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PubMed2005

The use of a recombinant baculovirus expressing a chitinase from the hard tick Haemaphysalis longicornis and its potential application as a bioacaricide for tick control.

Baculoviruses are specific insect pathogens used as selective biological insecticides on lepidopteran insects. We have tested a recombinant baculovirus expressing a chitinase gene for its efficacy as a tick bioacaricide. The recombinant Autographa californica multiple nuclear polyhedrosis virus expressing a chitinase enzyme (AcMNPV-CHT1) from the hard tick, Haemaphysalis longicornis, was constructed and found to have a novel bioacaricidal effect against ticks. The recombinant baculovirus was used to express the chitinase enzyme in Spodoptera frugiperda (Sf9) insect cells. Topical application of the supernatant harvested from the insect cell culture was found to cause mortality in nymphal ticks of H. longicornis. High temperature (>30 degrees C) and infrared radiation affected the chitinase enzyme activity and recombinant baculovirus infectivity by reducing the speed of tick killing by 60%. A mixture of recombinant virus and chitinase was found to kill ticks faster (p < 0.01) than pure chitinase and recombinant virus alone. Thus, the recombinant virus showed a synergistic effect with the foreign chitinase gene. In order to reduce the excessive use and cost of acaricides, it was found that a mixture of recombinant virus and flumethrin could halve the dose of the chemical acaricide used. These findings are important for the safe use of the recombinant virus expressing chitinase as a bioacaricide against ticks.

Animals↗
PubMed2002

Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by confocal laser scanning microscopy.

A comparative study of the co-localisation of three different families of neuropeptides, viz. allatostatins of the Y/FXFGL-NH(2) type, Manduca sexta allatostatin (Mas-AS) and allatotropin, in the frontal ganglion of lepidopteran larvae has been carried out by means of immunocytochemistry and confocal laser scanning microscopy. The simultaneous application of three types of fluorochrome-conjugated antibodies reveals triple co-localisation in an anterodorsal pair of neurones in the frontal ganglion of the noctuids Heliothis virescens and Lacanobia oleracea. There is no evidence of differential axonal transport, since all parts of these neurones show complete co-localisation of all three peptides. Prominent axons of the ganglionic neurones project in the recurrent nerve to the foregut and stomodeal valve. Over the crop, lateral and sub-lateral branches follow the course of circular muscle fibres and terminate in varicosities. All three neuropeptides have previously been shown to be myoregulatory on the foregut; the Y/FXFGL-NH(2) allatostatins and Mas-AS are inhibitory, whereas allatotropin is excitatory. The morphological evidence of co-localisation of physiologically antagonistic peptides within the same terminals suggests that an extremely complex mechanism controls the contractile activities of the foregut. A posterodorsal pair of neurones in the frontal ganglion have prominent axons projecting via the frontal connectives to the brain and in the recurrent nerve to the stomodeal valve where extensive branching suggests control over the valve movements. Studies of another noctuid, Spodoptera frugiperda, and the sphingid, M. sexta, show interesting variations in the co-localisation phenomenon.

Animals↗
PubMed2004

Effects of elevated atmospheric CO2 on the nutritional ecology of C3 and C4 grass-feeding caterpillars.

It is plausible that the nutritional quality of C3 plants will decline more under elevated atmospheric CO2 than will the nutritional quality of C4 plants, causing herbivorous insects to increase their feeding on C3 plants relative to C4 plants. We tested this hypothesis with a C3 and C4 grass and two caterpillar species with different diet breadths. Lolium multiflorum (C3) and Bouteloua curtipendula (C4) were grown in outdoor open top chambers at ambient (370 ppm) or elevated (740 ppm) CO2. Bioassays compared the performance and digestive efficiencies of Pseudaletia unipuncta (a grass-specialist noctuid) and Spodoptera frugiperda (a generalist noctuid). As expected, the nutritional quality of L. multiflorum changed to a greater extent than did that of B. curtipendula when grown in elevated CO2; levels of protein (considered growth limiting) declined in the C3 grass, while levels of carbohydrates (sugar, starch and fructan) increased. However, neither insect species increased its feeding rate on the C3 grass to compensate for its lower nutritional quality when grown in an elevated CO2 atmosphere. Consumption rates of P. unipuncta and S. frugiperda were higher on the C3 grass than the C4 grass, the opposite of the result expected for a compensatory response to the lower nutritional quality of the C4 grass. Although our results do not support the hypothesis that grass-specialist insects compensate for lower nutritional quality by increasing their consumption rates more than do generalist insects, the performance of the specialist was greater than that of the generalist on each grass species and at both CO2 levels. Mechanisms other than compensatory feeding, such as increased nutrient assimilation efficiency, appear to determine the relative performance of these herbivores. Our results also provide further evidence against the hypothesis that C4 grasses would be avoided by insect herbivores because a large fraction of their nutrients is unavailable to herbivores. Instead, our results are consistent with the hypothesis that C4 grasses are poorer host plants primarily because of their lower nutrient levels, higher fiber levels, and greater toughness.

