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Enhanced in vivo activity of peptidase-resistant analogs of the insect kinin neuropeptide family.

The diuretic/myotropic insect kinin neuropeptides, which share the common C-terminal pentapeptide core FX(1)X(2)WG-NH(2), reveal primary (X(2)-W) and secondary (N-terminal to F) sites of susceptibility to peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Analogs designed to enhance resistance to tissue-bound peptidases, and pure insect neprilysin and ACE, demonstrate markedly enhanced in vivo activity in a weight gain inhibition assay in H. zea, and strong in vivo diuretic activity in the housefly (M. domestica). The peptidase-resistant insect kinin analog pQK(pQ)FF[Aib]WG-NH(2) demonstrates a longer internal residence time in the housefly than the native muscakinin (MK), and despite a difference of over 4 orders of magnitude in an in vitro Malpighian tubule fluid secretion assay, is equipotent with MK in an in vivo housefly diuretic assay. Aminohexanoic acid (Ahx) is shown to function as a surrogate for N-terminal Lys, while at the same time providing enhanced resistance to aminopeptidase attack. Peptidaese-resistant insect kinin analogs demonstrate enhanced inhibition of weight gain in larvae of the agriculturally destructive corn earworm moth. Potent peptidase resistant analogs of the insect kinins, coupled with an increased understanding of related regulatory factors, offer promise in the development of new, environmentally friendly pest insect control measures.

Animals↗

Vitelline membrane biogenesis in Drosophila requires the activity of the alpha-methyl dopa hypersensitive gene (I(2)amd) in both the germline and follicle cells.

The vitelline membrane of Drosophila eggs is composed of a family of proteins which are cross linked into an insoluble matrix with an overlying waxy layer that prevents desiccation. We present here three sets of experiments which show that integrity of the vitelline membrane requires the activity of the alpha methyl dopa hypersensitive (I(2)amdH or amd) gene in both egg chambers and follicle cells. We show that loss of amd activity either by dietary administration of inhibitors or in genetic mosaics of either the germ line or follicle cells leads to production of defective vitelline membranes and by in situ hybridization, that amd is expressed in both nurse cells and follicle cells. The amd gene product is the first non-structural protein gene described whose activity is required for vitelline membrane biosynthesis. Given its unique role in insects and its demonstrated sensitivity to dietary inhibitors, the amd gene product poses an attractive target for insect control.

Animals↗

[The effect of infestation by mixed culture of Beauveria bassiana and Paecilomyces farinosus on reduction in numbers of experimental culture Blattella germanica L].

In this paper results of infestation of Blatella germanica L with mixed culture of insecticidal fungi belonging to one two species are presented. In the first stage of experiment the insects were infested first with B. bassiana spores and later with P. farinosus spores (strains L and P) was used as first and B. bassiana as second with time sequence of only 24 and 72 hours. Out of one species cultures tests were done alternately with strains L and P of P. farinosus. The control insects were always infested with fungi applied to cockroach in the first place. From comparison of data it results that irrespective of the time that elapsed from application of the first pathogen to the time of application of the second pathogen the number of dead individuals was always higher in experimental series than in control i.e. when only one pathogen was applied. After 30 days of experiment the highest mortality in females amounted to, 3.3% and in males--80.0% whereas in the control it amounted 6.6% and 20.0%. Respectively, after feeding on diet infested only with one pathogen. After 50 years the highest mortality amounted to 20% in females and 100% in males with control of 30% and 53.3% in meals and females, respectively. When the sequence of pathogen application was reversed, mortality after 30 day amounted to 53.3% in females and 36.7% in meals with corresponding numbers in controls beginning 26.6% and 26.6%, respectively. After elapse of 50 days the highest mortality in females was 90% and in males--100% with control showing mortality of 36.7% and 63.3% respectively. From comparison of numerical data complied in Table 1 it results that most advantageous time span between first and second infestation with fungi is 48 h. Mortality of cockroaches infested with fungi of two different species was higher than in insects infested with one species of fungi.

