Freeze-fracture evidence of gel-phase lipid in membranes of senescing cowpea cotyledons

The structural details of membrane organization in germinating and senescing cotyledons of cowpea (Vigna unguiculata (L.) Walp.) were studied by thin section and freeze-fracture electron microscopy. Germination- and senescence-related changes in the ultrastructure of parenchymal cells of cowpea cotyledons, as detected in thin sections, closely resemble those described for other leguminous seeds. Additionally, electron-dense deposits associated with the membranes, particularly the plasmalemma and endoplasmic reticulum, were seen to increase with advancing senescence. Freeze-fracture electron microscopy demonstrated that the membranes of cotyledons of 2-d-old seedings appear to be normal, with evenly dispersed intramembranous particles. However by 4 d, small areas or domains of the plasmalemma were free of intramembranous particles. These particle-free areas increased in both size and number as senescence progressed. We interpret these particle-free areas to be structural evidence for lateral phase separations of the membrane lipids into microdomains of gel-phase lipid from which intrinsic membrane proteins are excluded. Our results support wide-angle X-ray diffraction studies which have demonstrated the presence of gel-phase lipids in senescing bean cotyledons.Abbreviations EF exoplasmic fracture - ER endoplasmic reticulum - ESR electron-spin resonance - IMP(s) intramembranous particle(s) - PF protoplasmic fracture     

The relevance of seed membrane lipids to imbibitional chilling effects

Imbibitional chilling injury during rehydration of seeds is particularly marked in soybean but not in pea. Following the previous reports that the deleterious effects of chilling on soybean germination are probably mediated at least in part through a loss of membrane integrity, differences in composition of membrane lipid components extracted from pea (chilling-insensitive) and soybean (chilling-sensitive) were examined. Comparative compositional analysis of phospholipids, fatty acids and free sterols revealed few disparities between the two species. When the membrane lipids were re-formed into liposomes, little difference in permeability was found with respect to KCl, glucose or glycerol, even at chilling temperatures. It is suggested that the difference in chilling sensitivity between pea and soybean is not related to compositional differences in the major lipid components of the seed membranes.     

Characterization of water distribution and activities of enzymes during germination in magnetically-exposed maize (Zea mays L) seeds

Magnetic seed treatment is one of the physical pre-sowing seed treatments to enhance the performance of crop plants. In our earlier experiment, we found significant increase in germination and vigour characteristics of maize (Zea mays L.) seeds subjected to magnetic fields. Among various combinations of magnetic field (MF) strength and duration, best results were obtained with MF of 100 mT for 2 h and 200 mT for 1 h exposure. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions. Thus, in the present study, changes in water uptake during imbibition and its distribution and activities of germinating enzymes during germination were investigated in maize seeds exposed to static magnetic fields of 100 and 200 mT for 2 and 1 h respectively by nuclear magnetic resonance (NMR) spectroscopy. The magnetically-exposed seed showed higher water uptake in phase II and III than unexposed seed. The longitudinal relaxation time T1 of seed water showed significantly higher values and hence greater molecular mobility of cellular water in magnetically-exposed seeds as compared to unexposed. Component analysis of T2 relaxation times revealed the early appearance of hydration water with least mobility and higher values of relaxation times of cytoplasmic bulk water and hydration water in magnetically-exposed over unexposed seeds. Activities of alpha-amylase, dehydorgenase and protease during germination were higher in magnetically-exposed seeds as compared to unexposed. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions.     

Myelin labeled with mercuric chloride. Asymmetric localization of phosphatidylethanolamine plasmalogen

We have recorded modified X-ray diffraction patterns to 15 Å spacing from sciatic nerves treated with mercuric chloride (HgCl₂) at concentrations of 0.5 to 32 mm in water or in saline. The observed changes in repeat period and in the intensities of the low-order reflections indicate closer packing of membranes at their cytoplasmic surfaces after treatments with HgCl₂. In addition, HgCl₂ at 0.25 mm or more prevents swelling in water at the extracellular boundaries. By comparing the distinctive diffraction patterns from nerves treated under different conditions with HgCl₂, we have interpreted the changes in intensities of the higher order X-ray reflections and have calculated electron density profiles of the modified membranes. The most striking difference between membrane profiles before and after treatment with HgCl₂ is the large increase in electron density in the region of the lipid headgroup peak in the cytoplasmic half of the bilayer. The magnitude and location of this increase suggests labeling of myelin lipid. To examine this possibility, we analyzed the lipids from mercury-treated sciatic nerves.Thin-layer chromatography of lipids extracted from nerves treated with HgCl₂ shows a marked decrease of phosphatidylethanolamine, which exists in myelin primarily as plasmalogen. At the same time, a new spot identified as lysophosphatidylethanolamine appears. An identical result was obtained by treating extracted lipids with HgCl₂, suggesting that the same sites of interaction are present in the intact membrane as in the dispersed lipids. Previous studies on plasmalogens indicate that mercury adds to the β-carbon of the α,β-unsaturated ether group to produce a lyso-lipid and an aldehyde with bound mercury (Norton, 1959). From a correlation of our X-ray structural analysis and the chemical studies, we conclude that phosphatidylethanolamine plasmalogen is preferentially localized in the cytoplasmic half of the myelin membrane bilayer.     