Air↗
PubMed2004

Plant chemical defense against herbivores and pathogens: generalized defense or trade-offs?

Plants are often attacked by multiple enemies, including pathogens and herbivores. While many plant secondary metabolites show specific effects toward either pathogens or herbivores, some can affect the performance of both these groups of natural enemies and are considered to be "generalized defense compounds". We tested whether aucubin and catalpol, two iridoid glycosides present in ribwort plantain (Plantago lanceolata), confer in vivo resistance to both the generalist insect herbivore Spodoptera exigua and the biotrophic fungal pathogen Diaporthe adunca using plants from P. lanceolata lines that had been selected for high- and low-leaf iridoid glycoside concentrations for four generations. The lines differed approximately three-fold in the levels of these compounds. Plants from the high-selection line showed enhanced resistance to both S. exigua and D. adunca, as evidenced by a smaller lesion size and a lower fungal growth rate and spore production, and a lower larval growth rate and herbivory under both choice and no-choice conditions. Gravimetric analysis revealed that the iridoid glycosides acted as feeding deterrents to S. exigua, thereby reducing its food intake rate, rather than having post-ingestive toxic effects as predicted from in vitro effects of hydrolysis products. We suggest that the bitter taste of iridoid glycosides deters feeding by S. exigua, whereas the hydrolysis products formed after tissue damage following fungal infection mediate pathogen resistance. We conclude that iridoid glycosides in P. lanceolata can serve as broad-spectrum defenses and that selection for pathogen resistance could potentially result in increased resistance to generalist insect herbivores and vice versa, resulting in diffuse rather than pairwise coevolution.

Adaptation, Physiological↗
PubMed2005

Induced plant responses to multiple damagers: differential effects on an herbivore and its parasitoid.

Herbivore-induced plants responses can affect the preference and performance of herbivores and their natural enemies. These responses may vary depending on the identity and number of herbivore species feeding on the plant so that when herbivores from different guilds feed on plants, the interactions between plants, herbivores, and natural enemies may be disrupted. Tomato plants were damaged either by the caterpillar Spodoptera exigua, or the aphid Macrosiphum euphorbiae, or damaged by both herbivores, or undamaged controls. We measured the preference and performance of S. exigua and its parasitoid Cotesia marginiventris, and activity of proteinase inhibitors (PI) as an indicator of induced resistance. Compared to undamaged plants, caterpillar damage reduced the number of eggs laid by S. exigua adults, reduced growth, consumption, and survival of larval S. exigua and C. marginiventris, and increased activity of PIs 43%; but did not increase attraction of C. marginiventris. While pupal mass of S. exigua was not affected, the pupal mass of C. marginiventris decreased on caterpillar-damaged plants compared to controls. In contrast, plants damaged by aphids were preferred for oviposition by S. exigua, and had increased larval consumption and survival, compared to controls. Aphid feeding did not affect the preference or performance of C. marginiventris, or PI activity, compared to controls. While oviposition was deterred on caterpillar-damaged plants, plants damaged by both herbivores received the same amount of oviposition as controls. The attraction of C. marginiventris to plants damaged by caterpillars and aphids was increased compared to controls. However, plants damaged by both herbivores had similar PI activity, larval growth and survival of S. exigua and C. marginiventris, as plants singly damaged by caterpillars. Overall, the preference component for both the herbivore and parasitoid was more strongly affected by damage due to multiple herbivores than the performance component.

