Photostabilized titanium dioxide and a fluorescent brightener as adjuvants for a nucleopolyhedrovirus

Titanium dioxide (TiO₂)reflects ultraviolet light, and so could beexpected to protect the occlusion bodies (OBs)of nucleopolyhedroviruses (NPVs) fromdegradation by sunlight. However, in thepresence of sunlight and water, TiO₂catalyzes the formation of hydrogen peroxide,which can degrade OBs. We tested microfineTiO₂ that had been photostabilized(particles were coated to prevent catalyticactivity), as a UV protectant for the OBs ofthe NPV of Helicoverpa zea (Boddie). Inthe absence of UV, activity of the OBs wasreduced by nonphotostabilized TiO₂ but wasunaffected by photostabilized TiO₂ or byzinc oxide (ZnO). None of these materialsinfluenced larval feeding rates. Undersimulated sunlight, photostabilizedTiO₂ protected the OBs to a greater degreethan did ZnO. Photostabilized TiO₂ wascompatible with a viral enhancer, thefluorescent brightener Blankophor HRS. Undersimulated sunlight, both materials increasedactivity of the OBs, relative to OBs withneither material, in a largely additive manner. In bioassays of foliage collected from fieldplots of lima bean plants sprayed with OBs withor without one or both of these materials,TiO₂ increased persistence of the OBs, butBlankophor HRS had no significant effect.     

Viral-enhancing activity of an optical brightener for Spodoptera littoralis (Lepidoptera: Noctuidae) nucleopolyhedrovirus

The effect of an optical brightener on the insecticidal activity of a Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) nucleopolyhedrovirus (SpliNPV) were examined in three instars of S. littoralis. LD₅₀ values of the SpliNPV were reduced from 33 to <5, 73 to 5.7 and 342 321 to 288 412 occlusion bodies for second, third and fourth instars, respectively, by the addition of 1% Tinopal UNPA-GX. Relative potencies were >66, 12.8, and 1.2 for second-. third- and fourth-instar S. littoralis larvae, respectively. Relative mortality between the treatments with and without the brightener decreased from third- to fourth-stage larvae. In terms of speed of kill, the ST₅₀ values of the baculovirus-infected larva were reduced from 210 to 159, 213 to 147, and 207 to 165 h for second-, third- and fourth-instar larvae, respectively, by the addition of the optical brightener at biologically equivalent doses.     

Optical brighteners as ultraviolet protectants and as enhancers in pathogenicity of Spodoptera litura (Fabricius) (Lep., Noctuidae) nucleopolyhedrovirus

Seven optical brighteners were tested using diet disc bioassays in the laboratory to determine their effects on the protection and activity of Spodoptera litura (Fabricius) (Lep., Noctuidae) nucleopolyhedrovirus ( Sl MNPV) against 4th instar S. litura larvae. All brighteners tested were found to be effective UV protectants and five brighteners (Blankophor BBH, Blankophor RKH, Blankophor P167, Blankophor HRS and Tinopal LPW) enhanced viral activity by 5.53–11.08 times. Viral activity was increased with the concentration of brighteners. The time required for the virus to kill larvae was reduced by 24–27% at 1% concentration of brighteners. Some infectivity of the virus was found in the older larvae, which were resistant to Sl MNPV, by adding the brighteners. On the basis of data obtained in the present study we discuss the synergistic action of optical brighteners with Sl MNPV.     

Green tea extracts as ultraviolet protectants for the beet armyworm,Spodoptera exigua,nucleopolyhedrovirus

The addition of a caffeinated green tea, Camellia sinensis L., filtrate (1%) to the nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner), provided almost complete protection following UVB irradiation (30 min) in laboratory tests. There were few differences in UV protection when extracts were prepared at 27 or at 90°C. Moreover, few differences in UV protection were demonstrated following infusion times of 5, 15, 30, and 60 min at 90°C. At a 1% concentration, decaffeinated and caffeinated green teas were equally effective as UV protectants. At lower concentrations (0.1, 0.01, and 0.001%) caffeinated green tea provided greater UV protection (UVB/UVB 30, 60 min). Virus/tea extracts (caffeinated), under field conditions at 1 and 5%, were ineffective as UV screens. At a 10% concentration, some UV protection was provided and UV protection further increased in a concentration-dependent manner.     