Animals↗

Field tests on managing resistance to Bt-engineered plants.

Several important crops have been engineered to express toxins of Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers planted nearly 8 million hectares (nearly 20 million acres) of transgenic Bt crops approved by the EPA. Bt-transgenic plants can greatly reduce the use of broader spectrum insecticides, but insect resistance may hinder this technology. Present resistance management strategies rely on a "refuge" composed of non-Bt plants to conserve susceptible alleles. We have used Bt-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in our field tests indicate that a "separate refuge" will be more effective at conserving susceptible larvae than a "mixed refuge" and would thereby reduce the number of homozygous resistant (RR) offspring. Our field tests also examined the strategy of spraying the refuge to prevent economic loss to the crop while maintaining susceptible alleles in the population. Results indicate that great care must be taken to ensure that refuges, particularly those sprayed with efficacious insecticides, produce adequate numbers of susceptible alleles. Each insect/Bt crop system may have unique management requirements because of the biology of the insect, but our studies validate the need for a refuge. As we learn more about how to refine our present resistance management strategies, it is important to also develop the next generation of technology and implementation strategies.

Animals↗

Dependence of the entomopathogenic fungus, Beauveria bassiana, on high humidity for infection of Rhodnius prolixus.

The impact of relative humidity (RH) on the infective potential of the isolate Bb INRA 297 of Beauveria bassiana (Bals.) Vuillemin (Deuteromycotina: Hyphomycetes) against first instar nymphs of Rhodnius prolixus Stål. (Hemiptera: Reduviidae) was determined. Fungus-treated insects were exposed to RHs ranged from 75 to 100% at 25 degrees C. Results clearly showed a threshold of humidity at ca. 96% for high and rapid mortality. After initial exposure to increasing periods of 97% (4, 8, 16, 24, 36 and 48 h) and subsequent transfer to constant lower RHs (43, 53, 75 and 86%) at a constant 25 degrees C, an incubation of at least 48 h at 97% RH was necessary to kill all insects. On changing RHs of 97/75% and different regimes of temperature (15/28 degrees C, 20/25 degrees C, 25/28 degrees C, and 25/35 degrees C), at least 72 h of initial exposure at 97% RH for the 15/28 degrees C regime, 48 h for the 20 degrees/25 degrees C and 25/28 degrees C regimes and 36 h for 25/35 degrees C were needed to kill all insects over a 6-day incubation time. Delayed exposure to favorable moisture condition (97% RH), significantly affected infection for up to a 3-day delay within the various temperature-humidity regimes tested.

Animals↗

Entomopathogens as insecticides.

Entomopathogens, diseases of insects, are suggested as a possible new generation of safe, selective insecticides. Over a thousand pathogens have been isolated from insects. Many of these, associated with major insect pests, are potential candidates for development into microbial insecticides. Phases in the development of a microbial insecticide are discussed as well as factors (production, safety, efficacy, registration, non-technical) which may influence this development.

Bacteria↗

Effectiveness of different emulsifiers for neem oil against the western flower thrips (Thysanoptera, Thripidae) and the warehouse moth (Lepidoptera, Pyralidae).

The neem tree produces highly specified acting insecticides mainly in its seeds. By pressurizing or extracting the seeds an insecticide oil can be manufactured. For successful application emulsifiers are needed to render the oil soluble in water. The heavy oil has to be stable in emulsion, but on the other hand the surfactant should not reduce the ecological property of the neem oil. The emulsifiers Lutensol TO10, Emulan ELP, Rimulgan and Tween 80 and for comparison the formulation NeemAzal-T/S were tested in their emulsion stability, as well as in their insecticidal effects towards two different insect pests: The western flower thrips Frankliniella occidentalis and the ware house moth Ephestia elutella. The emulsifiers were applied purely, and in different contents mixed in neem oil. Data showed significant differences of mortality and development on the tested pests. Lutensol TO10 and Emulan ELP caused spontaneous mortality on the western flower thrips and an additive efficacy when mixed with neem oil. Rimulgan led to mortality of the larvae of the warehouse moth. NeemAzal showed in both bioassays the highest efficacy of 95% mortality.