Analysis of Variance↗
PubMed2005

Within-plant variation in induced defence in developing leaves of cotton plants.

According to optimal defence theory (ODT) plants should invest in stronger defence in the most valuable parts, such as reproductive or young tissue. Cotton plants are known to express high resistance to herbivore feeding in the developing leaves at the top of the plant after herbivore-damage. Cotton plants also have developing leaves on side shoots from the nodes all along the plant. This allowed us to investigate within-plant distribution of defence between younger tissues at different locations on the plant. We found that all developing leaves showed increased resistance to feeding by larvae of the generalist moth Spodoptera littoralis after herbivore damage to leaves of the plant. An increase in the concentration of secondary compounds, terpenoid aldehydes, was found in developing leaves both at the top of the plant and on the side shoots. However, the resistance was stronger in the top leaves than in the side shoot leaves, showing that there is intra-plant variation in the induced response between different leaves of the same age. After the initial damage, larval feeding mainly occurred on the older, fully developed true leaves. Furthermore, the herbivore-induced resistance in the developing leaves reduced upward movement of feeding larvae on the plant and reduced the feeding on the upper parts of the plant over a period of at least 6 days. The plant thus benefits from defending all developing leaves by directing feeding to older, less valuable leaves and lower parts of the plant. The observed distribution of defence within cotton plants supports ODT.

Age Factors↗
PubMed2005

Is palatability of a root-hemiparasitic plant influenced by its host species?

Palatability of parasitic plants may be influenced by their host species, because the parasites take up nutrients and secondary compounds from the hosts. If parasitic plants acquired the full spectrum of secondary compounds from their host, one would expect a correlation between host and parasite palatability. We examined the palatability of leaves of the root-hemiparasite Melampyrum arvense grown with different host plants and the palatability of these host plants for two generalist herbivores, the caterpillar of Spodoptera littoralis and the slug Arion lusitanicus. We used 19 species of host plants from 11 families that are known to contain a wide spectrum of anti-herbivore compounds. Growth of M. arvense was strongly influenced by the host species. The palatability of the individual host species for the two herbivores differed strongly. Both A. lusitanicus and S. littoralis discriminated also between hemiparasites grown with different host plants. There was no correlation between the palatability of a host species and that of the parasites grown on that host, i.e., hemiparasites grown on palatable host species were not more palatable than those grown on unpalatable hosts. We suggest an interacting pattern of specific effects of chemical anti-herbivore defences and indirect effects of the hosts on herbivores through effects on growth and tissue quality of the parasites.

Animals↗
PubMed2005

Members only: induced systemic resistance to herbivory in a clonal plant network.

The stoloniferous herb Trifolium repens was used to study the expression of induced systemic resistance (ISR) to the generalist caterpillar Spodoptera exigua in interconnected ramets of clonal fragments. The ISR was assessed as caterpillar preference in dual choice tests between control and systemically induced plants. The ISR was detected in young ramets, after inducing older sibling ramets on the same stolon by a controlled herbivore attack. However, older ramets did not receive a defense induction signal from younger ramets unless the predominant phloem flow was reversed by means of basal shading. This provides evidence for the notion that in T. repens the clone-internal expression of ISR is coupled to phloem transport and follows source-sink gradients. The inducibility of the genotypes was not linked to their constitutive ability to produce cyanide, implying the absence of a trade-off between these two defense traits. To our knowledge, this is the first study that explores ISR to herbivory in the context of physiological integration in potentially extensive clonal plant networks.

Animals↗
PubMed2003

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Animals↗
PubMed2003

Rice stripe virus 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells.

The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9 K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9 K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9 K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9 K protein antiserum. Moreover, electron-dense masses labeled with 23.9 K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9 K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.

Animals↗
PubMed2006

Replication of Heliothis virescens ascovirus in insect cell lines.

