Baculoviruses in populations of western spruce budworm.

Population studies of western spruce budworm, Choristoneura occidentalis, revealed that a baculovirus, ChocNPV, was widespread in outbreak populations over a broad geographical area of British Columbia, Canada although the rate of mortality was usually low (<5%). Elevated levels of ChocNPV-related mortality (≈20%) were found when western spruce budworm populations reached high densities (≈300 larvae per kg of Douglas-fir foliage) and contributed to declines in population densities in these areas. A subsample from budworm collections examined using a multiplex-PCR assay showed ChocNPV was the most prevalent virus but also often occurred in combination with a granulovirus, ChocGV and a cypovirus, CoCPV.

A plant-inspired robot with soft differential bending capabilities.

We present the design and development of a plant-inspired robot, named Plantoid, with sensorized robotic roots. Natural roots have a multi-sensing capability and show a soft bending behaviour to follow or escape from various environmental parameters (i.e., tropisms). Analogously, we implement soft bending capabilities in our robotic roots by designing and integrating soft spring-based actuation (SSBA) systems using helical springs to transmit the motor power in a compliant manner. Each robotic tip integrates four different sensors, including customised flexible touch and innovative humidity sensors together with commercial gravity and temperature sensors. We show how the embedded sensing capabilities together with a root-inspired control algorithm lead to the implementation of tropic behaviours. Future applications for such plant-inspired technologies include soil monitoring and exploration, useful for agriculture and environmental fields.

Microfungal variations relative to post-fire changes in soil environment.

Soils of three Picea mariana-Cladina stellaris woodland sites, aged 2, 104 and 137 years after fire, were sampled at 3 cm intervals to 9 cm depth for soil microfungi. Mortierella, Mucor, Penicillium and Trichoderma were the most frequently isolated genera. The species composition of the microfungi remained much the same across the age gradient. Direct and indirect gradient analyses of the microfungal species and 14 soil environmental factors indicate that the frequencies of occurrence of the microfungi are influenced by environmental factors primarily related to the build-up of the organic layer and its destruction by fire.

Reproductive strategies and adaptations for survival among obligatory microsporidian and fungal parasites of mosquitoes: a comparative analysis of Amblyospora and Coelomomyces.

Amblyospora (Microsporida: Amblyosporidae) and Coelomomyces (Chytridiomycetes: Blastocladiales) have independently evolved a diverse array of unique and highly specialized mechanisms that have allowed them to more fully exploit their mosquito hosts and the aquatic environment that their hosts inhabit. Amblyospora and Coelomomyces both have complex life cycles that include obligatory development in an intermediate microcrustacean host and 2 mosquito generations for completion. Amblyospora is polymorphic with 3 separate and distinct developmental sequences, asexual and sexual reproduction, and aspects of both vertical (transovarial) and horizontal transmission. Infective stages of Coelomomyces are motile, a temporal gating mechanism coordinates gamete release, and, even though there is no transovarial transmission, infection of primary host ovaries is important in dissemination of the fungus to new habitats. The intent of this review is to examine how these and other strategies and adaptations facilitate parasite reproduction within the host(s) and enhance transmission and survival between hosts.

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.

Development and structure of the resting sporangium wall in Coelomomyces dodgei and modification during dehiscence.

The mature resting sporangium (RS) wall of Coelomomyces dodgei (Chytridiomycetes; Blastocladiales) consists of three principal layers: (I) an outer pigmented layer (1.8-2.2 microns) that contains polysaccharide, (II) a middle electron translucent layer (1.3-1.6 microns) comparatively free of polysaccharide, and (III) an inner layer (125 nm) rich in polysaccharide that surrounds the meiospores. These layers develop successively between the hyphal coat of the RS initial and its plasma membrane. Prior to meiospore formation, a convex discharge plug that later becomes the dehiscence vesicle forms beneath the inner layer along the preformed dehiscence slit in the outer wall. As dehiscence begins, the RS wall opens along the dehiscence slit, exposing the middle layer which then becomes the rigid outer refractile layer above the dehiscence vesicle. During dehiscence, the discharge plug swells out through the slit, and expands to form the dehiscence vesicle. Meiospores become active as the vesicle expands and are released through tears in the vesicle that result from continued expansion and lysis. Melanized areas observed along the dehiscence slit of RS that initiated dehiscence in the larval hemocoel indicate that the host's defense system is functional but does not recognize the parasite's hyphal coat as foreign, possibly because it lacks any significant level of polysaccharide.