Formulation with an optical brightener does not increase probability of developing resistance to Spodoptera frugiperda nucleopolyhedrovirus in the laboratory

Stilbene-derived optical brighteners can markedly enhance the insecticidal activity of certain baculoviruses. We evaluated the influence of an optical brightener on the rate at which Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) developed resistance to nucleopolyhedrovirus (SfMNPV). Two laboratory colonies of S. frugiperda were inoculated with an LC50 of SfMNPV, in the absence or presence of the optical brightener Tinopal LPW (0.1%), over a period of two and 11 generations, in the first and second experiment, respectively. Compared with the initial susceptibility of the insect colony, resistance ratios of 11- and 12-fold were observed after two generations of treatment with SfMNPV + Tinopal LPW and SfMNPV alone. Similar, but variable degrees of resistance were observed in the long-term experiment with resistance ratios of 8- to 35-fold after seven to 11 generations. The presence of Tinopal LPW alone, or in mixtures with SfMNPV, did not cause any systematic change in insect resistance in either experiment. At the end of the long-term experiment, debilitating effects on pupal weight, adult fecundity, and longevity were observed in the insects exposed to Tinopal LPW alone or in mixtures with SfMNPV, but the pattern of such effects among treatments differed in each generation. We conclude that optical brighteners are unlikely to affect the rate of development of resistance to nucleopolyhedroviruses applied as biological insecticides.     

Tinopal AN as a selective agent for the differentiation of phytopathogenic and saprophytic Pseudomonas species

The selective toxicity of the respiratory inhibitor Tinopal AN (1,1-bis (3, N -5–dimethylbenzoxazol-2-yl) methine p -toluene sulphonate) towards phytopathogenic bacteria was investigated further and in general was confirmed using more than 160 additional strains of Pseudomonas spp. The mechanism(s) of the resistance shown by saprophytic fluorescent pseudomonads were studied to elucidate the differences between resistant saprophytic and sensitive phytopathogenic Pseudomonas species. Damage to, or partial removal of the cell wall of Tinopal AN-resistant Pseudomonas aeruginosa , resulted in a marked Tinopal AN-sensitivity, as judged by the ability of Tinopal AN to inhibit oxygen uptake. Furthermore, removal of part of the lipo-polysaccharide (LPS) component of the outer membrane also resulted in sensitivity. Mutants of Ps. aeruginosa with modified outer cell walls were tested for their reactions towards Tinopal AN, and two cell wall lipopolysaccharide (LPS) mutants of Escherichia coli (env Al) and Salmonella typhimurium (rfa) were, in contrast to the wild-type strains, found to be sensitive towards Tinopal AN. The results therefore suggest that the resistance of saprophytic pseudomonads towards Tinopal AN is due (at least in part) to the selectively permeable properties of the outer membrane of the cell wall. The usefulness of Tinopal AN for screening potentially phytopathogenic strains of Pseudomonas was confirmed.     

Evidence for a role for L-proline as a salinity protectant in the cyanobacterium Nostoc muscorum

Nostoc muscorum required an active proline oxidase in order to assimilate exogenous proline as a source of fixed nitrogen. A mutant strain (Ac^r) resistant to growth inhibition by L-azetidine-2-carboxylate (AC) was found to be deficient in proline oxidase activity, and to be a proline overaccumulator. Proline overaccumulation, resulting either from mutational acquisition of the Ac^r phenotype or from salinity-inducible uptake of exogenous proline, conferred enhanced salinity tolerance in this cyanobacterium.     

