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.     

Experimental study of the routes of transmission of Microsporidia in blood-sucking mosquitoes from southwestern Siberia

Experiments were conducted on the artificial infection of larvae of 8 species of bloodsucking mosquitoes (Aedes diantaeus, A. punctor, A. cinereus, A. flavescens, A. aegypti, Culex pipiens, Anopheles messeae, Anopheles atroparvus) with spores of Amblyospora opacita var. sibirica, Parathelohania costatus sp. n., P. obesa, P. anophelis. Negative results were obtained in all experiments.     

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.     

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)     

Microsporidia of the genus Parahtelohania (Microspora: Amblyosporidae) from blood-sucking mosquitoes of the genus Anopeles (Diptera: Culicidae) from the South of the West Siberia

Five new microsporidian species of the genus Parathelohania have been found in the fat body of blood-sucking mosquitoes collected in various water basins in the South of West Siberia: Parathelohania divulgata sp. n., P. formosa sp. n., P. sibirika sp. n., P. teguldeti sp. n., and P. tomski sp. n. Processes of merogony, sporogony, and ultrastructure of spores was were investigated by means light and electronic microscopy.     

Parasitic systems of Microsporidia: descriptions and terminology questions

Three parasitic systems of Microsporidia are described: the system of monoxenic Vairimorpha mesnili with paraxenic hosts presented lepidopteran and hymenopteran species; the system of dixenic Amblyospora sp. with metaxenic hosts presented bloodsucking mosquitoes and crustaceans and the system of Metchnikovella sp. as parasite of other obligate parasite. The last case is characterized by very intimate interrelations between hyperparasite (microsporidian species), obligate parasite--host of Microsporidia (gregarine) and hyperhost--host of gregarine (polychaeta). This hyperparasite system is exclusive case of parasitic systems. Parasitic and hyperparasitic systems reflects a population level of host-parasite interactions. On biocenotic level many other organisms such as predators, vectors and reservators of invasion stages of Microsporidia affect parasitic systems giving a chance to one of the members of the system (to the host or to the parasite). These organisms form epiparasitic system. In all cases of the parasitic systems there are two-way communications between parasites and their hosts. In systems on biocenotic level--parasitic consortium--members of epiparasitic systems acts on parasitic systems, but members of parasitic systems don't affect epiparasitic systems.     

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.     

Changes in the behaviour of Moina macrocopa (Crustacea: Cladocera) under the influence of Gurleya sp. (Microsporidia: Gurleyidae)

It is established, that infestation with Gurleya sp. (Microsporidia, Gurleyidae) changes the behaviour of its host Moina macrocopa (Straus, 1820), a crustacean inhabiting pools. Heavily infested host individuals attach to surface film of water before their death by senescence. As a consequence, their dead bodies filled with spores of the parasite adhere to objects dipped in water that probably facilitates the transfer of spores by birds and cattle to other pools. Weakly infested or uninfested individuals drown after the end of their ontogenesis. Distribution of the microsporidian spores by two different ways (with attached dead bodies of the hosts or with bottom sediments) makes invasion of a new host more probable.     

Prehibernation diet and reproductive condition of female Anopheles messeae in Sweden

1. Blood and sugar feeding in the mosquito Anopheles messeae Falleroni were investigated near Uppsala, Sweden, from 1981 to 1985, with emphasis on prehibernation diet. 2. Fructose, indicating plant feeding, was present in females of all ovarian stages, from April to October, and contributed to fat body development in non-gonoactive females before hibernation. 3. An.messeae males and females (inseminated and uninseminated) were observed feeding on floral nectaries of the plants Achillea millefolium L. and Tanacetum vulgare L. at night during August. 4. Gonotrophic dissociation started in some females in July, and by the end of September all females were inseminated and nulliparous but non-gonoactive. 5. Animal sheds were used as diurnal resting sites of non-gonoactive, prehibernating female An.messeae. Among recently fed mosquitoes in that group, 85% were gut positive for fructose and 15% for blood. Most of them showed negative host tropism, indicating that bloodfeeding is not a prerequisite for hibernation. 6. Survival rates of bloodfed (48 +/- 31 days) and non-bloodfed (42 +/- 21 days) females, collected from a cattle shed in September and kept caged without food or water in a store house, were not significantly different. This indicates that bloodfeeding may occur facultatively before hibernation but does not affect longevity and survival. 7. If overwintering is possible for bloodfed An.messeae females, they would be more likely vectors of pathogens such as Batai virus.     

