Ultrastructural characterization and comparative phylogenetic analysis of new microsporidia from Siberian mosquitoes: evidence for coevolution and host switching

A survey of mosquito larvae infected with microsporidia was conducted from 2005 to 2008 in the Tomsk, Kemerovo and Novosibirsk regions of western Siberia, Russia. Twenty-one morphologically and genetically unique species of microsporidia were isolated from nine species of Anopheles, Aedes, Culex and Ochlerotatus mosquitoes including: (1) 14 proposed new species of Amblyospora (A. bakcharia, A. baritia, A. bogashovia, A. chulymia, A. hristinia, A. jurginia, A. kazankia, A. mavlukevia, A. mocrushinia, A. modestium, A. salairia, A. severinia, A. shegaria, and A. timirasia); (2) a newly proposed genus and species, Novothelohaniaovalae and; (3) six species of Amblyospora (A. flavescens, A. kolarovi, A. rugosa), Parathelohania (P. divulgata and P. tomski) and Trichoctosporea (T. pygopellita) from which gene sequences had not been previously obtained. Detailed ultrastructure of meiospores revealed unique cytological features associated with the length, arrangement and ratio of broad to narrow coils of the polar filament, comparative thickness of the exospore and endospore, and overall size of each species reaffirming their value in distinguishing taxonomic relationships. SSU rDNA sequences obtained from each species of microsporidia were unique when compared with GenBank entries. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and Neighbor Joining analyses yielded similar topologies with a high degree of congruence between parasite and host at the generic level. Species that parasitize Aedes/Ochlerotatus and Culex mosquitoes segregate into distinct monophyletic groupings mirroring their host phylogeny, while species from Anopheles mosquitoes group as a sister clade basal to the entire group of mosquito-parasitic microsporidia as their Anopheles hosts cluster as a sister clade to the entire group of culicine mosquitoes. This provides strong evidence for host-parasite coevolution by descent at the generic level and limited host lineage switching between unrelated taxa. Among parasites of Aedes/Ochlerotatus and Anopheles mosquitoes, we found several instances where a single mosquito species serves as a host for two or more related species of microsporidia, an observation consistent with host switching and independent parasite speciation. Among the microsporidian parasites of Culex mosquitoes, we found only one parasite per host indicating a higher degree of host specificity and less host switching among parasites of this genus. Findings suggest a degree of host-parasite co-speciation with host switching occurring occasionally when the "normal" host is unavailable in the aquatic ecosystem. Frequency of host switching seems to be occurring in proportion to host relatedness and does not cross generic boundaries in this system.     

Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae)

Amblyospora species and other aquatic Microsporidia were isolated from mosquitoes, black flies, and copepods and the small subunit ribosomal RNA gene was sequenced. Comparative phylogenetic analysis showed a correspondence between the mosquito host genera and their Amblyspora parasite species. There is a clade of Amblyospora species that infect the Culex host group and a clade of Amblyospora that infect the Aedes/Ochlerotatus group of mosquitoes. Parathelohania species, which infect Anopheles mosquitoes, may be the sister group to the Amblyospora in the same way that the Anopheles mosquitoes are thought to be the sister group to the Culex and Aedes mosquitoes. In addition, by sequence analysis of small subunit rDNA from spores, we identified the alternate copepod host for four species of Amblyospora. Amblyospora species are specific for their primary (mosquito) host and each of these mosquito species serves as host for only one Amblyospora species. On the other hand, a single species of copepod can serve as an intermediate host to several Amblyospora species and some Amblyospora species may be found in more than one copepod host. Intrapredatorus barri, a species within a monotypic genus with Amblyospora-like characteristics, falls well within the Amblyospora clade. The genera Edhazardia and Culicospora, which do not have functional meiospores and do not require an intermediate host, but which do have a lanceolate spore type which is ultrastructurally very similar to the Amblyospora spore type found in the copepod, cluster among the Amblyospora species. In the future, the genus Amblyospora may be redefined to include species without obligate intermediate hosts. Hazardia, Berwaldia, Larssonia, Trichotuzetia, and Gurleya are members of a sister group to the Amblyospora clades infecting mosquitoes, and may be representatives of a large group of aquatic parasites.     

