Development,Ultrastructure, and Mode of Transmission of Amblyospora sp. (Microspora) in the Mosquito*

SYNOPSIS. Amblyospora sp. in Culex salinarius (Coquillett) is transovarially transmitted and has 2 developmental sequences, one in each host sex. In females, the entire life cycle is restricted to oenocytes which become greatly hypertrophied due to the multiplication of diplokaryotic cells during merogony and come to lie next to ovaries. Sporulation occurs only after a blood meal is taken and is shortly followed by infection of the oocytes and subsequent transmission to the next host generation. In the male host, infections spread from oenocytes to adipose tissue where diplokaryotic cells undergo a 2nd merogony. During this merognic cylce, the number of diplokaryotic cells greatly increases and the infection is spread throughout the body of the larval host. Sporulation is initiated with the physical separation of the 2 members of the diplokaryon and the simulatneous secretion of a pansporoblastic membrane. Subsequent meiotic division and morphogenesis result in the formation of 8 haploid spores enclosed with a pansporoblastic membrane. Buildup of spores and subsequent destruction of host adipose tissue prove fatal to the male host during the 4th larval stage.     

Life Cycle and Epizootiology of Amblyospora sp. (Microspora: Amblyosporidae) in the Mosquito,Aedes cantator1

In Aedes cantator, Amblyospora sp. is transovarially transmitted and has two developmental sequences. The life cycle is initiated in the adult female with the release of sporoplasms from binucleated spores not bounded by membranes, lying free within host oenocytes. Sporoplasms infect the developing oocytes and are transmitted to the filial generation when the eggs are laid. In some of the female progeny that hatch from infected eggs, diplokaryotic cells infect host oenocytes and divide by binary fission during merogony. Sporulation and spore formation do not occur until a blood meal is taken by the host and they coincide with the development and maturation of the oocytes to complete the cycle. In other female and all male progeny, pathogen development occurs within fat body tissue of the host where diplokaryotic cells divide by multiple fission during merogony to spread the infection. Sporulation in this developmental sequence is characterized by the secretion of an accessory membrane and the meiotic division of diplokaryotic sporonts, which result in the formation of octonucleated plasmodia that undergo cytokinesis to form eight haploid spores which are not perorally infectious to other mosquito larvae. There is no increase in the prevalence of either type of infection in field populations during juvenile development, indicating that there is no direct horizontal transmission of the pathogen within any one generation. Data obtained from laboratory rearings of infected progeny, however, show that infections cannot persist relying solely upon maternal-mediated transmission and that some other mode of transmission must be operative for continued maintenance of this microsporidium in A. cantator.     

Impact of a Novel, Feminising Microsporidium on its Crustacean Host

We describe the transmission and pathogenic effects of a novel, feminising microsporidium, probably a Nasema species, on its crustacean host Gammarus duebeni. The parasite prevalence in the field was high (46% of females were infected) and the parasite was transovarially transmitted to 91% of embryos of infected females. The impact of the parasite on the host was assessed by means of a host breeding experiment. The parasite feminised 66% of infected host young and was transovarially transmitted by these individuals to the next host generation. The parasite differed from other feminising microsporidia in G. duebeni in that early embryos had a high parasite burden (288 parasites per embryo) and the infection was pathogenic, causing a reduction in both the growth rate of young hosts and in adult size. This study suggests that feminising microsporidia are a diverse group in which a variety of host/pathogen relationships have evolved.     

Development, ultrastructure, and mode of transmission of Amblyospora sp. (Microspora) in the mosquito

Amblyospora sp. in Culex salinarius (Coquillett) is transovarially transmitted and has 2 developmental sequences, one in each host sex. In females, the entire life cycle is restricted to oenocytes which become greatly hypertrophied due to the multiplication of diplokaryotic cells during merogony and come to lie next to ovaries. Sporulation occurs only after a blood meal is taken and is shortly followed by infection of the oocytes and subsequent transmission to the next host generation. In the male host, infections spread from oenocytes to adipose tissue where diplokaryotic cells undergo a 2nd merogony. During this merogonic cycle, the number of diplokaryotic cells greatly increases and the infection is spread throughout the body of the larval host. Sporulation is initiated with the physical separation of the 2 members of the diplokaryon and the simultaneous secretion of a pansporoblastic membrane. Subsequent meiotic division and morphogenesis result in the formation of 8 haploid spores enclosed with a pansporoblastic membrane. Buildup of spores and subsequent destruction of host adipose tissue prove fatal to the male host during the 4th larval stage.     