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.     

Identification of mosquito-parasitic microsporidia, Amblyospora rugosa and Trichoctosporea pygopellita (Microsporidia: Amblysporidae), from Acanthocyclops venustus and Acanthocyclops reductus (Copepoda: Cyclopidae), based on small subunit rDNA analysis

Identical small subunit rDNA sequences were obtained for microsporidia Amblyospora rugosa from blood-sucking mosquitoes larvae Ochlerotatus cantans, O. cataphylla and copepods Acanthocyclops venustus, as well as for Trichoctosporea pygopellita from mosquitoes larvae Ochlerotatus cyprius, O. excrucians and copepods Acanthocyclops reductus. The data on molecular phylogeny and ecological researches show that in Siberia mosquito-parasitic microsporidia of the genera Amblyospora and Trichoctosporea have complex life cycle involving likely intermediate hosts, Acanthocyclops copepods. Life cycle of parasites is synchronized with phenology of their hosts. The phylogenetic analyses shows, that genus Trichoctosporea should be transferred from the family Thelohaniidae to the family Amblyosporidae.     

Crepidulospora--nomen novum for the junior generic homonym (preoccupied generic name) Crepidula

Crepidulospora nom. nov. is a replacement generic name for the genus Crepidula Simakova, Pankova et Issi, 2003 based on the type species Crepidula beklemishevi Simakova, Pankova et Issi, 2003 (Microsporida) from Anopheles beklemishevi. The name proposed by Simakova et al., 2003 is a preoccupied name, because it was already used for the gastropode Crepidula Lamarque 1899 (Echinospirida, Calibraeidae), a parasite of Mytilus. A valid name of the type species of the genus is now Crepidulospora beklemishevi (Simakova, Pankova, Issi, 2003) comb. n. Crepidulospora Simakova, Pankova et Issi nom. non. Type species: Crepidula beklemishevi Simakova, Pankova, Issi, 2003. Diagnosis. Sporogony is octosporoblastic. Sporogonal stages are in direct contact with host cell cytoplasm. 8 uninucleate spores, 4.2 x 2.2 mkm, are sandals-like. Polar tube is anisofilar, with 6-7 coils (2 + 4-5). Polaroplast is three-partite, with broad vesicular, vesicular and lamellar compartments. Microsporidia ilnfects larval adipose tissues. Type host: Anopheles beklemishevi (Diptera, Culicidae).     

Systemic reorganization of the architectonics of polytene chromosomes in the onto- and phylogenesis of malaria mosquitoes

Cardinal differences in the spatial organization of polytene chromosomes in the nuclei of salivary gland cells (malphigian tubules) and nurse ovarian cells in malarial mosquitoes of Anopheles maculipennis complex are found. The chromosomes have typical chromocentric organization in salivary gland cells and malphigian tubules. In contrast to these cells, the trophocytes have no single chromocentre and some of their chromosomes are rigidly attached to the nucleus envelope with the centromeric parts or with the particular loci. The loci-specificity of the X-chromosome intercalary parts' attachment to the nucleus envelope is found in Anopheles messeae.     

Microsporidians in bloodsucking Aedes (O.) cataphylla mosquitoes in Ukraine]

The paper reports the results of studies on Microsporidia from the bloodsucking mosquito Aedes cataphylla for the period from 1974 undertaken in the Kiev and Chernigov Provinces of the Ukraine. A new species, Amblyospora cataphyllus sp. n., is described from larvae of the mosquito. Living spores oval or elongate-oval, 6.3--7.5 x 4.5--5.0 (most commonly 6.8 x 4.8) microns, fixed spores oval, 5.6--6.9 x 3.8--4.8 microns. Mucocalyx becomes distinct around spores in 10 to 15 days after the preparation is made. Spontaneously ejected polar tube 125 microns long. Exospore smooth or undulate, 170 to 310 nm thick, endospore 60 to 160 nm. Polaroplast elongated and curved, its 1/3 is composed of closely situated plates and 2/3 of more widely arranged ones. Polar tube anisophyllous, forms 18 to 20 spires in a spore, of which 3 to 5 spires form basal area and 14 to 16 form distal area (of them 1-2 most thick spires form intermediate area). Nucleus large, in a shape of half-ring. In living spores posterior vacuole crescentic, in fixed ones it is strongly deformed together with hind pole of spores. Sporogony of Microsporidia takes place in larvae of mosquitoes of both sexes. Infection extensiveness of IV stage larvae is 10%.     