Life cycle of Amblyospora indicola (Microspora: Amblyosporidae), a parasite of the mosquito Culex sitiens and of Apocyclops sp. Copepods

The life cycle of Amblyospora indicola, a parasite of the mosquito Culex sitiens, was revealed by field observations and laboratory infection experiments conducted in Australia. In northern Queensland, infected C. sitiens larvae were often found breeding in association with two cyclopoid copepods: Apocyclops dengizicus and an undescribed species of the same genus. The latter species was found to be an intermediate copepod host of this microsporidium whereas A. dengizicus was not. One complete cycle of the parasite extends over two mosquito generations (by transovarial transmission from females with binucleate spores to their eggs) and by horizontal transmission between mosquitoes and copepods. The latter involves horizontal transmission from mosquitoes to copepods via meiospores produced in larval fat body infections and horizontal transmission from copepods to mosquitoes via uninucleate spores produced within infected copepods. Uninucleate clavate spores were formed in Apocyclops sp. nov. copepods 7-10 days after exposure to larval meiospores and were infectious to larvae of a microsporidian-free colony of C. sitiens. The development of A. indicola within mosquito larvae exposed to infected copepods is similar to that of A. dyxenoides infecting C. annulirostris. It proceeds from stages with a single nucleus to diplokaryotic binucleate cells in oenocytes. These stages persist through pupation to adult emergence after which time a proportion of male mosquitoes and female mosquitoes may develop binucleate spores without the need for a blood meal. A proportion of both male and female larval progeny of infected females with binucleate spores develop patent fat body infections via transovarial transmission and die in the fourth larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epizootiology of Amblyospora connecticus (Microsporida) in field populations of the saltmarsh mosquito, Aedes cantator, and the cyclopoid copepod, Acanthocyclops vernalis

The natural ecology of a heterosporous microsporidium, Amblyospora connecticus was investigated at three different salt marsh habitats during 1986-1989. The parasite has a well-defined seasonal transmission cycle that occurs regularly each year and intimately involves the primary mosquito host, Aedes cantator, and the intermediate copepod host, Acanthocyclops vernalis. In the spring, the microsporidium is horizontally transmitted from the copepod, where it appears to overwinter, to the mosquito via the ingestion of haploid spores produced in the copepod. Mosquitoes develop a benign infection, and females transmit the microsporidium transovarially to their progeny via infected eggs. Oviposition occurs during the summer and infected eggs hatch synchronously in the fall causing widespread epizootics. Infected larvae die, and the cycle is completed when meiospores are released into the pool and subsequently are eaten by A. vernalis, which reappears in the fall and early winter. Amblyospora connecticus thereby persists by surviving in one of two living hosts throughout most of its life cycle rather than in the extra-corporeal environment. This represents an important survival strategy for A. connecticus as results show the salt marsh habitat to be a relatively unstable environment that is subject to periodic flooding and drying. The adaptive significance of utilizing an intermediate host in the life cycle is discussed as it directly facilitates transmission and enhances survival of the microsporidium.     

Epizootiology of Amblyospora connecticus (Microsporida) in Field Populations of the Saltmarsh Mosquito, Aedes cantator, and the Cyclopoid Copepod, Acanthocyclops vernalis

The natural ecology of a heterosporous microsporidium, Amblyospora connecticus was investigated at three different salt marsh habitats during 1986–1989. The parasite has a well-defined seasonal transmission cycle that occurs regularly each year and intimately involves the primary mosquito host, Aedes cantator, and the intermediate copepod host, Acanthocyclops vernalis. In the spring, the microsporidium is horizontally transmitted from the copepod, where it appears to overwinter, to the mosquito via the ingestion of haploid spores produced in the copepod. Mosquitoes develop a benign infection, and females transmit the microsporidium transovarially to their progeny via infected eggs. Oviposition occurs during the summer and infected eggs hatch synchronously in the fall causing widespread epizootics. Infected larvae die, and the cycle is completed when meiospores are released into the pool and subsequently are eaten by A. vernalis, which reappears in the fall and early winter. Amblyospora connecticus thereby persists by surviving in one of two living hosts throughout most of its life cycle rather than in the extra-corporeal environment. This represents an important survival strategy for A. connecticus as results show the salt marsh habitat to be a relatively unstable environment that is subject to periodic flooding and drying. The adaptive significance of utilizing an intermediate host in the life cycle is discussed as it directly facilitates transmission and enhances survival of the microsporidium.     

