Interactions between an entomopathogenic microsporidium and the endoparasitoid Glyptapanteles liparidis within their host, the gypsy moth larva.

Interactions in the host-parasitoid-pathogen system, Lymantria dispar L. (Lep., Lymantriidae)-Glyptapanteles liparidis (Bouché) (Hym., Braconidae)-Vairimorpha sp. (Protista, Microspora), were investigated. Host selection experiments revealed that G. liparidis females did not discriminate between infected and uninfected host larvae for oviposition. Transmission of the microsporidium from infected to uninfected hosts by stinging female wasps could not be ascertained. Females that developed in infected L. dispar larvae did not transmit the pathogen via oviposition. Vairimorpha infection of the host negatively affected the performance of the braconid, when inoculation took place either before or after parasitization. Microsporidiosis of the host caused delayed development, reduced pupation and adult eclosion, reduction in size and weight, and reduction of adult longevity of G. liparidis. Parasitoids themselves were not systemically infected by Vairimorpha sp., but braconid larvae did ingest microsporidian spores at the end of their endoparasitic development and accumulated the undigested and ungerminated spores in the blind midgut. Negative effects of host infection on parasitoid larvae were detectable from the beginning of parasitoid larval development. Lethal time was reduced when L. dispar larvae were infected and parasitized, often at the expense of the parasitoid when G. liparidis were unable to complete endoparasitic development before the host died. Intensity of infection, measured as number of spores produced per milligram fresh weight of L. dispar larva, was slightly higher in parasitized and infected hosts than in unparasitized and infected hosts.

Interactions between a Nosema sp. (Microspora: nosematidae) and nuclear polyhedrosis virus infecting the gypsy moth, Lymantria dispar (Lepidoptera: lymantriidae).

Simultaneous and sequential per os inoculations of gypsy moth larvae with the Lymantria dispar nuclear polyhedrosis virus (LdNPV) and a Nosema sp. from Portugal demonstrated that the interaction of two pathogens during coinfection was variable, ranging from synergistic to antagonistic. Susceptibility of gypsy moth larvae to viral infection was unaffected by simultaneous and subsequent microsporidian infection. This resulted from the comparatively slow pathogenesis of the microsporidium when compared to the virus. Viral infectivity, however, increased 10-fold when larvae were preinfected with Nosema sp. per os, or through transovarial infection. Time to death decreased for larvae infected with both pathogens compared to larvae infected with the virus alone. Polyhedron production was significantly reduced by microsporidian infection preceding viral infection. In this infection sequence, larvae died at an earlier stage and were less than half the mass of cadavers infected with virus alone. The biological significance of these results on gypsy moth population dynamics and the implication for use of this Nosema sp. from Portugal in gypsy moth biological control are discussed in the context of viral epizootiology.

Physiological host specificity of microsporidia as an indicator of ecological host specificity.

For most groups of biological control agents the relationship between laboratory (physiological) host range and the host range in the field (ecological host range) has not been explored empirically. The objective of our study was to investigate this relationship using the North America gypsy moth, Lymantria dispar, as a model nontarget host for microsporidia from native North American Lepidoptera. The gypsy moth, L. dispar, a native of Europe, has been established in North America for nearly 130 years and presumably exposed to many species of microsporidia from sympatric native Lepidoptera. Nevertheless, microsporidia have never been observed in North American populations of L. dispar. We conducted traditional laboratory feeding experiments using microsporidia from 20 lepidopteran host species and 1 coleopteran host species against L. dispar. Microsporidia from 18 native hosts infected L. dispar larvae. Although some of the infections were not typical of infections in the indigenous natural hosts, mature spores were produced in most of these infections. Horizontal transmission experiments, based on exposure of uninfected L. dispar larvae to infected L. dispar larvae, demonstrated that the microsporidia were far more host specific than the direct feeding experiments suggested. Of the three microsporidian biotypes that were horizontally transmitted between the nontarget L. dispar larvae, all were transmitted at very low levels. The results of our experiments provide additional evidence that the ecological host specificity of terrestrial microsporidia is much narrower than the physiological host specificity. Our studies establish the validity of using nonindigenous insect species with long-term data sets on natural enemies associated with them as a tool for testing hypotheses about host specificity.

Phylogenetic position of Amblyospora Hazard & Oldacre (Microspora:Amblyosporidae) based on small subunit rRNA data and its implication for the evolution of the microsporidia.

Sequences of the small subunit rRNA genes of Amblvospora california 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.

Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms.

Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans (Enterocytozoon bieneusi, Nosema corneum, Septata intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi), reveals that human microsporidia are polyphyletic in origin. Septata intestinalis and E. hellem are very similar to the mammalian parasite E. cuniculi. Based on the results of our phylogenetic analysis, we suggest that S. intestinalis be designated Encephalitozoon intestinalis. Furthermore, analysis of our data indicates that N. corneum is much more closely related to the insect parasite Endoreticulatus schubergi than it is to other Nosema species. This finding is supported by recent studies which have shown a similarity between E. schubergi and N. corneum based on the origin and development of the parasitophorous vacuole. Thus these opportunistic microsporidian parasites can originate from hosts closely or distantly related to humans. Finally, the phylogeny based on small subunit ribosomal DNA sequences is highly inconsistent with traditional classifications based on morphological characters. Many of the important morphological characters (diplokaryon, sporophorous vesicle, and meiosis) appear to have multiple-origins.

Phylogenetic relationships among Vairimorpha and Nosema species (Microspora) based on ribosomal RNA sequence data.

A portion (approximately 350 nucleotides) of the large subunit ribosomal RNA (rRNA) 5' to the 580 region (Escherichia coli numbering) was sequenced using the reverse transcriptase dideoxy method and compared for several species of Nosema and Vairimorpha. Comparison among Nosema species suggests that this genus is composed of several unrelated groups. The group which includes the type species, Nosema bombycis, consists of closely related species found primarily in Lepidoptera. Other Nosema species sequenced (Nosema kingi, Nosema algerae, and Nosema locustae) do not appear to be closely related to each other or to the lepidopteran Nosema group. Comparison among the Vairimorpha species indicates that two distinct but very closely related groups exist. The Lymantria group consists of species isolated from the gypsy moth, Lymantria dispar, while the Vairimorpha necatrix group consists of species isolated from other Lepidoptera. Intergeneric comparison of the sequence data suggests that the lepidopteran Nosema species are closely related to the Vairimorpha species.

Use of cellular fatty acid analysis to characterize commercial brands of Bacillus thuringiensis var. israelensis.

The cellular fatty acid composition of Bacillus thuringiensis var. israelensis (B.t.i.) from 5 commercial brands (Vectobac, Acrobe, Skeetal, Bactimos and Teknar), as well as of the current International Standard for B.t.i. (IPS 82), was determined using a Hewlett-Packard Microbial Identification System. The original strain of B.t.i., B.t. var. kurstaki, B.t. var. thuringiensis, B.t. var. morrisoni and Bacillus sphaericus strain 2362 were used as outgroups. Acrobe, Bactimos, Teknar, Vectobac and IPS 82 consisted of the same strain. Skeetal represented a different strain than the other commercially produced B.t.i. Our results indicate that cellular fatty acid analysis can be used to distinguish among the forms of B.t.i. produced by various manufacturers.

Effects of Ascogregarina barretti (Eugregarinida: Lecudinidae) infection on Aedes triseriatus (Diptera: Culicidae) in Illinois.

Ascogregarina barretti (Vavra), a gregarine protozoan parasite, infected 23.6 and 5.0% of Aedes triseriatus (Say) adults that emerged from pupae collected from an east-central Illinois tire dump in 1990 and 1991, respectively. Infection significantly reduced the wing length of males and females in both years, but its effect was greatest in 1991. Females were approximately 1.7 times as likely to be infected as males. Infected pupae were 3.53 and 2.76 times more likely to die than uninfected pupae in 1990 and 1991, respectively. Wing length also was affected by location within the tire dump and by tire water volume. We conclude that the A. barretti infection was deleterious to Ae. triseriatus and that the effects of this pathogen may be moderated by environmental factors.

Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes.

The microsporidia are a group of unusual, obligately parasitic protists that infect a great variety of other eukaryotes, including vertebrates, arthropods, molluscs, annelids, nematodes, cnidaria and even various ciliates, myxosporidia and gregarines. They possess a number of unusual cytological and molecular characteristics. Their nuclear division is considered to be primitive, they have no mitochondria, their ribosomes and ribosomal RNAs are reported to be of prokaryotic size and their large ribosomal subunit contains no 5.8S rRNA. The uniqueness of the microsporidia may reflect their phylogenetic position, because comparative sequence analysis shows that the small subunit rRNA of the microsporidium Vairimorpha necatrix is more unlike those of other eukaryotes than any known eukaryote 18S rRNA sequence. We conclude that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.