Susceptibility of Aedes aegypti and Aedes albopictus larvae to Ascogregarina culicis and Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) from Florida

The susceptibility of Aedes aegypti to Ascogregarina culicis and Aedes albopictus to Ascogregarina taiwanensis was examined with mosquito and parasite strains from Tampa, FL. When each host was bioassayed with its natural gregarine, the infection intensity indicated that Ae. aegypti was 59% more susceptible to A. culicis (87 gamonts/larva) than Ae. albopictus to A. taiwanensis (47 gamonts/larva). Infections in single and mixed host populations exposed to 100 oocysts/larva of one and both parasites demonstrated that Ae. aegypti harbors higher A. culicis gamont loads than Ae. albopictus of A. taiwanensis. In dual gregarine exposures of single host populations, the A. culicis infection intensity in Ae. aegypti was reduced by approximately 50%. A. taiwanensis exhibited the same capability of infecting Ae. albopictus in single and dual exposures. In mixed host populations there were no cross infections, but A. taiwanensis in Ae. albopictus produced an infection intensity of approximately 70% lower than that of A. culicis in Ae. aegypti.     

Role of gregarine parasite Ascogregarina culicis (Apicomplexa: Lecudinidae) in the maintenance of Chikungunya virus in vector mosquito

Ascogregarina culicis and Ascogregarina taiwanensis are common gregarine parasites of Aedes aegypti and Aedes albopictus mosquitoes, respectively. These mosquito species are also known to transmit dengue and Chikungunya viruses. The sporozoites of these parasites invade the midgut epithelial cells and develop intracellularly and extracellularly in the gut to complete their life cycles. The midgut is also the primary site for virus replication in the vector mosquitoes. Therefore, studies were carried out with a view to determine the possible role of these gregarines in the vertical transmission of dengue and Chikungunya viruses from larval to adult stage. Experiments were performed by exposing first instar mosquito larvae to suspensions containing parasite oocysts and viruses. Since Ascogregarina sporozoites invade the midgut of first instar larvae, the vertical transmission was determined by feeding the uninfected first instar larvae on the freshly prepared homogenates from mosquitoes, which were dually infected with viruses and the parasite oocysts. Similarly, the role of protozoan parasites in the vertical transmission of viruses was determined by exposing fresh first instar larvae to the dried pellets of homogenates prepared from the mosquitoes dually infected with viruses and the parasite oocysts. Direct vertical transmission and the vertical transmission of CHIK virus through the oocyst of the parasites were observed in the case of Ae. aegypti mosquitoes. It is suggested that As. culicis may have an important role in the maintenance of CHIK virus during the inter-epidemic period.     

Comparison of the morphology of oocysts and the phylogenetic analysis of four Ascogregarina species (Eugregarinidae: Lecudinidae) as inferred from small subunit ribosomal DNA sequences

This study on the ultrastructure of the oocysts of four isolated species of Ascogregarina (A. taiwanensis (Lien and Levine) (Eugregarinidae: Lecudinidae) from Aedes albopictus (Skuse), A. culicis (Ross) (Eugregarinidae: Lecudinidae) from Aedes aegypti (L.), A. armigerei (Eugregarinidae: Lecudinidae) from Armigeres subalbatus (Coquillet), and Ascogregarina sp. (Eugregarinidae: Lecudinidae) from Ochlerotatus japonicus japonicus (Theobald)) using a scanning electron microscope revealed significant differences in size and in surface structure. The average length of the oocyst was greatest in A. armigerei (13.2+/-0.2 microm) (mean+/-SD) and least in A. culicis (8.8+/-0.4 microm). Oocysts were of moderate length in A. taiwanensis (9.9+/-0.6 microm) and in Ascogregarina sp. (10.7+/-1.1 microm) isolated from O. j. japonicus. The ultrastructure of the surface of the A. culicis oocyst was rough in texture with numerous dense spots and was easily distinguishable from the oocysts of the other three Ascogregarina spp. The maximum likelihood tree inferred from small subunit ribosomal DNA (SSU rDNA) sequences indicated that the four Ascogregarina spp. form a monophyletic cluster among other gregarine parasites. Within the Ascogregarina clade, A. culicis, A. taiwanensis, and Ascogregarina sp. from O. j. japonicus showed a close relationship, whereas A. armigerei was a distantly related species.     