Epizootiology of Hyalinocysta chapmani (Microsporidia: Thelohaniidae) infections in field populations of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae): a three-year investigation

The epizootiology, transmission dynamics and survival strategies employed by the microsporidium Hyalinocysta chapmani were examined in field populations of its primary mosquito host, Culiseta melanura and its intermediate copepod host, Orthocyclops modestus over a three-year period in an aquatic subterranean habitat. H. chapmani was enzootic and was maintained in a continuous cycle of horizontal transmission between each host. There were three distinct periods during the summer and fall when developing mosquito larvae acquired infections; each was preceded by or coincident with the detection of infected copepods. Results were corroborated in laboratory bioassays, wherein transmission was achieved in mosquito larvae that were reared in water and sediment samples taken from the site during the same time periods. The highest infection rates, ranging from 60% to 48%, were repeatedly observed during the first six weeks of larval development. These were coincident with the most sustained collections of infected copepods obtained during the year and highest levels of infection achieved in the laboratory transmission studies. The high prevalence rates of lethal infection observed in larval populations of C. melanura at this site are among the highest recorded for any mosquito-parasitic microsporidium and clearly suggest that H. chapmani is an important natural enemy of C. melanura. H. chapmani appears to overwinter in diapausing mosquito larvae but may also persist in copepods. The absence of vertical transmission in the life cycle of H. chapmani and the sole reliance on horizontal transmission via an intermediate host are unique survival strategies not seen among other mosquito-parasitic microsporidia. The epizootiological data suggest that this transmission strategy is a function of the biological attributes of the hosts and the comparatively stable environment in which they inhabit. The subterranean habitat is inundated with water throughout the year; copepods are omnipresent and C. melanura has overlapping broods. The spatial and temporal overlap of both hosts affords abundant opportunity for continuous horizontal transmission and increases the likelihood that H. chapmani will find a target host. It is hypothesized that natural selection has favored the production of meiospores in female host mosquitoes rather than congenital transfer of infection to progeny via ovarian infection as a strategy for achieving greater transmission success.     

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.     

Life cycle,ultrastructure and molecular phylogeny of Hyalinocysta chapmani (Microsporidia: Thelohaniidae), a parasite of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae)

The complete life cycle of the microsporidium Hyalinocysta chapmani is described from the primary mosquito host Culiseta melanura and the intermediate copepod host Orthocyclops modestus. Infections are initiated in larval C. melanura following the oral ingestion of uninucleate spores from infected copepods. Spores germinate within the lumen of the midgut and directly invade fat body tissue where all development occurs. Uninucleated schizonts undergo binary division (schizogony) followed by karyokinesis (nuclear division) to form diplokaryotic meronts. Merogony is by synchronous binary division. The onset of sporogony is characterized by the simultaneous secretion of a sporophorous vesicle and meiotic division of the diplokaryon resulting in the formation of eight ovoid meiospores enclosed within a sporophorous vesicle. Most infected larvae die during the fourth stadium and there is no evidence of a developmental sequence leading to vertical transmission. Hyalinocysta chapmani is horizontally transmitted to O. modestus via oral ingestion of meiospores. Infections become established within ovarian tissue of females and all parasite development is haplophasic. Uninucleate schizonts divide by binary division during an initial schizogonic cycle. Newly formed uninucleate cells produce a thin sporophorous vesicle and undergo repeated nuclear division during sporogony to produce a rosette-shaped, multinucleated sporogonial plasmodium with up to 18 nuclei. This is followed by cytoplasmic cleavage, sporogenesis, and disintegration of the sporophorous vesicle to form membrane-free uninucleate spores. Infected females eventually die and there is no egg development. The small subunit rDNA sequence of H. chapmani isolated from meiospores from C. melanura was identical to the small subunit rDNA sequence obtained from spores from O. modestus, corroborating the laboratory transmission studies and confirming the intermediary role of O. modestus in the life cycle. Phylogenetic analysis was conducted with closely related microsporidia from mosquitoes. Hyalinocysta chapmani did not cluster within described Amblyospora species and can be considered a sister group, warranting separate genus status.     

