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PubMedDate not supplied

A new approach to insect-pest control--combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity.

Voltage-sensitive sodium channels are responsible for the generation of electrical signals in most excitable tissues and serve as specific targets for many neurotoxins. At least seven distinct classes of neurotoxins have been designated on the basis of physiological activity and competitive binding studies. Although the characterization of the neurotoxin receptor sites was predominantly performed using vertebrate excitable preparations, insect neuronal membranes were shown to possess similar receptor sites. We have demonstrated that the two mutually competing anti-insect excitatory and depressant scorpion toxins, previously suggested to occupy the same receptor site, bind to two distinct receptors on insect sodium channels. The latter provides a new approach to their combined use in insect control strategy. Although the sodium channel receptor sites are topologically separated, there are strong allosteric interactions among them. We have shown that the lipid-soluble sodium channel activators, veratridine and brevetoxin, reveal divergent allosteric modulation on scorpion alpha-toxins binding at homologous receptor sites on mammalian and insect sodium channels. The differences suggest a functionally important structural distinction between these channel subtypes. The differential allosteric modulation may provide a new approach to increase selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective toxins. Thus, a comparative study of neurotoxin receptor sites on mammalian and invertebrate sodium channels may elucidate the structural features involved in the binding and activity of the various neurotoxins, and may offer new targets and approaches to the development of highly selective pesticides.

Amino Acid Sequence↗
PubMed1975

Unique pathways of sterol metabolism in the Mexican bean beetle, a plant-feeding insect.

Radiolabeled sterols, 14C-cholesterol, 14C-cholestanol, 3H-stigmasterol, 3H-stigmastanol, and 3H-sitosterol, were fed to larvae of the Mexican bean beetle, Epilachna varivestis Mulsant, bu coating soybean leaves. Free sterol and sterol ester fractions from treated insects were isolated and analyzed, and in each case nearly 30% or more of total radiolabeled sterols retained by the insect were found in the sterol ester fraction after 8 days. delta5-Dietary sterols were readily reduced to stanols, and C29-stanols thus produced were dealkylated to cholestanol. Significant amounts of labeled lathosterol were formed from delta5-C29 sterols; little, if any, radiolabeled cholesterol was detected in insects fed either of the labeled delta5-phytosterols, stigmasterol or sitosterol. Sterol metabolism of this insect thus differs considerably from that found for most phytophagous insects.

Animals↗
PubMed2002

The coagulation of insect hemolymph.

In contrast to both vertebrates and non-insect arthropods, little is known about the coagulation of hemolymph (hemostasis) in insects. We discuss the integration of the hemostatic response with other branches of the insect immune system. We also describe the present stage in the characterization of both soluble and cellular factors that contribute to hemostasis in insects. The factors of the well-characterized clotting cascades of vertebrates, primitive chelicerates and crustaceans are used to assess the implications of sequencing the whole Drosophila genome for searching candidate genes involved in hemostasis. Some striking similarities between blood clotting in vertebrates and the reaction of insect cells involved in hemolymph coagulation have implications for a phylogenetic comparison of hemostasis between divergent animal classes.

Animals↗
PubMed2004

The mechanisms of lift enhancement in insect flight.

Recent studies have revealed a diverse array of fluid dynamic phenomena that enhance lift production during flapping insect flight. Physical and analytical models of oscillating wings have demonstrated that a prominent vortex attached to the wing's leading edge augments lift production throughout the translational parts of the stroke cycle, whereas aerodynamic circulation due to wing rotation, and possibly momentum transfer due to a recovery of wake energy, may increase lift at the end of each half stroke. Compared to the predictions derived from conventional steady-state aerodynamic theory, these unsteady aerodynamic mechanisms may account for the majority of total lift produced by a flying insect. In addition to contributing to the lift required to keep the insect aloft, manipulation of the translational and rotational aerodynamic mechanisms may provide a potent means by which a flying animal can modulate direction and magnitude of flight forces for manoeuvring flight control and steering behaviour. The attainment of flight, including the ability to control aerodynamic forces by the neuromuscular system, is a classic paradigm of the remarkable adaptability that flying insects have for utilising the principles of unsteady fluid dynamics. Applying these principles to biology broadens our understanding of how the diverse patterns of wing motion displayed by the different insect species have been developed throughout their long evolutionary history.