Enhancement of nucleopolyhedrovirus infectivity against Mamestra brassicae (Lepidoptera: Noctuidae) by proteins derived from granulovirus and a fluorescent brightener

The synergistic enhancement of nucleopolyhedrovirus (NPV) infection by granuloviruses (GVs) is well documented; and a GV granule protein, named viral enhancin, has been identified as an active contributor to this effect. We detected the presence of two proteins with molecular mass of 93 and 108 kDa in granules of a GV isolated from Xestia c-nigrum (L.) (XecnGV) as candidates for enhancin, and we confirmed that at least the 108-kDa protein enhances the infectivity of Mamestra brassicae nucleopolyhedrovirus (MabrNPV). We tested the effect of virion-free proteins obtained from XecnGV granules (GVPs) on MabrNPV infection, and we made a comparison with an enhancing chemical, the stilbene-derived fluorescent brightener Tinopal. Bioassay was performed employing the diet contamination method, by using second instars of Mamestra brassicae (L.) (Lepidoptera: Noctuidae). The enhancing effects of GVPs (0.1 mg/g diet) and Tinopal (1 mg/g diet) were estimated to be 70.7-81.5-fold and 26.9-33.7-fold, respectively, as calculated from the LC50 values of MabrNPV with or without the additives. The additives reduced the lethal time of MabrNPV-infected larvae and they caused death at a younger instar. These results suggest that GVPs can enhance MabrNPV infection as effectively as Tinopal.     

Potential of Agrotis ipsilon nucleopolyhedrovirus for suppression of the black cutworm (Lepidoptera: Noctuidae) and effect of an optical brightener on virus efficacy.

Studies were performed in the laboratory, greenhouse and field to assess the potential of Agrotis ipsilon multicapsid nucleopolyhedrovirus (AgipMNPV) and a viral enhancing agent, M2R, for suppression of Agrotis ipsilon (Hufnagel). In laboratory droplet feeding bioassays, AgipMNPV was shown to be highly active against third-instar A. ipsilon. The optical brightener M2R significantly reduced LD50 estimates by approximately 160-fold, but had no direct effect on survival time estimates. In greenhouse trials, spray and bait formulations of AgipMNPV significantly reduced feeding damage to corn seedlings caused by third-instar A. ipsilon. In two sets of replicated field trials, bait formulations of AgipMNPV significantly reduced feeding damage to corn seedlings by third-instar A. ipsilon. However, there were no beneficial effects attributable to the inclusion of M2R in AgipMNPV formulations under greenhouse or field conditions. It seems likely that in an appropriately designed pest management program AgipMNPV could be used to suppress field populations of early and mid-instar A. ipsilon.     

Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide

The Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) has been registered and is commercially produced in China as a biopesticide to control the bollworm in cotton. However, the virus has a relatively slow speed of action. To improve its efficacy, recombinant HearNPVs were generated by deleting the ecdysteroid UDP-glucosyltransferase (egt) gene (HaCXW1 and HaLM2) or by inserting the insect-specific toxin gene AaIT in the egt locus (HaCXW2) of HearNPV using conventional recombination strategies in insect cell culture. The various recombinants remained genetically stable when cultured in HzAM1 insect cells. Bioassay data showed a significant reduction in the time required for all HearNPV recombinants to kill second instar H. armigera larvae. The LT(50) of the egt deletion recombinants HaCXW1 and HaLM2 was about 27% faster than that of wild-type HearNPV. The largest reduction in LT(50) was achieved by inserting the gene for the insect-specific neurotoxin, AaIT, in the egt locus, giving a reduction in LT(50) of 32% compared to wild-type HearNPV. The ability to genetically improve the properties of HearNPV as a biopesticide provides a further opportunity to develop this virus into a commercially viable product to control the bollworm in China.     

Use of a Stilbene Brightener,Tinopal LPW,as a Radiation Protectant for Steinernema carpocapsae

A stilbene fluorescent brightener, Tinopal LPW, was used as an ultraviolet (UV) protectant for the entomopathogenic nematode Steinernema carpocapsae (All strain). Irradiation of an aqueous suspension of nematodes produced a LC₅₀ in 15.7 minutes under a sunlamp and in 31.7 minutes in direct sunlight. Irradiation by both sunlamp and sunlight of a suspension of nematodes in Tinopal LPW did not reduce their biological activity as measured by their ability to parasitize wax moth larvae after exposure of 8 hours and 4 hours, respectively. Tinopal LPW appeared promising as a radiation protectant.     