Parasitism of Ascogregarina taiwanensis and Ascogregarina culicis (Apicomplexa: Lecudinidae) in larvae of Aedes albopictus and Aedes aegypti (Diptera: Culicidae) from Manaus, Amazon region, Brazil

The aim of the study was to determine the existence of Ascogregarina spp. in larvae of Aedes albopictus and Aedes aegypti collected in urban and suburban areas of Manaus, Amazon region, Brazil. Between May 2004 and July 2005, the mid-gut of 3rd and 4th instar larvae, collected in tire traps in six neighborhoods of Manaus, was examined for the presence of trophozoites of Ascogregarina. Coexistence of Ae. albopictus larvae infected by A. taiwanensis, and Ae. aegypti larvae by A. culicis, was detected in traps in the field. The percentage of Ae. albopictus larvae infected by A. taiwanensis ranged from 21% to 93.5% and of Ae. aegypti larvae infected by A. culicis from 22% to 95%. The mean infection intensity was similar in both species of Aedes. In traps located in Mauazinho, the replacement of Ae. aegypti by Ae. albopictus larvae was observed. In Manaus, Ae. albopictus larvae were parasitized by A. taiwanensis, and Ae. aegypti larvae by A. culicis. Infection rates were high when the species of Aedes were found separately.     

Oviposition by Aedes aegypti (Diptera: Culicidae) in relation to conspecific larvae infected with internal symbiotes.

Female mosquitoes use abiotic and biotic factors, such as the presence of heavy metals or parasites, to determine the acceptability of oviposition sites. Some biotic factors can originate from mosquito larvae. When mosquito larvae are starved or infected with trematode metacercaria, they render associated water unacceptable to ovipositioning females. Internal symbiotes are common in wild mosquito populations, and I tested whether or not larvae of Aedes aegypti infected with a gregarine (Ascogregarina taiwanensis), a yeast (Candida near pseudoglaebosa), or a trichomycete (Smittium morbosum) rendered their rearing water unacceptable to oviposting mosquitoes. Infections with S. morbosum had no effect on the acceptability of the associated rearing water when compared to rearing water from uninfected larvae. However, the rearing water from larvae infected with A. taiwanensis or C. near pseudoglaebosa was more acceptable to ovipositing females than was distilled water or rearing water from uninfected larvae. Ovipositing female mosquitoes either preferred or were neutral to rearing water from larvae with gut symbiotes.     

Electrophoretic differences in the isoenzymes of two mosquito gregarines, Ascogregarina barretti and A. geniculati

Cross-infection experiments demonstrated that Ascogregarina barretti, from Aedes triseriatus, completes its life cycle in Aedes geniculatus. Parasite numbers were comparable to infection with Ascogregarina geniculati, making the separation of these parasites by host preference difficult. However, electrophoresis readily distinguished isoenzymes from the two morphologically similar gregarine species. Different migration rates were obtained for isocitrate dehydrogenase 1 and 2, lactate dehydrogenase, and malate dehydrogenase. The migration rates were also different for parasite and host isoenzymes. When a single, heavily infected gut was subjected to electrophoresis the isocitrate dehydrogenase bands of each were clearly distinguishable on the same electrophoretic track. Electrophoresis appears to be a reliable method for resolving taxonomic complications of mosquito gregarines, a group often with wide host specificities and variable taxonomic characters.     