Development of Amblyospora campbelli (Microsporida: Amblyosporidae) in the mosquito Culiseta incidens (Thomson)

The complete life cycle of Amblyospora campbelli (Kellen and Wills, 1962) (Microsporida: Amblyosporidae) requires a two-host system involving the mosquito host, Culiseta incidens (Thomson), and an obligatory intermediate copepod host. The parasite has dimorphic spore development producing meiospores (haploid condition) and binucleated spores (diploid condition), either as an exclusive infection or simultaneously (within females only). This is the 1st known report of concurrent spore development within an adult mosquito host, and, therefore, shows the Amblyospora campbelli system to be uniquely different from other Amblyospora spp. cycles previously described. The significance of dimorphic spore development is discussed. In females, diplokaryotic meronts may invade oenocytes, causing a benign-type of infection. A blood-meal is required to initiate sporulation of the binucleate spore. The binucleate spore contains the sporoplasm involved in transovarial transmission. A 2nd sporulation sequence, primarily in adipose tissue, may involve both males and females. In this sequence, repeated merogonic division greatly increased the density of diplokaryotic meronts and generally involved most of the body of the host. Production of meiospores, unlike that for the binucleate spore, appeared to be spontaneous (i.e. no obligatory blood meal). Survivorship of male and female larval mosquitoes was nearly equal. Adult females spread the parasite in three ways: transovarial, transovum, and by meiospore deposition.     

Mosquitoes and other aquatic insects in fallow field biotopes and rice paddy fields

Fallow field biotopes that develop from abandoned rice fields are man‐made wetlands that provide new habitats for various aquatic animals. Although consideration of such biotopes generally focuses on their positive aspects, this study evaluated the negative aspects of establishing fallow field biotopes with regard to mosquito breeding sites. To determine whether fallow field biotopes become breeding habitats for vector mosquitoes, we evaluated mosquito fauna in fallow field biotopes and adjacent rice fields. We found larvae of Anopheles lesteri, Anopheles sinensis and Culex tritaeniorhynchus (all: Diptera: Culicidae) in the biotopes. Although abundances of mosquito larvae in the biotopes and rice fields were statistically similar, mosquito abundances in rice fields increased dramatically in August when the water level reduced after the rainy season. The abundance and variety of the mosquitoes' natural predators were greater in biotopes than in rice fields because the former are a permanent and stable aquatic environment. A generalized linear mixed model showed a negative effect of predator diversity on mosquito larvae abundance in both habitats. Although fallow field biotopes become breeding habitats for vector mosquitoes, establishing biotopes from fallow fields in order to protect various aquatic animals, including mosquito insect predators, may help to control mosquito breeding.     

Horizontal transmission of Parathelohania anophelis to the copepod, Microcyclops varicans, and the mosquito, Anopheles quadrimaculatus

The copepod Microcyclops varicans was infected with Parathelohania anophelis by unincleate meiospores from a field-collected fourth instar Anopheles quadrimaculatus larva. Large numbers of unincleate, pyriform spores developed in the copepod. These spores were fed to early instar A. quadrimaculatus larvae, infecting both males and females, resulting in the production of cylindrical, binucleate spores in the adult. These spores were responsible for vertical transmission, through the eggs, to the larvae. The original spore type collected from the field was found in the male progeny from the infected females. Another P. anophelis-infected mosquito colony was established by feeding spores from a single, field-collected, infected copepod to A. quadrimaculatus larvae. The microsporidium was continuously maintained by vertical transmission in newly established infected colonies.     

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)