A nuclear polyhedrosis virus from the obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera:Tortricidae).

A nuclear polyhedrosis virus (CrMNPV) has been isolated from larvae of the obliquebanded leafroller Choristoneura rosaceana (Harris) collected near St. Quentin in northwest New Brunswick, Canada. Restriction endonuclease digestion-gel electrophoresis of this virus, using a number of different endonucleases and whole CrMNPV genome Southern hybridization, have shown it to be different but similar to the equivalent virus from the spruce budworm Choristoneura fumiferana (Clem.) (CfMNPV). The polyhedrin gene of CrMNPV has been isolated, cloned, and sequenced and shown to have a predicted amino acid sequence only two amino acids different from the CfMNPV polyhedrin. In bioassays against spruce budworm larvae, CrMNPV had little pathogenicity to that host.

My child has hypospadias!

A child with a hypospadias anomaly needs a skilled surgeon to perform a delicate, complicated, sometimes staged, procedure successfully to prevent future psychological and physical problems.

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.

On thymidine incorporation by the cultured chick pineal gland.

Explanted chick pineal glands exhibited a cycle in thymidine incorporation when cultured either under a cycle of illumination or in constant darkness. This cycle appeared to be entrained to the light cycle under which the birds were maintained. The incorporation reflected gene replication in a small fraction of the cell population that was largely, but not entirely, located in the stroma of the gland. Glands cultured with colchicine for 28 h contained a very small number of cells showing metaphase chromosomes.

An unusual virus from the parasitic wasp Cotesia melanoscela.

Certain strains of the braconid parasitoid Cotesia melanoscela carry two different viruses within their ovaries, one of which (here designated CmV2) is apparently not a polydnavirus. Virus replication occurs in the ovarian calyx and in some other tissues of both male and female parasitoids; as yet, no replication has been observed in the testis, however. In addition, CmV2 is one of only two parasitoid viruses known to replicate in host insect larvae, and we not show that this virus is also capable of replicating in vitro; the virus is nevertheless nonpathogenic for gypsy moth larvae. The virus is not transmissible per os, either to host animals or to larvae of parasitoid strains lacking it. CmV2 is stably maintained within strains carrying it apparently by a vertical transmission mode involving the maternal line; transmission via the male germ line could not be demonstrated. While purification of the virus was not achieved, preliminary work allows us to suggest the genome consists of a single double-stranded DNA molecule of approximately 125 kb.

Identification and sequence analyses of the granulin gene of Choristoneura fumiferana granulovirus.

The nucleotide sequence of the granulin gene of Choristoneura fumiferana granulovirus (CfGV) was determined. The gene encodes a protein of 248 amino acids with a predicted Mr of 29.299 kDa. The granulin genes of Trichoplusia ni, Pieris brassicae and Cryptophlebia leucotreta granuloviruses showed homologies ranging from 76.7-80.5% for nucleotide sequences and 84.2-88.3% for amino acid sequences when compared to CfGV. The secondary structure of CfGV granulin protein, including the hydrophilic (polar) and hydrophobic (basic) regions, was predicted and found to be similar to other granulins. A very late baculovirus promoter motif, ATAAG, was found within the putative promoter region of the CfGV granulin gene.

A polydnavirus from the spruce budworm parasitoid, Tranosema rostrale (Ichneumonidae).

The calyx epithelium of the campoplegine wasp, Tranosema rostrale, contains typical ichneumonid polydnaviruses (PVs) that display an apparently uncommon association with the egg chorion. The latter structure features fine hair-like projections, longest around the egg's apices. In the lumen of the ovary, T. rostrale virus becomes lodged between these projections and forms a particulate coat around the egg. In the host, Choristoneura fumiferana, projections and associated virions are observed in close contact with basement membranes of fat body and muscle tissues, to which the eggs rapidly become attached following introduction into the host hemocoel. We discuss the implications of this unusual virus-chorion association in terms of immune protection, delivery of virus to specific host tissues, and the evolution of PVs.

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.