Animals↗
PubMed2003

The effectiveness of insect-pollination test to evaluate the level of self-incompatibility and their genetic analysis in radish (Raphanus sativus L.).

We have tried an insect-pollination self-incompatibility (SI) test to strictly evaluate the level of SI as a model for the actual F(1) seed production field using radish as experimental material. Twelve inbred lines, homozygous for the S-alleles, were used in the artificial self-pollination and the insect-pollination SI test. There was a positive correlation ( r = 0.606) between the results by these two methods. Some lines showed a low level of SI in the insect-pollination test despite showing a high level of SI in the artificial self-pollination test. On the other hand, no lines showing a low level of SI in artificial self-pollination had a high level of SI in insect-pollination. These results show that the insect-pollination SI test can be considered to be a more reliable and stricter method than the artificial self-pollination test with respect to an evaluation of SI levels. We have raised and analyzed an F(2) population and F(3) lines derived from an F(1) cross between a line showing a high level of SI (R00-04) and one showing a low level. The rate of self-seed settings of the F(2) population showed a binomial distribution. There were 39 high-level SI plants to 15 low-level SI plants. This result and F(3) progeny tests suggested that the high level of SI which R00-04 showed is controlled by a dominant gene.

Animals↗
PubMed1997

Effects of pH and sulfate on insects and protozoans inhabiting treeholes.

We used laboratory microcosms designed to simulate treeholes to study the effect of changing levels of chemicals found in precipitation in central Pennsylvania (H+ and SO4=) on the treehole insects Aedes triseriatus (a mosquito), Helodes pulchella (a helodid beetle), and Culicoides guttipennis (a ceratopogonid midge), and treehole protozoans. Protozoans were tested in both the presence and absence of insects. We hypothesized that the individual insect species would have differential tolerances to abiotic stresses, and that effects of low pH on protozoans would be especially strong. Survival of helodids was higher than mosquitoes and midges. Emergence and survival of mosquitoes and midges were lower at low pH. Densities of ciliates increased the most at high pH in the absence of insects. Densities of flagellates increased the most at low pH in the absence of insects. The presence of helodids, mosquitoes, and protozoans was associated with higher final [SO4=], as was low pH. Mosquitoes at low pH caused the largest increases in cation concentrations, and protozoans at high pH caused the largest decreases in cation concentrations. It appears that the biota of treeholes are affected by ionic changes in simulated tree stemflow that can be caused by anthropogenic atmospheric deposition. The species studied here were sometimes differentially affected by the common pollutants, [H+] and [SO4=]. The resulting changes in these discrete treehole communities may allow them to be useful bioindicators of the status of forest ecosystems altered by changing atmospheric chemistry.

Animals↗
PubMed2006

Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants.

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2-3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.

Animals↗
PubMed2000

Accumulation of a maize proteinase inhibitor in response to wounding and insect feeding, and characterization of its activity toward digestive proteinases of Spodoptera littoralis larvae.

The mpi gene encodes a maize proteinase inhibitor (MPI) protein whose mRNA accumulates in response to mechanical wounding. In this study, mpi gene expression in response to different types of damage was investigated. In mechanically damaged leaves of maize (Zea mays L.), mpi mRNA accumulation was affected by the degree of damage inflicted on the leaf. Consecutive wounds resulted in higher levels of mpi transcripts. The MPI protein was expressed in Escherichia coli and purified. Polyclonal antibodies were then produced and used to study MPI accumulation in insect-wounded and mechanically wounded maize leaves. When larvae of the lepidopteran insect Spodoptera littoralis were fed on maize leaves, MPI accumulated in tissues adjacent to the wound site. The level of inhibitor accumulation was higher in leaves chewed by larvae than in leaves that had been damaged mechanically. Longer feeding periods also resulted in higher levels of MPI accumulation. Additionally, the inhibitory properties of MPI toward mammalian and insect digestive serine proteinases were determined. Contrary to the majority of the plant proteinase inhibitors described, MPI is an inhibitor of mammalian elastase that only weakly inhibits mammalian chymotrypsin. However, both elastase and chymotrypsin-like activities from the larval midgut of S. littoralis were effectively inhibited by MPI. We discuss these results with regard to the function and evolution of plant proteinase inhibitors. The availability of a plant proteinase inhibitor which is able to inhibit the two types of insect digestive proteinase, elastase and chymotrypsin, might be useful for engineering protection against lepidopteran insect pests in transgenic plants.