Characterization of a coelomic gregarine parasite from Thitarodes pui (Lepidoptera: Hepialidae) in the Tibetan Plateau

Thitarodes pui, one of the host species of entomopathogenic fungus Ophiocordyceps sinensis, has great economic importance in the Tibetan Plateau. We report here, for the first time, a gregarine parasite found in the coelom of 7th instar and adults of T. pui. Gregarine gamonts (ovoid, ～15×8μm) underwent syzygy to produce reproductive gametocysts in T. pui larval hemolymph. All infected T. pui carried 2-17 mature gametocysts filled with numerous oocysts (lemon-shaped, 17.17±0.73×6.49±0.4μm). Transmission electron microscopy showed that these oocysts contained vacuoles of various sizes and amylopectin granules in the cytoplasm; scanning electron microscopy revealed a number of small bumps all over the surface of these oocysts. Small subunit ribosomal DNA sequence analysis showed a close relationship between the gregarine and the species of Ascogregarina (Eugregarinorida: Lecudinidae). Internal transcribed spacers and 5.8S ribosomal DNA from this gregarine exhibited 76% highest sequence identity with that from Ascogregarina culicis Ross.     

Laboratory susceptibility of Wyeomyia smithii (Diptera: Culicidae) to Ascogregarina taiwanensis (Apicomplexa: Lecudinidae)

Gregarines in the genus Ascogregarina are not known to develop in sabethine mosquitoes, but we successfully infected larvae of Wyeomyia smithii with Ascogregarina taiwanensis in the laboratory. Ascogregarina taiwanensis is a natural parasite of the exotic Asian tiger mosquito, Aedes albopictus. Only 18% to 70% of the W. smithii larvae had visible trophozoites, with a range of 1-92 per larva. Trophozoites persisted in the midgut for more than 37 d, and one adult female W. smithii had gametocysts in its Malphigian tubules, which indicated that A. taiwanensis might fully develop in W. smithii. After 50 d, gregarines were not found in W. smithii larvae.     

Prevalence and seasonality of Ascogregarina culicis (Apicomplexa: Lecudinidae) in natural populations of Aedes aegypti (Diptera: Culicidae) from temperate Argentina

Ascogregarina infections from South America were recently documented in Brazil and Argentina. The aim of this study was to report our recent findings on the prevalence and seasonality of Ascogregarina culicis in Aedes aegypti adults from temperate Argentina. Between December 2003 and May 2005, 391 females of Ae. aegypti were captured in two areas of Greater Buenos Aires. Overall prevalence of A. culicis was 21.2% (83/391), and a significant difference was observed between both areas (28.4% vs. 8%). Infected Ae. aegypti were found from November to May, with highest values in March (24-37%). Parasite prevalence and host abundance showed similar seasonal patterns. Our observations suggest a widespread infestation of A. culicis among Ae. aegypti populations from temperate Argentina.     

Molecular Characterization of Gregarines from Sand Flies (Diptera: Psychodidae) and Description of Psychodiella n. g. (Apicomplexa: Gregarinida)

ABSTRACT. Sand fly and mosquito gregarines have been lumped for a long time in the single genus Ascogregarina and on the basis of their morphological characters and the lack of merogony been placed into the eugregarine family Lecudinidae. Phylogenetic analyses performed in this study clearly demonstrated paraphyly of the current genus Ascogregarina and revealed disparate phylogenetic positions of gregarines parasitizing mosquitoes and gregarines retrieved from sand flies. Therefore, we reclassified the genus Ascogregarina and created a new genus Psychodiella to accommodate gregarines from sand flies. The genus Psychodiella is distinguished from all other related gregarine genera by the characteristic localization of oocysts in accessory glands of female hosts, distinctive nucleotide sequences of the small subunit rDNA, and host specificity to flies belonging to the subfamily Phlebotominae. The genus comprises three described species: the type species for the new genus— Psychodiella chagasi ( Adler and Mayrink 1961 ) n. comb., Psychodiella mackiei ( Shortt and Swaminath 1927 ) n. comb., and Psychodiella saraviae ( Ostrovska, Warburg, and Montoya-Lerma 1990 ) n. comb. Its creation is additionally supported by sequencing data from other gregarine species originating from the sand fly Phlebotomus sergenti . In the evolutionary context, both genera of gregarines from mosquitoes ( Ascogregarina ) and sand flies ( Psychodiella ) have a close relationship to neogregarines; the genera represent clades distinct from the other previously sequenced gregarines.     

