Occurrence of natural dixenic associations between the symbiont Photorhabdus luminescens and bacteria related to Ochrobactrum spp. in tropical entomopathogenic Heterorhabditis spp. (Nematoda, Rhabditida).
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Bacteria naturally associated with the symbiont Photorhabdus luminescens subsp. akhurstii were isolated from the entomopathogenic nematode Heterorhabditis indica. Bacterial isolates distinct from P. luminescens subsp. akhurstii were obtained from 33% of the samples. Fourteen bacterial isolates, from nematodes collected from three different Caribbean islands, were characterized by conventional phenotypic tests, restriction fragment length polymorphism and sequence analyses of PCR-amplified 16S rRNA genes (16S rDNAs). Isolates were grouped into three genotypes, each one being associated with one Caribbean island. Phenotypic characteristics and 16S rDNA analysis showed that the Photorhabdus-associated bacteria were closely related to Ochrobactrum anthropi for the group from Guadeloupe, and to Ochrobactrum intermedium for the two groups from the Dominican Republic and Puerto Rico. No pathogenicity of the Ochrobactrum spp. to the insects Galleria mellonella and Spodoptera littoralis (Lepidoptera) was detected. Since Ochrobactrum spp. are considered as human opportunist pathogens, the mass production of entomopathogenic nematodes for biological control requires strict vigilance. (+info)
Molecular evolution of the wingless gene and its implications for the phylogenetic placement of the butterfly family Riodinidae (Lepidoptera: papilionoidea).
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The sequence evolution of the nuclear gene wingless was investigated among 34 representatives of three lepidopteran families (Riodinidae, Lycaenidae, and Nymphalidae) and four outgroups, and its utility for inferring phylogenetic relationships among these taxa was assessed. Parsimony analysis yielded a well-resolved topology supporting the monophyly of the Riodinidae and Lycaenidae, respectively, and indicating that these two groups are sister lineages, with strong nodal support based on bootstrap and decay indices. Although wingless provides robust support for relationships within and between the riodinids and the lycaenids, it is less informative about nymphalid relationships. Wingless does not consistently recover nymphalid monophyly or traditional subfamilial relationships within the nymphalids, and nodal support for all but the most recent branches in this family is low. Much of the phylogenetic information in this data set is derived from first- and second-position substitutions. However, third positions, despite showing uncorrected pairwise divergences up to 78%, also contain consistent signal at deep nodes within the family Riodinidae and at the node defining the sister relationship between the riodinids and lycaenids. Several hypotheses about how third-position signal has been retained in deep nodes are discussed. These include among-site rate variation, identified as a significant factor by maximum likelihood analyses, and nucleotide bias, a prominent feature of third positions in this data set. Understanding the mechanisms which underlie third-position signal is a first step in applying appropriate models to accommodate the specific evolutionary processes involved in each lineage. (+info)
Detection and distribution patterns of telomerase activity in insects.
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Telomeres of most insects consist of pentanucleotide (TTAGG)n repeats, although the repeats are absent in Diptera and some other insect species, where the telomere regions are perhaps maintained without telomerase. To understand various and unusual telomere formation in insects, we have studied the characteristic features of a putative insect telomerase that has not been previously described. Using a modified telomeric repeat amplification protocol (TRAP), we first detected the telomerase activity in crickets, cockroaches and two Lepidopteran insects. The telomerase from crickets and cockroaches required dATP, dGTP and dTTP but not dCTP as a substrate and sequence analyses of the products of TRAP revealed that the (TTAGG)n repeats are synthesized by telomerase. The cockroach telomerase was detected both in somatic (fat body, muscle and neural tissues) and germ line (testis) cells, suggesting that expression of this enzyme is not regulated in a tissue-specific manner at an adult stage. While we detected high levels of telomerase activity in crickets and cockroaches, we could not detect activity in all tissues and cell cultures of the silkworm, Bombyx mori and in two Drosophila and one Sarcophaga cell lines. This supports the theory that Dipteran insects maintain their telomeres without telomerase. (+info)
Immunological detection of serpin in the fall webworm, Hyphantria cunea and its inhibitory activity on the prophenoloxidase system.
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We previously identified a serine type protease inhibitor (serpin) cDNA, using PCR-based differential display, in the fall webworm which was up-regulated following a bacterial challenge (Shin et al., 1998). The serpin cDNA was inserted into an expression vector and the serpin protein was expressed in Escherichia coli. In order to investigate the action of serpin in vivo, we examined the concentration of serpin protein in the larvae of Hyphantria cunea by Western blot analysis using a polyclonal antibody raised in a rabbit injected with recombinant serpin. H. cunea serpin was found mainly in the plasma with a molecular mass of 56.6 kDa on SDS-PAGE followed by Western blot analysis. The concentration of serpin in the plasma was slightly increased following bacterial challenge. A new 50.5 kDa (approx.) band was detected post E. coli and distilled water injection. Both E. coli and distilled water injection induced increased phenoloxidase (PO) activity in the plasma, although E. coli injection produced a larger increase in activity. Hyphantria serpin probably participates in negative regulation of the prophenoloxidase (proPO) cascade. Recombinant serpin inhibits PO activity in the hemocyte lysate fraction activated by LPS. There is a similarity between the P2-P2' region (NKFG) of the serpin reactive site loop and the S2-S2' region (NRFG) of the insect proPO maturation site. This indicates a form of competitive inhibition of serpin against a protease involved in the activation of proPO. A tyrosine residue in the P11 region of serpin, which is conserved in the S11 regions of all known proPOs maturation sites, provides further support for this hypothesis. (+info)
Filamentous actin is required for lepidopteran nucleopolyhedrovirus progeny production.