Characteristics of protectant synthesis of infective juveniles of Steinernema carpocapsae and importance of glycerol as a protectant for survival of the nematodes during osmotic dehydration

Two hypotheses on the synthesis of the protectants glycerol and trehalose of the infective juveniles (IJs) of Steinernema carpocapsae during osmotic dehydration were tested and utilised to evaluate the function and importance of glycerol on survival of the nematodes during osmotic dehydration. This was achieved by comparing the changes in survival, morphology, behaviour and levels of glycerol, trehalose and permeated compounds of the IJs dehydrated in seven hypertonic solutions at two temperature regimes: (1) 5 °C for 15 days; and (2) 23 °C for 1 day followed by 5 °C for another 14 days. The results substantiate both hypotheses tested: (1) the permeability of the IJs to various compounds, such as sucrose or ethylene glycol, when they are dehydrated in hypertonic solutions of these compounds; and (2) suppression of the synthesis of protectant glycerol but not trehalose when IJs are dehydrated at low temperature. The results also showed that: (1) although trehalose was the preferred dehydration protectant, glycerol played an important role in rapidly balancing the osmotic pressure when IJs were exposed in hypertonic solutions; (2) the presence of glycerol was essential for the IJs to survive and function properly even under moderate osmotic dehydration, especially when IJs were dehydrated in salt solutions; and (3) some exogenous compounds permeated into IJs during osmotic dehydration such as ethylene glycol, may function in the same way as glycerol and significantly improve the survival and function of the IJs. The results indicate that each of the protectants glycerol and trehalose has a specific function and neither is replaceable by the other.     

In vivo production of recombinant protein by a baculovirus vector inoculated perorally to the prefinal instar larvae of Bombyx mori L. (Lep., Bombycidae) aided by an optical brightener, Tinopal UNPA-GX

Abstract: Budded particles of nucleopolyhedrovirus (NPV) were found to infect perorally the 4th (prefinal) instar larvae of Bombyx mori L. that were treated by an optical brightener, Tinopal UNPA-GX (Tinopal). Host larvae were fed a diet containing 0.3% (w/w) Tinopal on day 1 in the 4th instar and then fed a diet contaminated by budded particles of NPV (1.0 × 10⁶ TCID₅₀ U/larva) that was pathogenic to B . mori (BmNPV) on day 2 (inoculation schedule 1). Another set of host larvae was fed a diet containing BmNPV budded particles (2.5 × 10⁶ TCID₅₀ U/larva) together with 0.3% (w/w) Tinopal on day 1 in the 4th instar (inoculation schedule 2). Host larvae treated by both schedules died of viral infection. The operation of schedule 2 is simpler than that of schedule 1, although the former required higher doses of the virus for satisfactory infection. We inoculated a baculovirus vector carrying human serum albumin (HSA) gene into 4th instar B . mori larvae by schedule 1. Recombinant HSA was detected in the homogenate of host larvae 4 days after inoculation. The peroral inoculation of BmNPV budded particles aided by Tinopal may thus lead to industrial pharmaceutical production using a baculovirus vector for large numbers of insect hosts.     

The effect of mixtures of Bacillus thuringiensis-based insecticide and multiple nucleopolyhedrovirus of Lymantria dispar L. in combination with an optical brightener on L. dispar larvae

BioControl - This study evaluated the efficacy of the commercially available insecticide Lepidocide based on Bacillus thuringiensis var. kurstaki and Lymantria dispar multiple nucleopolyhedrovirus...     