Amino Acid Sequence↗
PubMed2006

Consequences of pre-dispersal damage by insects for the dispersal and recruitment of mangroves.

Herbivores may enhance plant recruitment, but such positive interactions may be overlooked in favour of obvious negative effects of herbivory on propagules. My objective was to determine whether larval insects that feed and develop within fruit of the mangrove Avicennia marina act as mutualist herbivores by increasing the dispersal of propagules without affecting their viability and emerging successfully as adults following dispersal of the propagule by water. Surveys revealed that frugivory is common throughout the mangrove forest, and fruit had up to six exit holes where larvae had emerged as adults. Larval insects did not affect the flotation of propagules with pericarps, a thin structure that provides buoyancy for dispersal by water. In contrast, after simulating germination by removing the pericarp, the majority of propagules with three exit holes floated on average for 20 h longer than those without exit holes, which sank immediately. Based on this evidence that frugivory could increase the dispersal potential of propagules, I predicted that propagules consumed by larval insects should disperse farther than undamaged propagules, and this was tested by quantifying the potential viability of propagules stranded on beaches at increasing distances (up to 20 km) from mangrove forests. Flies and moths emerged as adults after being transported tens of kilometres within mangrove propagules, revealing a novel mode of dispersal. Proportionally fewer potentially viable propagules were supplied to beaches at increasing distances from mangrove forests, however, indicating that larval insects negatively affect recruitment and are thus not acting as mutualist herbivores. Nevertheless, when transported back to the mangrove forest, seedlings established from propagules damaged by larval insects and stranded on beaches. Therefore, although frugivory does not preclude mangrove recruitment, its negative effects in the pre-dispersal environment may be intensified with increasing dispersal distance, thus limiting the long-distance supply of propagules and recruitment of mangroves.

Animals↗
PubMed2006

Insect neuronal cultures: an experimental vehicle for studies of physiology, pharmacology and cell interactions.

The current status of insect neuronal cultures is discussed and their contribution to our understanding of the insect nervous system is explored. Neuronal cultures have been developed from a wide range of insect species and from all developmental stages. These have been used to study the morphological development of insect neurones and some of the extrinsic factors that affect this process. In addition, they have been used to investigate the physiology of sodium, potassium and calcium channels and the pharmacology of acetylcholine and GABA receptors. Insect neurones have also been grown in culture with muscle and glial cells to study cell interactions.

Animals↗
PubMed2005

Differences in effects of pyrrolizidine alkaloids on five generalist insect herbivore species.

The evolution of the diversity in plant secondary compounds is often thought to be driven by insect herbivores, although there is little empirical evidence for this assumption. To investigate whether generalist insect herbivores could play a role in the evolution of the diversity of related compounds, we examined if (1) related compounds differ in their effects on generalists, (2) there is a synergistic effect among compounds, and (3) effects of related compounds differed among insect species. The effects of pyrrolizidine alkaloids (PAs) were tested on five generalist insect herbivore species of several genera using artificial diets or neutral substrates to which PAs were added. We found evidence that structurally related PAs differed in their effects to the thrips Frankliniella occidentalis, the aphid Myzus persicae, and the locust Locusta migratoria. The individual PAs had no effect on Spodoptera exigua and Mamestra brassicae caterpillars. For S. exigua, we found indications for synergistic deterrent effects of PAs in PA mixtures. The relative effects of PAs differed between insect species. The PA senkirkine had the strongest effect on the thrips, but had no effect at all on the aphids. Our results show that generalist herbivores could potentially play a role in the evolution and maintenance of the diversity of PAs.