Transovarian transmission of Nosema plodiae in the Indian-meal moth, Plodia interpunctella

Twenty-five adult female Plodia interpunctella infected with Nosema plodiae laid 856 eggs in laboratory tests; 12.5% of the eggs were infected transovarially. The highest level of transmission by an individual female was 14 infected eggs of 27 laid (51.8%); the lowest level of transmission observed was 1 of 43 eggs (2.3%). All stages of N. plodiae were not transmitted with equal frequency; moreover, most eggs harbored predominently only the trophozoite stage of the pathogen. Approximately 80% of the infected eggs contained trophozoites almost exclusively; about 13.1% contained about an equal number of spores and trophozoites, and about 6.5% contained mainly spores. Histological observations indicated that infections may be initiated in nurse cells and subsequently transferred to associated oocytes.     

Vertical transmission and overwintering of microsporidia in the gypsy moth, Lymantria dispar

Vertical transmission and the overwintering success of three different microsporidia infecting Lymantria dispar (Lepidoptera: Lymantriidae) larvae were investigated. Endoreticulatus schubergi, a midgut pathogen, was transmitted to offspring via female and male via the egg chorion (transovum transmission). Between 8% and 29% of the emerging larvae became infected. No spores of E. schubergi were found in surface-washed eggs. Nosema lymantriae, a microsporidium that causes systemic infections, was transovarially transmitted. Between 35% and 72% of the progeny were infected. Vairimorpha disparis, a fat body pathogen, was not vertically transmitted. The infectivity of spores that overwintered in cadavers of infected L. dispar varied by species, placement in the environment, and weather conditions. Spores of E. schubergi were still infective after an eight month exposure period of cadavers on the ground. Spores of N. lymantriae and V. disparis remained highly infective only when cadavers overwintered under a more or less continuous snow cover for four months.     

Life cycle of a new species of Amblyospora (Microspora: Amblyosporidae) in the mosquito Aedes taeniorhynchus

A new species of Microspora, Amblyospora polykarya, is described from the mosquito Aedes taeniorhynchus. The parasite is transovarially transmitted for one generation only. Spores in adult females extrude binucleate sporoplasms which infect developing eggs. Merogony occurs in larval oenocytes with diplokaryotic stages in early instars giving rise to plasmodia with many diplokarya. Plasmodia undergo cytokinesis to form diplokaryotic sporonts. In fat body cells, these sporonts secrete pansporoblastic membranes and undergo two nuclear divisions to form octonucleate sporonts. Cytokinesis and differentiation result in uninucleate spores in packets of eight. These spores are not transmissible per os and are of different morphotype from those in adult females. Infected larvae die in the fourth stadium.     

Invasion success of Fibrillanosema crangonycis, n.sp., n.g.: a novel vertically transmitted microsporidian parasite from the invasive amphipod host Crangonyx pseudogracilis

Parasitism is known to be an important factor in determining the success of biological invasions. Here we examine Crangonyx pseudogracilis, a North American amphipod invasive in the United Kingdom and describe a novel microsporidium, Fibrillanosema crangonycis n.sp., n.g. The primary site of infection is the female gonad and the parasite is transovarially transmitted to the eggs. PCR screening reveals a female bias in the distribution of parasites (96.6% of females, N=29; 22.2% of males, N=27), which is indicative of host sex ratio distortion. The morphological and molecular characterisations of this new microsporidium place it outside all currently established genera. On the basis of these differences, we erect the new genus Fibrillanosema n.g. While F. crangonycis is morphologically identical to uncharacterised microsporidia from populations of North American amphipods, it is distinct from microsporidia found in European populations of amphipods. These data support the hypothesis that vertically transmitted parasites may be selectively retained during invasion events. Furthermore where vertical transmission is combined with host sex ratio distortion these parasites may directly enhance host invasion success through increased rates of population growth.     

