mosquito host and a copepod intermediate host. Meiospores of A. albifasciati were infectious per os to adult females of the copepod Mesocyclops annulatus. All developmental stages in the copepod had unpaired nuclei, with sporulation involving the formation of a sporontogenic interfacial envelope and the production of a second type of uninucleate spore. These spores, formed in the ovaries of M. annulatus, were large, pyriform, and measured 10.4 x 4.8 microm. They infected Aedes albifasciatus larvae when ingested to initiate a sequence that involves schizogony and gametogony and ends with plasmogamy and nuclear association to form diplokaryotic meronts. Oval binucleate spores (9.3 x 3.1 microm) are formed in the adult mosquito and are responsible for vertical transmission to the filial generation.     

A new microsporidian species Glugea mesocotti sp. n. (Microsporidia:Glugeidae) from Mesocottus haitej (Scorpaeniformes:Cottidae)

A new microsporidian parasite of a freshwater fish Mesocottus haitej from the Amur River basin of Russia is described using light microscopy. The numerous whitish xenomas, round or oval, up to 3.0 mm large were found to be located in subcutaneous tissue of the body and mouth cavity, in the intestine and other internal organs. The formol fixed spores are elongate oval, measuring 4.8 (4.5-5.0) x 2.3 (2.2-2.5) microm in a wet smears. Posterior vacuole occupies about half of the spores. Sporophorous vesicles measuring up to 13 microm contain a great number of spores.     

Efficacy of Bacillus sphaericus 2362 against larvae of Anopheles gambiae under laboratory and field conditions in West Africa

A flowable concentrate of Bacillus sphaericus (Neide) strain 2362 was applied against Anopheles gambiae Giles s.l. mosquito larve in small plot field trials in Bobo-Dioulasso area. Burkina-Faso. Third and fourth instar larvae were controlled for 10-15 days with a dosage of 10 g/m2, 3-10 days with 1 or 0.1 mg/m2, and 2 days with 0.01 g/m2. Complete elimination of larval populations required 1 x 10(2) to 2 x 10(3) viable spores/ml in the larval feeding zone. After treatment, the total numbers of viable spores decreased in the ponds, due to ingestion of spores by non-target as well as target organisms and/or loss of viability of some spores by sunlight. This formulation was less effective against An. gambiae than against Culex quinquefasciatus Say larvae, both in laboratory bioassays and under field conditions.     

Testicular myxosporidiasis in the flat-backed toad, Bufo maculatus (Amphibia: Bufonidae), from Cameroon, Africa.

Macroscopic cysts measuring less than or equal to 860 x 500 microns were found in the testes of a flat-backed toad, Bufo maculatus, collected in Cameroon, West Africa. On histologic examination, the cysts contained numerous spores of a Myxobolus sp. (Myxozoa: Myxobolidae). Spores in fixed tissues measured 9.2 microns long, 8.9 microns wide, and 4.0 microns thick; the range of values for length, width, and thickness were 8.8 to 9.6 microns, 8.6 to 9.4 microns, 3.6 to 4.4 microns, respectively (n = 20). The shape index (length/width) was 1.03, and ranged from 1.00 to 1.09. Pathology was limited to a slight constriction of adjacent seminiferous tubules by the cysts. No host inflammatory response was noted. This myxozoan is distinct from all other members of the genus infecting anurans and is assigned the name Myxobolus bufonis sp. n.     

Fine structure and sporogonic development of a Vavraia sp. (Microsporida: Pleistophoridae) in the biting midge Culicoides edeni (Diptera: Ceratopogonidae)

A microsporidium with ultrastructural characteristics of the genus Vavraia was found in the fat body of an adult specimen of Culicoides edeni (Diptera: Ceratopogonidae) collected in northern Florida. The sporogonial stages developed within sporophorous vesicles, which contained variable numbers of oval spores at maturity. The wall of the sporophorous vesicle was composed of two electron-dense outer layers and an electron-lucent intermediate layer. Sporonts contained haplokaryotic nuclei and divided by rosette formation. Mature spores had anisofilar polar filaments and measured 3.8 +/- 0.28 microns in length and 2.2 +/- 0.16 microns in width in thick sections of resin-embedded material. This is the first report of a Vavraia sp. from a species of Culicodes.     