Animals↗
PubMed2005

Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors.

Protease inhibitors have been proposed as potential defense molecules for increased insect resistance in crop plants. Compensatory over-production of insensitive proteases in the insect, however, has limited suitability of these proteins in plant protection, with very high levels of inhibitor required for increased plant resistance. In this study we have examined whether combined used of two inhibitors is effective to prevent this compensatory response. We show that leaf-specific over-expression of the potato PI-II and carboxypeptidase inhibitors (PCI) results in increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae in homozygote tomato lines expressing high levels (>1% the total soluble proteins) of the transgenes. Leaf damage in hemizygous lines for these transformants was, however, more severe than in the controls, thus evidencing a compensation response of the larvae to the lower PI concentrations in these plants. Development of comparable adaptive responses in both insects suggests that insect adaptation does not entail specific recognition of the transgene, but rather represents a general adaptive mechanism triggered in response to the nutritional stress imposed by sub-lethal concentrations of the inhibitors. Combined expression of defense genes with different mechanisms of action rather than combinations of inhibitors may then offer a better strategy in pest management as it should be more effective in overcoming this general adaptive response in the insect.

Animals↗
PubMed1985

An excitatory and a depressant insect toxin from scorpion venom both affect sodium conductance and possess a common binding site.

Two insect selective toxins were purified by gel-permeation and ion-exchange chromatographies from the venom of the scorpion, Leiurus quinquestriatus quinquestriatus, and their chemical and pharmacological properties were studied. The first toxin (LqqIT1) induces a fast excitatory contraction paralysis of fly larvae and is about 40 times more toxic than the crude venom. It is a polypeptide composed of 71 amino acids, including 8 half-cystines and devoid of methionine and tryptophan, with an estimated molecular weight of 8189 and a pI value of 8.5. The second toxin (LqqIT2) induces a slow depressant, flaccid paralysis of fly larvae. It is composed of 72 amino acids, including 8 half-cystines, is devoid of proline methionine and histidine, and has an estimated molecular weight of 7990 and a pI value of 8.3. The contrasting symptomatology of these toxins is interpreted in terms of their effects on an isolated axonal preparation of the cockroach in current and voltage clamp conditions. LqqIT1 (0.5-4 microM) induced repetitive firing of the axon which was attributable to two changes in the sodium conductance, a small increase in the peak conductance and a slowing of its turning off. LqqIT2 (1-8 microM) caused a blockage of the evoked action potentials, attributable to both a strong depolarization of the axonal membrane and a progressive suppression of the sodium current. Neither toxin affected potassium conductance. The two toxins differ mainly in their opposite effects on the activatable sodium permeability. In binding assays to a preparation of insect synaptosomal membrane vesicles, the two toxins were shown to competitively displace the radioiodinated excitatory insect toxin derived from the venom of the scorpion, Androctonus australis [( 125I]AaIT), which strongly resembles, in its chemistry and action, the LqqIT1 toxin. The present two toxins have demonstrated a strong affinity closely resembling the AaIT, with KD values of 0.4, 1.9, and 1.0 nM for LqqIT1, LqqIT2, and AaIT, respectively. These data suggest the possibility that the excitatory and depressant insect toxins share a common binding site associated with sodium channels in insect neuronal membranes.

Amino Acids↗
PubMed1983

Heterorhabditis spp., Neoaplectana spp., and Steinernema kraussei: interspecific and intraspecific differences in infectivity for insects.

The effectiveness of various dosages of different species/strains of nematodes was compared for Galleria mellonella and various pest insects that live in or pupate in soil. Neoaplectana feltiae (= carpocapsae), the only nematode species tested by most other workers, was never the most infective for any of the insect species tested and was least infective for two. All species/strains of nematode were able to kill insects of each species. The degree of infectivity of each of the nematode species/strains for different hosts varied considerably, and no one species/strain of nematode was the most infective for all insect species. This indicates the importance of testing a number of nematode species against any particular insect before commencing field evaluations for biological control.