Ultrastructure of Infection, Development and Gametocyst Formation of Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in Its Mosquito Host, Aedes albopictus (Diptera: Culicidae)

ABSTRACT. The life history of the protozoan parasite Ascogregarina taiwanensis in mosquito larvae ( Aedes albopictus , collected in southern Taiwan) was shown to consist of two consecutive stages—intracellular and extracellular. Light microscopy showed that most trophozoites moved into the Malpighian tubules and developed into giant trophozoites during the first day pupa. The locomotion may be associated with bristle-like ridges of the trophozoite. The stage for sexual reproduction, i.e. the gamete, was then formed by segmentation of the giant trophozoite and twisting off the anucleate extremities of the body. Sexual reproduction occurred via fertilization by fusion of two resulting gametes, presumably two opposed sexes. The fused gametes finally generate the formation of the gametocyst, within which oocysts develop by budding from the cytoplasmic mass. This type of sexual reproduction has not been reported previously in any gregarine protozoa. We here proposed it as a new hypothesis for further elucidation of the protozoan reproduction.     

A parasite's modification of host behavior reduces predation on its host

Parasite modification of host behavior is common, and the literature is dominated by demonstrations of enhanced predation on parasitized prey resulting in transmission of parasites to their next host. We present a case in which predation on parasitized prey is reduced. Despite theoretical modeling suggesting that this phenomenon should be common, it has been reported in only a few host–parasite–predator systems. Using a system of gregarine endosymbionts in host mosquitoes, we designed experiments to compare the vulnerability of parasitized and unparasitized mosquito larvae to predation by obligate predatory mosquito larvae and then compared behavioral features known to change in the presence of predatory cues. We exposed Aedes triseriatus larvae to the parasite Ascogregarina barretti and the predator Toxohrynchites rutilus and assessed larval mortality rate under each treatment condition. Further, we assessed behavioral differences in larvae due to infection and predation stimuli by recording larvae and scoring behaviors and positions within microcosms. Infection with gregarines reduced cohort mortality in the presence of the predator, but the parasite did not affect mortality alone. Further, infection by parasites altered behavior such that infected hosts thrashed less frequently than uninfected hosts and were found more frequently on or in a refuge within the microcosm. By reducing predation on their host, gregarines may be acting as mutualists in the presence of predation on their hosts. These results illustrate a higher‐order interaction, in which a relationship between a species pair (host–endosymbiont or predator–prey) is altered by the presence of a third species.     

Cloning, purification, and nucleotide-binding traits of the catalytic subunit A of the V1VO ATPase from Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is commonly infected by the gregarine parasite Ascogregarina taiwanensis, which develops extracellularly in the midgut of infected larvae. The intracellular trophozoites are usually confined within a parasitophorous vacuole, whose acidification is generated and controlled by the V(1)V(O) ATPase. This proton pump is driven by ATP hydrolysis, catalyzed inside the major subunit A. The subunit A encoding gene of the Aedes albopictus V(1)V(O) ATPase was cloned in pET9d1-His(3) and the recombinant protein, expressed in the Escherichia coli Rosetta 2 (DE3) strain, purified by immobilized metal affinity- and ion-exchange chromatography. The purified protein was soluble and properly folded. Analysis of secondary structure by circular dichroism spectroscopy showed that subunit A comprises 43% alpha-helix, 25% beta-sheet and 40% random coil content. The ability of subunit A of eukaryotic V-ATPases to bind ATP and/or ADP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the FCS data indicates that the ADP-analogues bind slightly weaker to subunit A than the ATP-analogues. Tryptophan fluorescence quenching of subunit A after binding of different nucleotides provides evidence for secondary structural alterations in this subunit caused by nucleotide-binding.     

Cost of co-infection controlled by infectious dose combinations and food availability

To what extent the combined effect of several parasite species co-infecting the same host (i.e. polyparasitism) affects the host’s fitness is a crucial question of ecological parasitology. We investigated whether the ecological setting can influence the co-infection’s outcome with the mosquito Aedes aegypti and two parasites: the microsporidium Vavraia culicis and the gregarine Ascogregarina culicis. The cost of being infected by the two parasites depended on the interaction between the two infectious doses and host food availability. The age at pupation of the mosquito was delayed most when the doses of the two parasites were highest and little food was available. As infectious dose increases with the parasites’ prevalence and intensity of transmission, the cost of being co-infected depends on the epidemiological status of the two parasite species.     

