Cytology of Leidyana canadensis (apicomplexa: eugregarinida) in Lambdina fiscellaria fiscellaria larvae (lepidoptera: geometridae).

The eugregarine Leidyana canadensis infects the larval gut of the eastern hemlock looper, Lambdina fiscellaria fiscellaria. Guts of infected larvae were chemically fixed, embedded in epoxy resin, and sectioned for light and electron microscopy to describe the cytology of L. canadensis and its pathology in the larval host. Oocysts of L. canadensis are ingested by larval hemlock looper. Trophozoites emerge from the oocysts, pass through the peritrophic membrane into the ectoperitrophic space, and attach to the epithelium of the midgut by means of an apical epimerite. The epimerite does not actually penetrate the affected epithelial cell; instead, it causes an invagination of the plasma membrane of the cell. The center of the epimerite contains membrane cisternae, and mitochondria line its periphery. Microtubules and mitochondria in the host cell cytoplasm surround the epimerite. At the light microscopic level, there appeared to be septa between the epimerite and the protomerite and between the protomerite and the deutomerite; however, in the electron microscope, no septa were evident. Only differences in the concentrations and nature of the inclusions in the cytoplasms of these three regions were apparent. The deutomerite contains a single nucleus in the central-posterior area. After an undetermined period, the epimerite detaches from the host gut epithelium and is withdrawn into the protomerite, and the trophozoites float freely in the ectoperitrophic space before differentiating into gamonts. Division of the single, large nucleus into numerous small nuclei appears to occur prior to syzygy. Gamonts pair and a cyst wall is laid down around them, forming a gametocyst. Oocysts are extruded from mature gametocysts, in chains, through sporoducts.

Choristoneura fumiferana granulovirus: sequence analysis and 5' characterization of ORF891.

A gene located immediately upstream of the granulin gene of Choristoneura fumiferana (ChfuGV) granulovirus was identified, sequenced and named ORF891. The determined, putative open reading frame (ORF) of 891 bp encodes an estimated 34.6 kDa protein. The 5' end transcript of the gene was mapped and analysed. A putative promoter region organization of ChfuGV ORF891 contains a consensus late baculovirus promoter element, TAAG, and two putative early TATA boxes similar to the promoters of ORF909 of Cryptophlebia leucotreta granulovirus (ClGV). Sequence comparisons of ChfuGV ORF891 with ClGV ORF909 and Cydia pomonella granulovirus (CpGV) ORF124R showed respective homologies of 60.9 and 63.9% for nucleotides and 46.3% and 49.3% for amino acids. Homology of ChfuGV ORF891 with ME53 ORF of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was 68.2% for nucleotides but a total lack of homology for amino acid sequences. Two zinc finger motifs are also associated with ChfuGV ORF891.

Insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment.

Attempts to develop baculovirus-based insecticides by insertion of genes encoding enzyme inhibitors, neuropeptides or toxins have met with some success. However, it is often difficult to ensure correct processing or secretion of the encoded peptides. Here we tested a simpler strategy by insertion of an antisense fragment of a host gene to block translation of a protein essential for larval growth and development. We selected the c-myc gene for two main reasons: (i) its protein is known to be well conserved in evolution and to have multiple essential functions during development; and (ii) c-myc family genes have yet to be characterized in insects, thus blockage of essential genes by anti-sense transcripts from a strong virus promoter could provide a sensitive test for the existence of myc-like gene products. An appropriate fragment of the human c-myc gene was inserted downstream from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus and tested in bioassays on Spodoptera frugiperda larvae. Western blot analysis with a human c-myc antibody revealed an endogenous protein band which bound specifically to these antibodies. This band disappeared more rapidly from cells infected with the antisense c-myc recombinant virus than from those infected with c-myc-negative virus. Results of bioassays showed that the antisense construct stopped feeding as soon as the polyhedrin promoter-driven transcripts accumulated, followed shortly by death of the larvae. These results suggest that c-myc-like protein(s) exist in insects and that the antisense strategy is an effective approach to virus insecticide productions.

A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.

The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.