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.     

Epizootiology of Amblyospora stimuli (Microsporidiida: Amblyosporidae) infections in field populations of a univoltine mosquito, Aedes stimulans (Diptera: Culicidae), inhabiting a temporary vernal pool.

The epizootiology of the microsporidium Amblyospora stimuli was studied in natural populations of a univoltine mosquito, Aedes stimulans, inhabiting a temporary vernal pool over an 18-year period. The yearly prevalence of benign oenocytic infections in adult females was variable, ranging from 1.0 to 9.6% (mean = 5.1%). The yearly prevalence of transovarially transmitted meiospore infections in larval populations was consistently lower but less variable, ranging from 1.3 to 5.9% (mean = 3.5%). Meiospore infections in F(1)-generation larvae were significantly correlated with infections in parental-generation females, thus suggesting that larval infection rates could be substantially increased if methods were available to facilitate transmission of A. stimuli to a larger portion of the female population via inundative or inoculative release of infected copepods. No correlation was found when infections in filial-generation adult females were measured against meiospore infections in larvae from the preceding year. Analysis of yearly prevalence data using Fine's Fundamental Vertical Transmission Equation revealed low rates of horizontal transmission from the intermediate copepod host to female larvae in most years, ranging from 0.1 to 8.7% (mean = 3.1%). A. stimuli is enzootic, persists at a very low level, and has minimal impact on Ae. stimulans populations at this site. The low incidence rate of horizontal transmission to larvae appears to be due largely to a paucity of copepods and is a major factor that limits the abundance and subsequent proliferation of A. stimuli in Ae. stimulans populations at this locale. Results support the view that host-parasite cospeciation is an important mechanism of evolution in this group of mosquito/copepod microsporidia.     

Laboratory experiments on infection rates of Amblyospora dyxenoides (Microsporida: Amblyosporidae) in the mosquito Culex annulirostris

Laboratory observations were made of the microsporidian parasite Amblyospora dyxenoides in its natural mosquito host, Culex annulirostris. There were no differences in the numbers of eggs laid and in the proportions which hatched between infected and uninfected females, indicating that the parasite did not affect fecundity. Unlike other species of Amblyospora which have been studied the development of binucleate spores in adult mosquitoes increase with age of the host in both sexes and in females it proceeds independently of egg development and blood feeding. The same trend is apparent for adult mosquitoes which acquired the infection in the larval stage by horizontal transmission from the intermediate copepod host as well as for mosquitoes which acquired oenocytic infections by transovarial transmission. There was considerable variation in the proportion of mosquitoes which became infected after exposure to A. dyxenoides infected copepods. Infections in larval progeny of female mosquitoes infected via spores produced in copepods ranged from 0 to 100% in individual batches and averaged 45.6% with meiospore infections, 19.3% with oenocytic infections, with the remaining 35.7% being uninfected. Similar variability was observed in the progeny of infected female mosquitoes in the second generation after exposure to infected copepods. During experiments in which the microsporidium was maintained in C. annulirostris through 9 successive transovarially transmitted cycles (by selectively rearing the progeny of females infected with binucleate spores after an initial exposure to infected copepods) the proportion of infected progeny with oenocytic infections increased from 25 to around 50% whereas the incidence of meiospore infections declined from 50 to 10%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intermediate host for an Amblyospora sp. (microspora) infecting the mosquito, Culex annulirostris