Efficacy of some plants as a post-harvest protectant against some major stored pests

The toxicity of vapours of the essential oil of Ferula gummosa, Elettaria cardamomum and Salvia officinalis on the adults and larvae of some stored product pests was investigated. The bioassays were carried out in 70 mL vials containing 10 individuals of each insect. The LC₅₀ values of fumigant bioassay after 24 h were calculated. Results indicated that the effect of the essential oil of F.gummosa was stronger than E. cardamomum, S. officinalis on stored pests. Also fumigant toxicity of S. officinalis on Sitophilus oryzae was similar to that of Sitophilus granarius adults. On the other hand, R. officinalis had a good effect on adults of Tribolium castaneum and larvae of Ephestia kuehniella. According to our results and good effect of these compounds, they will be a safe replace for chemical compounds in the future.     

Postlarval chromatophores as an adaptation to ultraviolet radiation

It is now well established that ultraviolet radiation (UVR) may have detrimental, even lethal effects on zooplankters. Unlike copepods and other holoplankters, which may avoid UVR by undergoing diel vertical migration, larvae of many decapod crustaceans and fishes recruit to adult populations by remaining in near-surface waters during the daytime. Consequently, they are exposed to biologically damaging UVR. A possible adaptation in these larvae is chromatophores, which may absorb UVR by expanding in high light environments. The supposition is that expanded chromatophores more effectively absorb UVR, but there is some fitness cost to having expanded chromatophores in low light environments. Since the ratio of visible light to UVR in the water column changes as result of season, latitude, dissolved organic carbon, and a host of other factors, the benefits of chromatophores would be maximized if they responded specifically to UVR. The purpose of this study was to determine whether the chromatophores of crab postlarvae (megalopae) could expand in response to UVR. Megalopae of two species of crabs (Cancer oregonensis, Telmessus cheiragonus) were collected from large surface-swarms during mid-day as they recruited onshore in early May 1998 at Friday Harbor, Washington, USA. Dark-adapted megalopae (held in the dark for 8 h before experiments) were exposed to UVR (UVBR+UVAR, 280-400 nm), UVAR (320-400 nm), and light (400-1700 nm) in the laboratory. Chromatophores expanded after only minutes of exposure to UVR, UVAR, and light for both species. Two alternative hypotheses may explain why both harmful and comparatively benign wavelengths stimulated chromatophores to rapidly expand. First, larvae may not distinguish among different wavelengths, which, if true, would increase the vulnerability of these larvae to intensifying UVBR due to ozone depletion. Second, chromatophores have functions other than blocking UVR, such as crypsis and thermoregulation, and must respond to light for these other functions to operate.     

Does the mycotoxin citrinin function as a sun protectant in conidia from Penicillium verrucosum

Our results demonstrate high concentrations of the UV absorbing mycotoxin citrinin in the outer layer of spores from three citrinin-producing strains of Penicillium verrucosum, which is released in an aqueous environment. An important function of the toxin could be to act as a sun protectant in order to create favorable conditions during the initial germination process. When spores from these strains of P. verrucosum were examined by confocal microscopy, a clearly visible fluorescent layer associated with the cell wall was observed. The strains were grown on agar plates, and the mycelial mat was washed with saline. This suspension contained at least 95% of the spores and particulate material, which was removed by filtration after counting the conidia. An aliquot of this filtrate was extracted and citrinin was purified by high pressure liquid chromatography. The absorbance at 319 nm was used to calculate the amount of UV absorbing material released from the spores. Based on the spore numbers in the suspension of the saline extract, we estimated that this material released was 1.4–4.1 pg per spore or 8–24% of the spore weight. Citrinin (and minor amounts of ochratoxin A and some other unidentificable fluorescent compounds) were observed in the filtrate when subjected to thin layer chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optical tweezers as a probe for oligodeoxyribonucleotide structuration

The aim of this work is to investigate if the optical tweezers (OT) are suitable as a diagnostic tool for monitoring the oligodeoxyribonucleotide (ODN) structural behavior in solution. Preliminary experiments, performed on the quadruplex formed by the ODN sequence TGGGGT, showed that the OT can be used as a probe for ODN structuration by monitoring the medium viscosity changes associated with ODN folding-unfolding processes.