Life cycle and description of Amblyospora camposi n. sp. (Microsporidia: Amblyosporidae) in the mosquito Culex renatoi (Diptera, Culicidae) and the copepod Paracyclops fimbriatus fimbriatus (Copepoda, Cyclopidae)

The life cycle of Amblyospora camposi n. sp. is described from the mosquito Culex renatoi and the copepod Paracyclops fimbriatus fimbriatus collected in the leaf axils of the plant Eryngium cabrerae in Argentina. Meiospores of A. camposi (5.8 x 4.1 microm) were infectious per os to female adults of the copepod P. f. fimbriatus. All developmental stages in the copepod had unpaired nuclei, with sporulation involving the formation of a sub-persistent, sporontogenic, interfacial envelope and the production of a second type of uninucleate spore. These spores, formed in the ovaries of P. f. fimbriatus, were large, pyriform, and measured 10.70 x 3.85 microm. When ingested they infected C. renatoi larvae to initiate a sequence that involves schizogony and gametogony and ends with plasmogamy and nuclear association to form diplokaryotic meronts. Oblong ovate binucleate spores (7.86 x 2.96 microm) are formed in the adult mosquito and are responsible for vertical transmission to the filial generation. This is the first report of an Amblyospora species from a mosquito that inhabits the small-water bodies held in parts of terresterial plants (phytotelmata).     

Ameson hybomitrae sp. n. (Microsporidia, Pereziidae) from gadflies of Karelia

Microsporidia of the genus Ameson were recorded from larvae of horseflies of the genus Hybomitra in Karelia. Earlier these Microsporidia were recorded from crustaceans. The infection extensiveness ranges from 5.1 to 10.5%. The parasites develop in musculature, fat body and salivary glands of the host. The new species has uninucleate, single-located egg- and pear-shaped spores. The ultrafine structure of developmental stages and spores is studied.     

Amblyospora trinus N. sp. (Microsporida: Amblyosporidae) in the Australian mosquito Culex halifaxi (Diptera: Culicidae)

A new species of Amblyospora, a parasite found in wild populations of the predacious Australian mosquito Culex halifaxi, was investigated with light and electron microscopy. This species was found to be heterosporous with two concurrent sporulation sequences in the host larvae, both arising from diplokaryotic meronts and ending with haploid spores. One sequence was dominant and involved meiosis to produce eight thick-walled, broadly oval meiospores in a sporophorous vesicle (SV). The other sequence involved nuclear dissociation to produce lanceolate, thin-walled spores in a subpersistent SV. Horizontal transmission to the mosquito host, by one or both of two distinctly different pathways (one via an intermediate host, the other by cannibalism of infected individuals) and by vertical transmission, are postulated but have not been demonstrated. A new species, Amblyospora trinus, is proposed and its affinities to other heterosporous microsporidia in mosquitoes are discussed.     

Amblyospora trinus N. Sp. (Microsporida: Amblyosporidae) in the Australian Mosquito Culex halifaxi (Diptera: Culicidae)

ABSTRACT. A new species of Amblyospora , a parasite found in wild populations of the predacious Australian mosquito Culex halifaxi , was investigated with light and electron microscopy. This species was found to be heterosporous with two concurrent sporulation sequences in the host larvae, both arising from diplokaryotic meronts and ending with haploid spores. One sequence was dominant and involved meiosis to produce eight thick-walled, broadly oval meiospores in a sporophorous vesicle (SV). The other sequence involved nuclear dissociation to produce lanceolate, thin-walled spores in a subpersistent SV. Horizontal transmission to the mosquito host, by one or both of two distinctly different pathways (one via an intermediate host, the other by cannibalism of infected individuals) and by vertical transmission, are postulated but have not been demonstrated. A new species, Amblyospora trinus , is proposed and its affinities to other heterosporous microsporidia in mosquitoes are discussed.     

Ultrastructural Analysis of Spores and Parasporal Crystals Formed by Bacillus sphaericus 2297

Bacillus sphaericus 2297, growing from a boiled, relatively nontoxic spore inoculum, increased about 30-fold in toxicity for mosquito larvae during early exponential growth but showed an approximately 1,000-fold toxicity increase during the late-exponential phase, as spores began to appear in the culture. The development of spores in the bacterial cells was accompanied by the formation of parasporal crystals. These parasporal crystals appeared during stage III as the forespore septum engulfed the incipient forespore. The paraspores were separated from the forespores by a branch of the exosporium across the cell. Measurements of the parasporal substructure revealed a 6.3-nm distance between the striations. When spores and paraspores were fed to mosquito larvae and the larvae were fixed 15 min after feeding, it was found that the spores remained relatively unchanged but that the matrix of the paraspores was dissolved. After dissolution of the paraspore matrix, a meshlike envelope remained which retained the paraspore shape and which was often in contact with the cross-cell portion of the exosporium. The parasporal crystals may be a source of the mosquito larval toxin in this strain of B. sphaericus, but proof will require their isolation from other cellular components.     