Animals↗

Characterization in relation to development of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, in Lepidoptera.

Cytochrome P450 enzymes are involved in a number of steps in ecdysteroid (moulting hormone) homeostasis in insects. We report the cloning and characterization of an ecdysteroid agonist-responsive cytochrome P450, CYP18A1, from the cotton leafworm, Spodoptera littoralis. Northern blot analysis showed that the mRNA transcript was expressed at times of increasing ecdysteroid titre in final instar S. littoralis larvae and was induced by the ecdysteroid receptor agonist, RH-5992, in midgut and fat body. In addition, transcript expression was also detected in the prothoracic glands, a major ecdysteroid biosynthetic tissue, in both S. littoralis and the tobacco hornworm, Manduca sexta, at a time of increasing ecdysteroid titre. The exact significance of the temporal and spatial expression of CYP18A1 is unclear. The characterization of a P450 that is ecdysteroid agonist-responsive may provide a future target for exploitation in the development of novel insect control strategies.

Amino Acid Sequence↗

Transcriptional regulation in southern corn rootworm larvae challenged by soyacystatin N.

When fed on a diet containing a soybean cysteine protease inhibitor soyacystatin N (scN), southern corn rootworm larvae exhibited increased mortality and reduced growth rate. scN impacted mortality in a dose-dependent manner, and its effect on insect growth was more severe at early developmental stages. Insects that survived from continuous exposure to the inhibitor at doses ranging from 0.1% to 0.5% had less reduction in body weight during later developmental stages. This insensitivity as insects developed was not observed in the insect group fed on diet containing 0.05% scN, the lowest dose tested. Thus, individuals that survived the higher dose treatments may have had higher fitness under dietary inhibitory challenge. Subtractive hybridization and cDNA microarray analyses identified 29 transcript species responsive to scN. Southern corn rootworm larvae over-expressed cysteine and aspartic proteases to compensate for inhibition of digestion. Induction of a peritrophin gene suggested that strengthening the peritrophic membrane plays a role in coping with protease inhibitors. scN down-regulated genes encoding proteins involved in insect metabolism and development, reflecting the insect's ability to reallocate resources to prioritize its defense response. Further, protease and the peritrophin genes were also developmentally regulated, which may explain the lower toxicity in older larvae than in neonates when first encountering dietary scN. Multiple regulatory mechanisms of counter defense-related genes may allow insects to evade the effect of plant defense proteins, and impose an obstacle to biotechnology-based insect control.

Animals↗

cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation.

The insect kinin neuropeptides have been implicated in the regulation of water balance, digestive organ contraction, and energy mobilization in a number of insect species. A previous solution conformation study of an active, restricted-conformation cyclic analog, identified two possible turn conformations as the likely active conformation adopted by the insect kinins at the receptor site. These were a cisPro type VI beta-turn over C-terminal pentapeptide core residues 1-4 and a transPro type I-like beta-turn over core residues 2-5, present in a ratio of 60:40. Synthesis and evaluation of the diuretic activity of insect kinin analogs incorporating a tetrazole moiety, which mimics a cis peptide bond, identifies the active conformation as the former. The discovery of a receptor interaction model can lead to the development of potent agonist and antagonist analogs of the insect kinins. Indeed, in this study a tetrazole analog with D stereochemistry has been shown to demonstrate partial antagonism of the diuretic activity of natural insect kinins, providing a lead for more potent and effective antagonists of this critical neuropeptide family. The future development of mimetic agonists and antagonists of insect kinin neuropeptides will provide important tools to neuroendocrinologists studying the mechanisms by which they operate and to researchers developing new, environmentally friendly pest insect control strategies.

Animals↗