Experiments conducted in Australia are described which show that there is an intermediate host involved in the life cycle of an Amblyospora sp. infecting the mosquito, Culex annulirostris. Microsporidian spores produced in the cyclopoid copepod, Mesocyclops albicans, are infectious to laboratory colony C. annulirostris larvae and produce developmental stages identical to those of natural Amblyospora infections in this mosquito. These larval infections result in uninucleate spores in progeny mosquitoes which, in turn, are infectious to M. albicans. Subsequent studies conducted with Amblyospora, copepods, and mosquitoes in the United States to replicate and reconfirm results obtained in Australia were successful. This is the first substantiated evidence of the involvement of intermediate hosts in the life cycles of microsporidia, and the discovery now provides us with information needed to evaluate these organisms as biological control agents for mosquitoes and other disease vectors.     

Ultrastructural Characterization of Meiospores of Six New Species of Amblyospora (Microsporida: Amblyosporidae) from Northern Aedes (Diptera: Culicidae) Mosquitoes

ABSTRACT. The ultrastructure of meiospores of six different species of Amblyospora found infecting larval mosquitoes of Aedes abserratus, Aedes aurifer, Aedes cinereus, Aedes excruciates, Aedes sticticus , and Aedes stimulans are described. Meiospores of all species exhibited characteristics typical for the genus Amblyospora including: a single nucleus, a large posterior vacuole, a thick undulating exospore and thinner endospore, a bipartite and lamellate polaroplast with more tightly stacked membranes in the proximal region, and an anisofilar polar filament with distal coils reduced in thickness. Distinct differences were found in the arrangement and number/ ratio of coils formed by the broad basal and narrow distal portions of the polar filament. These differences, when quantified and averaged, were unique from all other mosquito-parasitic species that have been examined ultrastructurally. Information on parasite development, natural field prevalence and transmission to suspected intermediate copepod hosts is presented. The creation of six new species, Amblyospora abserrati, Amblyospora auriferi, Amblyospora cinerei, Amblyospora excrucii, Amblyospora stictici , and Amblyospora stimuli is proposed. Synonymies and complete host lists of all forms and species of Amblyospora described from mosquitoes are given.     

Epizootiological studies of Amblyospora albifasciati (Microsporidiida: Amblyosporidae) in natural populations of Aedes albifasciatus (Diptera: Culicidae) and Mesocyclops annulatus (Copepoda: Cyclopidae) in a transient floodwater habitat

The epizootiology of the microsporidium Amblyospora albifasciati was studied in natural populations of its definitive host, a multivoltine, neotropical, floodwater mosquito, Aedes albifasciatus, and its intermediate copepod host, Mesocyclops annulatus, in an ephemeral floodwater habitat during a 12-month period. A. albifasciati was enzootic in mosquitoes. Vertically (transovarially) transmitted meiospore infections occurred regularly and were detected in five of eight larval broods but the prevalence of infection was always low, ranging from 0.5 to 6.9% with an overall average of 0.7%. Horizontal transmission of A. albifasciati infection from copepods to mosquitoes was nominal and limited. It was detected at levels of 6.4 to 20% in larval Ae. albifasciatus populations on two occasions, the month of August and late September through early October. The low levels of horizontal transmission of infection to mosquito larvae appeared to be the principal limiting factor that prevented the proliferation of A. albifasciati in Ae. albifasciatus populations. Copepod populations were abundant from May through September and weekly prevalence rates of A. albifasciati averaged over 50% (range = 5.8 to 100%). The moderately high infection rates in M. annulatus copepods were inconsistent with the low prevalence of meiospore infection in Ae. albifasciatus mosquito larvae. Results suggest that either meiospores of A. albifasciati produced in the mosquito host are highly infectious to copepods or they are long-lived and remain viable within the pool as long as some standing water is present. Observations further indicate that A. albifasciati has a significant detrimental impact on M. annulatus copepod populations but minimal impact on larval populations of Ae. albifasciatus at this site.     