Proteome of Aedes aegypti in response to infection and coinfection with microsporidian parasites

Hosts are frequently infected with more than one parasite or pathogen at any one time, but little is known as to how they respond to multiple immune challenges compared to those involving single infections. We investigated the proteome of Aedes aegypti larvae following infection with either Edhazardia aedis or Vavraia culicis, and coinfections involving both. They are both obligate intracellular parasites belonging to the phylum microsporidia and infect natural populations of Ae. aegypti. The results found some proteins only showing modified abundance in response to infections involving E. aedis, while others were only differentially abundant when infections involved V. culicis. Some proteins only responded with modified abundance to the coinfection condition, while others were differentially abundant in response to all three types of infection. As time since infection increased, the response to each of the single parasite infections diverged, while the response to the E. aedis and coinfection treatments converged. Some of the proteins differentially abundant in response to infection were identified. They included two vacuolar ATPases, proteins known to have a role in determining the infection success of intracellular parasites. This result suggests microsporidia could influence the infection success of other intracellular pathogens infecting vector species of mosquito, including viruses, Plasmodium and Wolbachia.     

Morphological and molecular characterizations of the Gregarina sp. (Apicomplexa: Protozoa) parasitizing on Phaedon brassicae (Coleoptera: Chrysomelidae)

A gregarine parasite (Eugregarinida: Gregarinidae) was observed in the population of daikon leaf beetle, Phaedon brassicae Baly, (Coleoptera: Chrysomelidae) in Korea. Gregarines are well known species-specific parasites of various Arthropoda. This Gregarina sp. also confirmed a species-specific parasite in P. brassicae. Based on 1.727 kb of 18S rDNA sequence (FJ481523), this gregarine species was grouped in eugregarine and a 5.258 kb of full length rDNA replicon was cloned (JF412715). We also observed interaction of trophozoite or gamonto of gregarine and epithelium of a host midgut using a light microscope and a scanning electron microscope. Although the developmental period of the infected host is delayed half a day in every larval stage, there was no significant difference in the developmental period of P. brassicae whether Gregarina sp. was infected or not. Gregarina sp. was a kind of facultative parasite from P. brassicae. This is the first report of a gregarine parasite in P. brassicae.     

Intestinal expression of H+ V-ATPase in the mosquito Aedes albopictus is tightly associated with gregarine infection

Vacuolar ATPase (V-ATPase) is a family of ATP-dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V-ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V-ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full-length sequence of mRNA of V-ATPase subunit A, which contains a poly-A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V-ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V-ATPase-energized high-pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V-ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development.     

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.     

The life cycle of Ascogregarina taiwanensis (Apicomplexa:Lecudinidae).

A number of features, including large size of the trophozoite, host-specificity, developmental synchrony and evident migration behavior, of Ascogregarina taiwanensis make it suitable as a model for studies of parasite-host relationships. In this article, Wei June Chen describes the life cycle of As. taiwanensis; and raises some areas for future work, including the interaction between the parasite and its host, inhibition of sexual reproduction by non-natural hosts and their mutual effects during the development.     

Proteome of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis

We report on the modification of the Aedes aegypti larval proteome following infection by the microsporidian parasite Vavraia culicis. Mosquito larvae were sampled at 5 and 15 days of age to compare the effects of infection when the parasite was in two different developmental stages. Modifications of the host proteome due to the stress of infection were distinguished from those of a more general nature by treatments involving hypoxia. We found that the major reaction to stress was the suppression of particular protein spots. Older (15 days) larvae reacted more strongly to infection by V. culicis (46% of the total number of spots affected; 17% for 5 days larvae), while the strongest reaction of younger (5 days) larvae was to hypoxia for pH range 5-8 and to combined effects of infection and hypoxia for pH range 3-6. MALDI-TOF results indicate that proteins induced or suppressed by infection are involved directly or indirectly in defense against microorganisms. Finally, our MALDI-TOF results suggest that A. aegypti larvae try to control or clear V. culicis infection and also that V. culicis probably impairs the immune defense of this host via arginases-NOS competition.