Aquatic tetrasporoblastic microsporidia from caddis flies (Insecta, Trichoptera): characterisation, phylogeny and taxonomic reevaluation of the genera Episeptum Larsson, 1986, Pyrotheca Hesse, 1935 and Cougourdella Hesse, 1935

Seven microsporidian species infecting caddis fly larvae, corresponding to conventional genera Episeptum, Pyrotheca and Cougourdella were studied using light and electron microscopy. Parts of their small subunit, ITS and large subunit ribosomal RNA genes were sequenced and compared with sequences of rDNA obtained from syntype slides of Cougourdella polycentropi Weiser 1965 and Pyrotheca sp. from Hydropsyche pellucidula. All studied caddis fly microsporidia form a closely related group. Their developmental stages in trichopteran hosts are restricted to fat body cells and oenocytes and have isolated nuclei. In late merogony, uninucleate meronts and binucleate plasmodia are formed. In sporogony a sporogonial plasmodium with four nuclei gives rise by rosette-like budding to four sporoblasts within a non-persistent sporophorous vesicle. Sporoblasts mature into pyriform to lageniform spores. The shape and size of spores, the number of polar filament coils, the structure of the polaroplast and of the exospore, together with morphometric characters present a set of markers unique for respective species. Four new species are established. The new genus Paraepiseptum is proposed to replace the tetrasporoblastic Pyrotheca and Cougourdella species from caddis flies. The genus Episeptum is redefined. Field and laboratory examinations as well as the phylogenetic position within the aquatic clade of microsporidia suggest that the life cycle of trichopteran microsporidia probably involves an alternate (copepod?) host and (or) transovarial transmission.     

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.     

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.     

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.     

Toxoplasma gondii: Congenital transmission in a hamster model

The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non-invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n = 168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated. Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.     

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)     

Nosema pyrausta infection in Macrocentrus grandii, a braconid parasite of the European corn borer, Ostrinia nubilalis

Macrocentrus grandii which develop within Nosema pyrausta-infected larvae of the European corn borer, Ostrinia nubilalis, develop direct systemic infections from the ingestion of spores at the time of larval emergence from the host. Infections adversely affect pupal development and adult longevity. Infected females are unable to transmit the microsporidian to additional corn borer hosts. Pathogen development in the parasite host appears identical to its development in the corn borer host and mature spores show no morphological differences in size or shape when observed at the ultrastructural level. The prevalence of infection in natural parasite populations is 53.8% and closely parallels the 56.7% prevalence of infection in corn borer populations. Results suggest N. pyrausta may play a significant role in limiting M. grandii populations when levels of N. pyrausta in corn borers are high.     

Host specificity of microsporidia pathogenic to the gypsy moth, Lymantria dispar (L.): Field studies in Slovakia

Several species of microsporidia are important chronic pathogens of Lymantria dispar in Europe but have never been recovered from North American gypsy moth populations. The major issue for their introduction into North American L. dispar populations is concern about their safety to native non-target insects. In this study, we evaluated the susceptibility of sympatric non-target Lepidoptera to two species of microsporidia, Nosema lymantriae and Vairimorpha disparis, isolated from European populations of L. dispar and applied in field plots in Slovakia. Application of ultra low volume sprays of the microsporidia maximized coverage of infective spores in a complex natural environment and, thus, exposure of non-target species to the pathogens. Of 653 non-target larvae collected from plots treated with V. disparis in 2002, 18 individual larvae representing nine species in four families were infected. These plots were monitored for two subsequent seasons and V. disparis was not recovered from non-target species. Of 2571 non-target larvae collected in N. lymantriae-treated sites, one larva was found to be infected. Both species of microsporidia, particularly N. lymantriae, appear to have a very narrow host range in the field, even when an inundative technique is used for their introduction. V. disparis infections in L. dispar exceeded 40% of recovered larvae in the treated study sites; infection rates were lower in sites sprayed with N. lymantriae. Several naturally-occurring pathogens were recorded from the non-target species. The most common pathogen, isolated from 21 species in eight families, was a microsporidium in the genus Cystosporogenes.     