Phylogenetic Position of Amblyospora Hazard & Oldacre (Microspora: Amblyosporidae) Based on Small Subunit rRNA Data and Its Implication for the Evolution of the Microsporidia

ABSTRACT. Sequences of the small subunit rRNA genes of Amblyospora californica and an Amblyospora sp. from Culex salinarius were determined. These sequences were compared phylogenetically with 16 other microsporidia. The results suggest Amblyospora forms a sister taxon to the rest of the microsporidia examined. The basal position of Amblyospora is discussed with respect to the evolution of microsporidian life cycles. These sequences represent the longest microsporidian small subunit rRNA genes sequenced to date, 1,359 and 1,358 bp, respectively. Structural features and GC content (49% for both) are comparable to those of other microsporidia which have been sequenced.     

Influence of temperature on microsporidia infections in a natural population of Simulium pertinax Kollar, 1832 (Diptera; Simuliidae).

During the studies involving the correlation between the water temperature of the breeding site of Simulium pertinax larvae and the infection prevalence by microsporidia, developed in the Andorinhas river, Magé, RJ, weekly samples of blackfly larvae were taken within a two-year period (2001-2002 and 2003-2004), and it was noticed that the infections by Amblyospora sp. were more prevalent when compared to infections by Polydispyrenia sp. in larvae. It was also observed that the infections do not follow the same pattern, since the genus Amblyospora was recorded almost every month during the study with the exception of December, 2001. In the results of correlation between the environmental water temperature and the microsporidia infection rates, it was observed that for the first period studied, there was a high negative correlation, while during the second period there was absence correlation. On the other hand, the Amblyospora sp. infection rates prove that the correlation was high and significant in the first period, but was not significant in the second sampling period and Polydispyrenia sp. showed absence correlation in both periods.     

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%).     

Verification of Intermediate Hosts in the Life Cycles of Microsporidia by Small Subunit rDNA Sequencing

Small subunit rDNA sequences were obtained from field-collected Amblyospora connections (Microsporida: Amblyosporidae) spores isolated from the mosquito, Aedes cantator (Diptera: Culicidae), and from field collected spores isolated from the putative intermediate host, Acanthocyclops vernalis (Copepoda: Cyclopidae). The ribosomal DNA sequences of the spores isolated from the two hosts were identical. These findings corroborate previous laboratory transmission studies and validate the intermediary role of A. vernalis in the life cycle of this microsporidium. These data represent the first comparative sequence analysis of a microsporidium isolated from its definitive and intermediate hosts. The results demonstrate the effectiveness of using rDNA sequence data for screening potential intermediate hosts. Unlike laboratory transmission tests, which can take months or years to complete, this technique can be completed in days and can be performed on a single infected organism.     

Coelomomyces dodgei: Establishment of an in vivo laboratory culture

An in vivo laboratory culture of the fungus Coelomomyces dodgei (Chytridiomycetes: Blastocladiales) was established, using the copepod Cyclops vernalis as an intermediate host and the larvae of the mosquito Anopheles quadrimaculatus as the definitive host. The culture was perpetuated by infecting copepods and mosquitoes using separate procedures. Copepods were infected by being combined with dehiscing sporangia. Patently infected copepods, which contained either light amber, bright orange, or both light amber and bright orange mycelia, were collected daily beginning 12 days later. Mosquitoes were infected by combining 100 first-instar larvae for 48 hr with a mixture of 12 infected copepods, four of each of the above types. The mean rate of infection for the first 100 trials was 41%. When groups of 100 first-, second-, third-, and fourth-instar larvae were exposed to a similar mixture of infected copepods for 48 hr, the mean rates of infection were 37.4, 27.0, 17.8, and 2.4%, respectively. Observations and experimental evidence suggest that the differentially pigmented mycelia found in infected copepods are gametophytes which develop into gametangia that subsequently release gametes of opposite mating types, the light amber gametangia producing female and the bright orange gametangia producing male gametes. Zygotes resulting from the fusion of these gametes lead to the infection of mosquito larvae. Thus, C. dodgei appears to have an Euallomyces type of life cycle with sporophyte and gametophyte generations alternating between mosquito and copepod hosts, respectively, with differentially pigmented sexual structures present in the gametophyte phase.