Development of the insect pathogenic alga Helicosporidium.

This study examined the morphogenesis and replication dynamics of the different life stages (cysts, filamentous cells, vegetative cells) of Helicosporidium sp., a non-photosynthetic, entomopathogenic alga. The isolate (SjHe) used originated from an infected black fly larva. Filamentous cell transformation into vegetative cells and autosporulation during vegetative cell replication were observed under controlled in vitro conditions. The transformation process was initiated by a partial swelling of the filamentous cell along with the reorganization of the nuclear material. Two subsequent nuclear and cell divisions resulted in the release of 4 rod-shaped daughter cells, which divided into oval to spherical vegetative cells. These underwent several cycles of autosporogenic cell division. Multiple-passaged vegetative cell cultures formed non-motile, adherent cell clusters (palmelloid colonies). Vegetative replication dynamics were also observed in 2 experimental noctuid hosts, Spodoptera exigua and Helicoverpa zea. The average density of helicosporidial cells produced per microliter hemolymph exceeded cell concentrations obtained in vitro by 15- and 46-fold in S. exigua and H. zea, respectively. Cyst morphogenesis was only observed in the hemolymph, whereas no cysts differentiated at various in vitro conditions.

Differential expression of chitin synthase (CHS) and glucan synthase (FKS) genes correlates with the formation of a modified, thinner cell wall in in vivo-produced Beauveria bassiana cells.

During infection (in vivo), the entomopathogenic fungus Beauveria bassiana produces yeast-like cells that are surrounded by modified cell walls. These modifications have been related to the fungus ability to limit recognition by the host defense system. The composition of the in vivo cell wall was analyzed using a combination of cytochemical and molecular techniques. The in vivo cell walls still contained both chitin and 1,3-beta-glucan, but they were significantly thinner than in vitro cell walls (50-60 nm versus 100-160 nm, respectively). The difference in cell wall thickness was correlated with transcriptional regulation of cell wall-related genes: quantitative RT-PCR reactions demonstrated that B. bassiana chitin synthase (CHS) and glucan synthase (FKS) genes are down regulated in vivo. These analyses indicate that in vivo-triggered phenotypic modifications, including cell wall adjustments, are controlled by molecular mechanisms that include regulation of gene expression at the transcriptional level.

Review of microsporidia-mosquito relationships: from the simple to the complex.

Microsporidia in mosquitoes can be divided into two categories based on their life cycles and host-parasite relationships. Some species of microsporidia exhibit simple life cycles with one spore type responsible for oral (horizontal) transmission. They affect only one generation of the mosquito and are not usually host or tissue specific. Brachiola algerae and Vavraia culicis are examples of species isolated from mosquitoes with relatively straightforward life cycles (one spore type) and simple host-parasite relationships. B. algerae and a close relative of V. culicis have also been isolated from a vertebrate (human) host but sources for these infections are unknown. In contrast to B. algerae and V. culicis, polymorphic (heterosporous) microsporidia in mosquitoes are characterized by complex life cycles involving multiple spore types responsible for horizontal and vertical transmission. They affect two generations of the mosquito and some involve an obligate intermediate host. These microsporidia are generally very host and tissue specific with complex developmental sequences comprised of unique stages and events. The microsporidium Edhazardia aedis is a pathogen of Aedes aegypti and does not require an intermediate host. The developmental cycle of E. aedis is characterized by four sporulation sequences, two in the parental host and two in the filial generation. Recent speculation relative to the source of B. algerae human infection have implicated infected mosquitoes and raised concerns about the safety of mosquito microsporidia in general. The subject of this review is to compare and contrast three species of microsporidia from mosquitoes, two with broad host ranges (B. algerae and V. culicis) and one specific to mosquitoes (E. aedis). This review describes features that distinguish mosquito-parasitic microsporidia with simple life cycles and broad host ranges from truly mosquito-specific microsporidian parasites with complex life cycles.

A picorna-like virus from the red imported fire ant, Solenopsis invicta: initial discovery, genome sequence, and characterization.

We report the first discovery and genome sequence of a virus infecting the red imported fire ant, Solenopsis invicta. The 8026 nucleotide, polyadenylated, RNA genome encoded two large open reading frames (ORF1 and ORF2), flanked and separated by 27, 223, and 171 nucleotide untranslated regions, respectively. The predicted amino acid sequence of the 5' proximal ORF1 (nucleotides 28 to 4218) exhibited significant identity and possessed consensus sequences characteristic of the helicase, cysteine protease, and RNA-dependent RNA polymerase sequence motifs from picornaviruses, picorna-like viruses, comoviruses, caliciviruses, and sequiviruses. The predicted amino acid sequence of the 3' proximal ORF2 (nucleotides 4390-7803) showed similarity to structural proteins in picorna-like viruses, especially the acute bee paralysis virus. Electron microscopic examination of negatively stained samples from virus-infected fire ants revealed isometric particles with a diameter of 31 nm, consistent with Picornaviridae. A survey for the fire ant virus from areas around Florida revealed a pattern of fairly widespread distribution. Among 168 nests surveyed, 22.9% were infected. The virus was found to infect all fire ant caste members and developmental stages, including eggs, early (1st-2nd) and late (3rd-4th) instars, worker pupae, workers, sexual pupae, alates ( male symbol and female symbol ), and queens. The virus, tentatively named S. invicta virus (SINV-1), appears to belong to the picorna-like viruses. We did not observe any perceptible symptoms among infected nests in the field. However, in every case where an SINV-1-infected colony was excavated from the field with an inseminated queen and held in the laboratory, all of the brood in these colonies died within 3 months.

A nucleopolyhedrovirus from Uranotaenia sapphirina (Diptera: Culicidae).

In this report we present data on biology, gross pathology, ultrastructure, and host range studies of a naturally occurring nucleopolyhedrovirus from the mosquito Uranotaenia sapphirina (UrsaNPV). Development of this virus was restricted to nuclei of epithelial cells in posterior midgut and distal gastric caecum. Occlusion bodies contained numerous singly enveloped rod-shaped virions. Early occlusion bodies were irregularly shaped and seemed to subsequently coalesce to form larger polyhedra. Mature occlusion bodies had a unique dumbbell shape, and lacked a polyhedron envelope and crystalline structure. Developmental and structural features of UrsaNPV were generally similar to other mosquito NPVs, with major differences in occlusion body shape and size. Transmission tests showed that only members of Uranotaenia (Ur. sapphirina and Ur. lowii) were susceptible to this virus. Transmission was facilitated by magnesium. Field collected Ur. sapphirina larvae had a relatively high rate of dual infections with UrsaNPV and UrsaCPV (cypovirus).