Nosema tyriae n.sp. and Nosema sp., microsporidian parasites of Cinnabar moth Tyria jacobaeae.

Nosema tyriae n.sp. was found in 63% of a population of Cinnabar moth larvae (Tyria jacobaeae). The infection was found in the gut wall, silk glands, and fat body and was probably generalized but appeared to be of low pathogenicity. Merogony and sporogony were by binary fission of diplokaryotic stages. Fresh spores were elongate, slightly pointed at the anterior end, and measured 4.7 x 2.0 microm. Ultrastructural features of special interest were 20-nm tubules connecting the surface of sporonts with host cell cytoplasm and, in the spores, a deeply domed polar sac, polaroplast consisting of closely packed longitudinally arranged membranes and loosely packed horizontally arranged membranes, and 10.5-14 coils of the polar tube in a single rank. The 16S rRNA genes of N. tyriae and Nosema bombycis from silkworms, Bombyx mori, differed by only six nucleotides and N. tyriae spores gave a moderately positive reaction with a monoclonal antibody raised to N. bombycis. N. tyriae was infective to B. mori but was less virulent than N. bombycis. However, no amplification product was obtained by PCR using N. tyriae DNA and primers considered to be specific for N. bombycis. Also, the spores of the two species are of entirely different shapes. A second diplokaryotic microsporidium, Nosema sp., found as a light infection in only one of the larvae had much smaller developmental stages and spores measuring 3.8 x 2.0 microm (fixed). Ultrastructurally it was distinguished by an abundance of dense membranes in cytoplasmic vesicles in both meronts and sporonts. Spores with up to 15 coils of the polar tube in irregular clusters or with about 12 coils in a single rank were observed in the tissues fixed from the one larva infected with this parasite. As this larva had been kept with N. tyriae-infected larvae for a few days before examination, it is possible that the two types of spores resulted from a double infection.     

The fine structure of Culicinomyces clavisporus invading mosquito larvae

Electron microscope observations were made of the Australian and U.S. strains of Culicinomyces clavisporus infecting mosquito larvae. The wall of the conidium is composed of an inner (primary) layer, an outer (secondary) layer, and an exterior coating of a mucopolysaccharide substance believed responsible for conidial adhesion to the host cuticle prior to germination and penetration. In some instances the wall of the conidium is ruptured during germination and new wall layers and mucoid coating form around the germ tube whereas in other specimens the conidial wall layers extend around the germ tube without fracturing. The most common invasion site is through the larval foregut following ingestion of conidia. The apex of the germ tube presses tightly against the surface of the foregut cuticle and the mucilaginous coating is stripped away. There is evidence to suggest that the host epicuticle, which disappears across the zone of contact with the germ tube, is utilized for nutrition of the invading fungus. A collar of cuticle forms around the germ tube apex and a narrow penetrant hyphae extends into the procuticle. It is believed that cuticular penetration is primarily enzymatic assisted by mechanical pressure. The penetrant hypha swells into an oval cell in the hypodermal region and vegetative hypha then invade the hemocoel. The cells of the hypodermis develop signs of degeneration presumably due to the secretion of toxic substances from the invading hyphae. Host reactions, involving melanization of the host tissues, are sometimes evident among the invading penetrant hyphae in the cuticle or in the hypodermal cells in contact with the fungus. Melanized capsules form around some of the hyphae within the hemocoel. These latter reactions do not directly involve host blood cells and are examples of “humoral encapsulation” similar to that described by other authors during invasion of pathogenic organisms into mosquito larvae and chironomid larvae.     

Differential effects of Bacillus sphaericus strain 2362 on Culex quinquefasciatus and its competitor Culex cinereus in West Africa

The larval susceptibility to Bacillus sphaericus strain 2362 of the non-man-biting mosquito Culex cinereus and the urban filariasis vector Culex quinquefasciatus, two competitor mosquitoes in polluted habitats, was compared. In the laboratory, both species ingested a similar amount of B. sphaericus spores when fed c. 2 x 10(5) spores per ml for 30 min. However, in the same experiment, third-instar larvae of Cx quinquefasciatus were reduced by 98% at 24 h exposure while Cx cinereus larvae were only reduced by 6% at 72 h. In the field, preimaginal populations of Cx cinereus ingested, within a week, more than 99% of the applied spores, but showed no significant decrease through 14 days in cesspools treated at 10 g/m2 of a flowable concentrate of B. sphaericus 2362, containing 2 x 10(10) spores/g. It is proposed that specific biological control of Cx quinquefasciatus could result from appropriate treatment of breeding-sites with larvicidal B. sphaericus and competitive displacement by Cx cinereus or other mosquitoes with larvae that are more tolerant of B. sphaericus.