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Autographa californica M nucleopolyhedrovirus (AcMNPV) is the prototypical member of the NUCLEOPOLYHEDROSIS: genus of the BACULOVIRIDAE:, a family of large, double-stranded DNA viruses that are highly diverse. Nucleocapsid morphogenesis of AcMNPV and others in the NUCLEOPOLYHEDROVIRUS: genus takes place within the nuclei of infected host cells. Previously, we showed that filamentous actin (F-actin) is essential for this process to occur in AcMNPV-infected cells, an unprecedented finding for a DNA virus that replicates within the nucleus. Because of the fundamental importance of this requirement to our understanding of virus-host interactions, and because of the diversity of viruses included within the Nucleopolyhedrovirus genus, we were compelled to determine whether the replication of other nucleopolyhedroviruses was also F-actin dependent. We report here that progeny virus production of six other lepidopteran nucleopolyhedroviruses, representing both phylogenetic groups I and II within the genus, is also F-actin dependent. The six viruses studied (Spodoptera frugiperda MNPV, Bombyx mori NPV, Orgyia pseudotsugata MNPV, Lymantria dispar MNPV, Anticarsia gemmatalis MNPV and Helicoverpa zea SNPV) were unable to produce progeny in the presence of either cytochalasin D or latrunculin A, two actin-binding agents that interfere with F-actin-dependent processes but differ in their modes of action. F-actin-dependent progeny morphogenesis, therefore, appears to be a characteristic common among viruses in this genus that have lepidopteran hosts. (+info)
Biocontrol of the sugarcane borer Eldana saccharina by expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA genes in sugarcane-associated bacteria.
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The cry1Ac7 gene of Bacillus thuringiensis strain 234, showing activity against the sugarcane borer Eldana saccharina, was cloned under the control of the tac promoter. The fusion was introduced into the broad-host-range plasmid pKT240 and the integration vector pJFF350 and without the tac promoter into the broad-host-range plasmids pML122 and pKmM0. These plasmids were introduced into a Pseudomonas fluorescens strain isolated from the phylloplane of sugarcane and the endophytic bacterium Herbaspirillum seropedicae found in sugarcane. The ptac-cry1Ac7 construct was introduced into the chromosome of P. fluorescens using the integration vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated cry1Ac7 gene were much higher under the control of the tac promoter than under the control of its endogenous promoter. It was also determined that multicopy expression in P. fluorescens and H. seropedicae of ptac-cry1Ac7 carried on pKT240 caused plasmid instability with no detectable protein expression. In H. seropedicae, more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nm(r) promoter on pML122 than in the opposite orientation and bioassays showed that the former resulted in higher mortality of E. saccharina larvae than the latter. P. fluorescens 14::ptac-tox resulted in higher mortality of larvae than did P. fluorescens 14::tox. An increased toxic effect was observed when P. fluorescens 14::ptac-tox was combined with P. fluorescens carrying the Serratia marcescens chitinase gene chiA, under the control of the tac promoter, integrated into the chromosome. (+info)
A unique 33-kD cysteine proteinase accumulates in response to larval feeding in maize genotypes resistant to fall armyworm and other Lepidoptera.
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Plants respond to insect feeding with a number of defense mechanisms. Using maize genotypes derived from Antiquan germ plasm that are resistant to Lepidoptera, we have demonstrated that a unique 33-kD cysteine proteinase accumulates in the whorl in response to larval feeding. The abundance of the proteinase increased dramatically at the site of larval feeding after 1 hr of infestation and continued to accumulate for as long as 7 days. The 33-kD cysteine proteinase was most abundant in the yellow-green portion of the whorl-the normal site of larval feeding and the tissue that has the greatest inhibitory effect on larval growth in bioassays. The proteinase was expressed in response to wounding and was found in senescent leaves. It may be a marker of programmed cell death. The gene coding for the proteinase, mir1, has been transformed into Black Mexican Sweet callus. When larvae were reared on callus expressing the proteinase, their growth was inhibited approximately 60 to 80%. The expression of a cysteine proteinase, instead of a cysteine proteinase inhibitor, may be a novel insect defense mechanism in plants. (+info)
Cross-resistance of pink bollworm (Pectinophora gossypiella) to Bacillus thuringiensis toxins.
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Two strains of pink bollworm (Pectinophora gossypiella) selected in the laboratory for resistance to Bacillus thuringiensis toxin Cry1Ac had substantial cross-resistance to Cry1Aa and Cry1Ab but not to Cry1Bb, Cry1Ca, Cry1Da, Cry1Ea, Cry1Ja, Cry2Aa, Cry9Ca, H04, or H205. The narrow spectrum of resistance and the cross-resistance to activated toxin Cry1Ab suggest that reduced binding of toxin to midgut target sites could be an important mechanism of resistance. (+info)