Ascoviruses (AVs) infect larvae of various insect pests belonging to the family Noctuidae. The result of AV infection in the hosts is cleavage of infected cells into vesicles, a unique feature of AV infection. Since insect cell lines facilitate the study of virus life cycles, attempts were made to analyze Heliothis virescens AV (HvAV3e) infection in several cell lines and compare cell pathology to larval infection. In this study, replication and cytopathological effects of HvAV3e on four different cell lines were investigated. HvAV3e replication was confirmed in three noctuid cell lines from Spodoptera frugiperda (Sf9) and Helicoverpa zea (BCIRL-Hz-AM1 and FB33). However, the virus did not replicate in the non-noctuid insect cell line from Pieris rapae (Pieridae). Despite replication of the virus in the three permissive cell lines, the cytopathological effects of the virus were significantly different from that of larval infection.

Animals↗
PubMed1998

Expression and cellular distribution of baculovirus-expressed bovine herpesvirus 1 (BHV-1) glycoprotein D (gD) sequences.

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus gD, represents a major component of the viral envelope and is a dominant immunogen. To study the antigenic properties of the different regions of gD, we have expressed the full-length gD encoding gene and overlapping fragments spanning various regions of the gD open reading frame in a baculovirus (Autographa californica nuclear polyhedrosis virus)--insect cell (Spodoptera frugiperda, SF-9) system. Maximum levels of expression for all proteins were obtained 48 to 72 h post infection of SF-9 cells by recombinant viruses. Full-length and truncated recombinant gD proteins reacted specifically with anti-gD monospecific serum as determined by immunoprecipitation and immunoblotting, indicating that the proteins retained their antigenicity. However, based on the reactivity with a panel of gD-specific monoclonal antibodies (Mabs), the full-length recombinant gD lacked proper expression for two highly neutralizing linear epitopes identified by Mabs R54 and 9D6. The rest of the epitopes appeared to be preserved and antigenically unaltered. Immunofluorescence studies of recombinant baculovirus infected SF-9 cells using gD monospecific serum, revealed no direct correlation between cellular localization of the expressed proteins and their amino acid sequences.

Animals↗
PubMed1999

In vivo and in vitro phosphorylation of rice dwarf phytoreovirus Pns12 cytoplasmic nonstructural protein.

In vivo and in vitro phosphorylation and intracellular location of rice dwarf phytoreovirus Pns12, which is encoded by one of the twelve dsRNA genome segments, S12, and comprises 312 amino acids, was investigated. When [32P]phosphoric acid was incorporated into RDV-infected leafhopper cultured cells, labelled Pns12 was immunoprecipitated from the cells by a monospecific anti-Pns12 polyclonal antibody. Recombinant Pns12 was purified from Spodoptera frugiperda cells infected with AcRS12, a baculovirus recombinant carrying a full-length cDNA of RDV S12. Purified Pns12 was also demonstrated to be phosphorylated in vitro by a kinase activity present in host (rice, barley, wheat, leafhopper) cells and non-host (tobacco, spinach, white clover, S. frugiperda, mosquito, mammals) cells as well. Immunocytochemical studies showed Pns12 accumulated in the cytoplasm of infected cells, and frequently localized in a slightly electron-dense patch. These results demonstrated that RDV Pns12 was a cytoplasmic nonstructural phosphoprotein.

Animals↗
PubMed1999

Physical maps and virulence of Anticarsia gemmatalis nucleopolyhedrovirus genomic variants.

Seventeen plaque purified isolates of two viral preparations of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), were analyzed in terms of the genomic changes after digestion of their DNAs with HindIII and PstI restriction enzymes. The 1979 AgMNPV wild type preparation (AgMNPV-'79) resulted in six different variants and the 1985 viral commercial preparation (AgMNPV-'85), in eleven. The genomic variation of all the isolates was mapped showing that those from 1985 presented more heterogeneity with changes mapped in additional sites in comparison to the AgMNPV-'79 variants. Their virulence was compared by infecting two Lepidopteran cell lines, Spodoptera frugiperda (IPLB-SF-21AE) and Anticarsia gemmatalis (UFL-AG-286). The results indicated that there was some difference in virulence within the AgMNPV-'85 variants. This commercial preparation had been applied in soybean fields in Brazil over several years to control the velvetbean caterpillar defoliation.

Animals↗
PubMed2006

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells.

The advantage of using 13C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that 13C-[2] acetate or 13C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using 13C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/13C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.

Acetates↗
PubMed2004

Maize genes induced by herbivory and volicitin.