Animals↗
PubMed1990

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects.

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.

Animals↗
PubMed1991

A functional hepatitis B virus X protein produced in insect cells.

We have constructed a recombinant baculovirus from Autographa californica nuclear polyhedrosis virus, called AcX, that expresses the gene encoding the hepatitis B virus X protein in infected Spodoptera frugiperda (Sf21AE) insect cells. A 16.5-kDa monomer and a 33-kDa dimer of the X protein were detected in extracts from AcX-infected cells on immunoblots using a polyclonal anti-X antibody. The biological activity of the insect cell-produced X protein was assayed by fusing AcX-infected Sf21AE cells with African green monkey kidney (CV-1) cells and then transfecting the fused cells with the reporter plasmid pSV2cat. The expression of the cat gene in CV-1:Sf21AE(AcX) fusions was seven times higher than that derived from CV-1:Sf21AE(AcMNPV) fusions, indicating that the insect cell-produced X protein was functional. The transactivation function of the insect cell-produced X protein was also verified by scrape-loading nuclear extracts of AcX-infected Sf21AE cells into pSV2cat-transfected CV-1 cells. Treatment of the AcX-infected cell nuclear extracts with anti-X antisera prior to scrape-loading eliminated the transactivating activity of the extracts. We conclude that the insect cell-produced X protein was functionally identical to that generated in mammalian cells.

Animals↗
PubMed1996

Insect neurotransmission: neurotransmitters and their receptors.

The roles of acetylcholine, dopamine, octopamine, tyramine, 5-hydroxytryptamine, histamine, glutamate, 4-aminobutanoic acid (gamma-aminobutyric acid) and a range of peptides as insect neurotransmitters are evaluated in terms of the criteria used to identify transmitters. Of the biogenic amines considered, there is good evidence that acetylcholine, dopamine, octopamine, 5-hydroxytryptamine, and histamine should be considered to be neurotransmitters, but the case for tyramine is less convincing at the moment. The evidence supporting neurotransmitter roles for glutamate and gamma-aminobutyric acid at specific insect synapses is overwhelming, but much work remains to be undertaken before the full significance of these molecules in the insect nervous system is appreciated. Attempts to characterise biogenic amine and amino acid receptors using pharmacological and molecular biological techniques have revealed considerable differences between mammalian and insect receptors. The number of insect neuropeptides isolated and identified has increased spectacularly in recent years, but genuine physiological or biochemical functions can be assigned to very few of these molecules. Of these, only proctolin fulfills the criteria expected of a neurotransmitter, and the recent discovery of proctolin receptor antagonists should enable the biology of this pentapeptide to be explored fully.

Animals↗
PubMed1982

Power and heat measurement by direct calorimetry of individual insect response to allelo- and toxic compounds.

A microcalorimeter was constructed for the individual insect in order to measure the insect's power output as a function of time (the thermogram). The instrument has a figure of merit of 7 microW/microV. It includes a Peltier pumped thermoelectric control loop which protects from intruding ambient thermals, and holds baseline drift to less than 2 microV/day. the design is a conventional twin, or differential heat conduction calorimeter, with two insect-holding vessels of glass. The vessels are connected by a stopcock, to give the insect the option of crawling from the sample chamber to the reference chamber. Continuous, metered air flow is provided. A small pulse of compound may be born in, as vapor with the air flow, for the study of attractants, toxic compounds, anesthetics and allelochemicals. The insect's reaction to such compounds, appears in the thermogram. Cabbage looper larvae were examined in their irritative exothermic reaction to microgram amounts of benzene, and in their neutral behavior toward similar amounts of aliphatic hydrocarbons delivered as a single pulse of vapor. The male northern corn rootworm was monitored in his attraction to female rootworm extract ("pheromone') and his tendency to move upwind, or up airflow, toward the pheromone. The instrument enables discrimination of the transient metabolism of muscle use, from that of the resting state power which is probably the true basal metabolic rate power.

Animals↗