Benomyl: Effectiveness against the microsporidian Nosema heliothidis in the corn earworm,Heliothis zea

The fungicide benomyl was studied as a possible antimicrobial agent for obtaining Nosema heliothidis-free laboratory colonies of Heliothis zea. Newly hatched, transovarially infected larvae were placed on artificial diets containing 250, 500, or 1000 ppm benomyl. While late-stage larvae were found to be free of Nosema spores, low-level infections were found in pupae and newly emerged adults. The reduced intensity of infection in adults reared as larvae on treated diets was not correlated with a significant reduction in the efficiency of transovarian transmission. The chemical effect of benomyl was manifested by aberrant spores and vegetative stages and a rapid reduction in the number of microsporidian stages. However, small, isolated centers of infection in various host tissues resulted in a rapid resurgence of the microsporidiosis in pupae and adults. Thus, at the concentrations tested, benomyl was not effective in eliminating infection by N. heliothidis in H. zea. A discussion of the necessity for careful evaluation of the apparent suppression of microsporidioses by antimicrobial agents is also presented.     

Microsporidium goeldichironomi n. sp. and Microsporidium chironomi n. sp. (Microsporida: Apansporoblastina): Two new microsporidia from Florida chironomids

Two new species of Microsporida belonging to the genus Microsporidium are described. Microsporidium goeldichironomi n. sp. parasitizes the fat body of Goeldichironomus holoprasinus and Microsporidium chironomi n. sp. infects Chironomus attenuatus. Both microsporidia form uninucleate spores from rosette-shaped sporonts. M. goeldichironomi sporonts form 4, 6, 8, 10, 12, 16, and possibly more spores. Two shapes of spores are produced, oval, or slightly pyriform spores measuring 3.70 ± 0.09 × 2.49 ± 0.13 μm and pyriform spores measuring 3.74 ± 0.44 × 2.04 ± 0.17 μm. Electron micrographs show that both types of spores are uninucleate, have 8 to 11 polar filament coils and a lamellate polaroplast showing several distinct regions. M. chironomi spores are pyriform and are often joined at the posterior end in groups of two or four. They measure 4.12 ± 0.37 × 2.45 ± 0.26 μm. The spores are uninucleate, have six to seven polar filament coils and a lamellate polaroplast showing two distinct regions. Neither species can be transmitted per os and thus are assumed to be transovarially transmitted. No pansporoblastic membrane is present in either species.     

Biology and life-cycle of the microsporidium Kneallhazia solenopsae Knell Allan Hazard 1977 gen. n., comb. n., from the fire ant Solenopsis invicta

Thelohania solenopsae is a unique microsporidium with a life-cycle finely tuned to parasitizing fire ant colonies. Unlike other microsporidia of social hymenopterans, T. solenopsae infects all castes and stages of the host. Four distinctive spore types are produced: diplokaryotic spores, which develop only in brood (Type 1 DK spores); octets of octospores within sporophorous vesicles, the most prominent spore type in adults but never occurring in brood; Nosema-like diplokaryotic spores (Type 2 DK spores) developing in adults; and megaspores, which occur occasionally in larvae 4, pupae, and adults of all castes but predominantly infect gonads of alates and germinate in inseminated ovaries of queens. Type 2 DK spores function in autoinfection of adipocytes. Proliferation of diplokaryotic meronts in some cells is followed by karyogamy of diplokarya counterparts and meiosis, thereby switching the diplokaryotic sequence to octospore or megaspore development. Megaspores transmit the pathogen transovarially. From the egg to larvae 4, infection is inapparent and can be detected only by PCR. Type 1 DK spore and megaspore sequences are abruptly triggered in larvae 4, the key stage in intra-colony food distribution via trophallaxis, and presumably the central player in horizontal transmission of spores. Molecular, morphological, ultrastructural and life-cycle data indicate that T. solenopsae must be assigned to a new genus. We propose a new combination, Kneallhazia solenopsae.     

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.