In crop plants, both mechanical damage and insect attack trigger rapid changes in gene transcription. We investigated whether insect herbivory differs from a general wound response, and if so, is the induction specific to the pest/host plant interaction? Herbivory by beet armyworm (BAW; Spodoptera exigua) caterpillars on maize results in a unique pattern of volatile compounds not triggered by wounding alone that attracts the generalist parasitoid Cotesia marginiventris. Caterpillar-induced volatile emission can be mimicked when a component of the BAW oral secretions (N-(17-hydroxylinolenoyl)-L-glutamine) termed volicitin, is applied to wounded leaves. We identified genes that are affected by BAW feeding by comparing volicitin treatment with wounding alone. We compared cDNAs from these two populations by isolating genes from a subtractive library and using reverse northerns. Virtual northern blots confirmed these results and further showed that BAW infestation affected the expression of these genes. In some cases, BAW feeding inhibited the expression of volicitin-induced genes, suggesting the role of additional bioactive components in caterpillar regurgitate. Transcripts involved in volatile production are increased by volicitin and BAW infestation treatments, and are also detectable at low levels in mechanically wounded leaves. Finally, we identified three new sesquiterpene cyclase genes that are induced by volicitin.

Animals↗
PubMed2006

Fungal infection reduces herbivore-induced plant volatiles of maize but does not affect naïve parasitoids.

Plants attacked by insects release volatile compounds that attract the herbivores' natural enemies. This so-called indirect defense is plastic and may be affected by an array of biotic and abiotic factors. We investigated the effect of fungal infection as a biotic stress agent on the emission of herbivore-induced volatiles and the possible consequences for the attraction of two parasitoid species. Maize seedlings that were simultaneously attacked by the fungus Setosphaeria turcica and larvae of Spodoptera littoralis emitted a blend of volatiles that was qualitatively similar to the blend emitted by maize that was damaged by only the herbivore, but there was a clear quantitative difference. When simultaneously challenged by fungus and herbivore, the maize plants emitted in total 47% less of the volatiles. Emissions of green leaf volatiles were unaffected. In a six-arm olfactometer, the parasitoids Cotesia marginiventris and Microplitis rufiventris responded equally well to odors of herbivore-damaged and fungus- and herbivore-damaged maize plants. Healthy and fungus-infected plants were not attractive. An additional experiment showed that the performance of S. littoralis caterpillars was not affected by the presence of the pathogen, nor was there an effect on larvae of M. rufiventris developing inside the caterpillars. Our results confirm previous indications that naïve wasps may respond primarily to the green leaf volatiles.

Animals↗
PubMed2007

Scopolamine in Brugmansia suaveolens (Solanaceae): defense, allocation, costs, and induced response.

Brugmansia suaveolens (Solanaceae) contains tropane alkaloids (TAs), which can act as chemical defenses. Selective pressures might modulate the allocation of alkaloids within the plant, as postulated by optimal-defense theory. By tracing scopolamine, the most abundant TA in this species, we found that scopolamine in an artificial diet, in concentrations similar to those in leaves of B. suaveolens, increased mortality and prolonged developmental time of the larvae of the generalist noctuid moth Spodoptera frugiperda. A diet of undamaged leaves of B. suaveolens also showed a large negative effect on the growth of larvae of S. frugiperda compared to a diet of leaves of Ricinus communis, a species that did not have negative effects on this moth; more valuable plant parts, such as young leaves, flowers, and unripe fruits with seeds, have higher scopolamine concentrations than other tissues; leaves of B. suaveolens increase their content of scopolamine after artificial damage. The highest induction was found 24 hr after the damage, and after that, scopolamine content decreased to constitutive levels. This increase represented a cost, because in another experiment, a treatment with methyl jasmonate, an elicitor hormone, increased scopolamine production 9.5-fold and decreased leaf growth 2.3-fold; a diet of artificially damaged leaves of B. suaveolens showed a negative effect on the growth of larvae of S. furgiperda compared to undamaged leaves, suggesting that damage by herbivores induces resistance. Our data are in line with the optimal-defense theory, but experiments in the field with herbivores that share an evolutionary history with B. suaveolens must be undertaken to understand the dynamics of TA allocation in response to herbivory.

Acetates↗