Water binding and phase structures for different Acholeplasma laidlawii membrane lipids studied by deuteron nuclear magnetic resonance and x-ray diffraction

Water binding capability and phase structures for different lipid species extracted from Acholeplasma laidlawii A membranes have been studied using deuteron nuclear magnetic resonance and low-angle X-ray diffraction.The dominating membrane lipids are monoglucosyldiglyceride and diglucosyldiglyceride and each of them takes up limited amounts of water (bound plus trapped), i.e., up to 13% (w/w), whereas the phospholipids and phosphoglycolipids have larger hydration capacities.Addition of magnesium and calcium ions, but not sodium ions, to the diglucosyldiglyceride increases the hydration capability. This increase is accompanied by the formation of a metastable liquid crystalline phase and a hysteresis effect for the transition temperature.Large differences in water deuteron quadrupole splitting were observed between mono- and diglucosyldiglyceride. Both ²H nuclear magnetic resonance and low-angle X-ray diffraction studies on lipids containing biosynthetically incorporated ω-d₃-palmitic acid clearly indicate the existence of a reverse hexagonal phase structure for the monoglucosyldiglyceride and lamellar structures for the diglucosyldiglyceride and the other membrane lipids.The low hydration capability of the large diglucosyldiglyceride polar head is discussed in terms of polar head configuration.Both mono- and diglucosyldiglyceride have several physical properties similar to those of phosphatidylethanolamine.     

Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration.

The intermolecular and interfibrillar spacings of collagen in bovine corneal stroma have been measured as a function of tissue hydration. Data were recorded from low- and high-angle x-ray diffraction patterns obtained using a high intensity synchrotron source. The most frequently occurring interfibrillar spacing varied from 34 nm in dry corneas to 76 nm at H = 5 (the hydration, H, is defined as the ratio of the weight of water to the dry weight). The most frequently occurring intermolecular Bragg spacing increased from 1.15 nm (dry) to approximately 1.60 nm at normal hydration (H approximately 3.2) and continued to increase only slowly above normal hydration. Most of the increase in the intermolecular spacing occurred between H = O and H = 1. Over this hydration range the interfibrillar and intermolecular spacings moved in tandem, which suggests that the initial water goes equally within and between the fibrils. Above H = 1 water goes preferentially between the fibrils. The results suggest that, even at normal hydration, water does not fill the interfibrillar space uniformly, and a proportion is located in another space or compartment. In dried-then-rehydrated corneas, a larger proportion of the water goes into this other compartment. In both cases, it is possible to postulate a second set or population of fibrils that are more widely and irregularly separated and therefore do not contribute significantly to the diffraction pattern.     

Lipid participation in intracellular freezing avoidance mechanisms of lettuce seed

Freeze-fracture electron microscopy was used to study water content related freezing resistance in Grand Rapids lettuce seeds. Consistent and recognizable conformational changes occurred in lipid-water phases of lettuce seeds at different moisture contents. In air-dry lettuce seed cotyledons, the lipids lying in spherical lipid bodies near the cell wall appeared amorphous, while the structure was crystalline above 20% water content. The lipid bodies interassociated into membrane bilayers in seeds containing 20 to 25% water. Such lyotropic phase transitions in membrane lipids during lettuce seed hydration are believed to contribute to the biphasic freezing behavior observed in lettuce seeds at different moisture contents and to provide a natural freezing tolerance mechanism for highly desiccated plant tissues such as seeds.     

On the position of the hydro-phobic/philic boundary in lipid bilayers

The sensitivity of calculated structural dimensions of hydrated lipids to the position of the hydrophobic/hydrophilic boundary is reviewed. The position of this boundary is critical in determining the extent of hydration and location of water in the bilayer. A calculation of the dimensions of the hydrophilic and hydrophobic parts of the phosphatidylcholine and ethanolamine bilayer from literature values of the x-ray long spacing shows that the choice of boundary in phospholipids is not arbitrary and is best placed at the average position of the first CH₂ group in the hydrocarbon chains. Calculated dimensions of the hydrocarbon core and the water accessible regions agree with neutron and x-ray diffraction measurements. Hydration differences between phosphatidylcholines and phosphatidylethanolamines are readily explained from derived estimates of the layers of water which cover these headgroups.     

Isolation of biochemical mutants using haploid mesophyll protosplasts of Hyoscyamus muticus

The question of whether membrane expansion, which is caused by anesthetics in animal systems, alters the lipid composition of plant cell membranes was investigated. We have measured the effects of several anesthetics on the relative amounts of the principal fatty acids from the polar lipids of barley (Hordeum vulgare L.) root membranes. Procaine, dibucaine, tetracaine, chloroform and, to a lesser degree, methanol increased the proportions of palmitic, stearic and oleic acids and decreased the proportions of linoleic and linolenic acids. Ethanol had no significant effect. Total amounts of the fatty acids from the polar lipids of roots in procaine solution decreased markedly so that all of the acids decreased in amount. The anesthetic was effective as soon as the roots were introduced to the solution and the changes progressed at constant rates for 6 h. Only the polar membrane lipids were altered; other lipids were not affected. Increased hydrostatic pressure of about 1.0 MPa largely prevented the anesthetic effects, including the decrease in the total amounts of the fatty acids. Hydrostatic pressure as high as 2 MPa had no effect per se on the membrane lipid composition. These results indicate that anesthetics cause expansion of the root membranes which results in the lipid changes. That a compositional change in the membrane lipids involves a conformational change such as expansion is an indication of the nature of the link between changes in the membrane lipids and changes in function of areas where hydrophilic ions permeate.Abbreviations 16:0 palmitic acid - 18:0 stearic acid - 18:1 oleic acid - 18:2 linoleic acid - 18:3 linolenic acid     

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T₂) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T₂ data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.     

Structural Study of the Interaction Between the Mitochondrial Presequence of Cytochrome c Oxidase Subunit IV and Model Membranes

The structural effect of the presequence of cytochrome oxidase subunit IV (p25) on multilamellar liposomes with different lipid compositions has been investigated using X-ray diffraction and electron microscopy. The presequence causes the disordering of the liposomes containing negatively charged lipids, without destabilizing the bilayer structure or destroying the multilamellar nature of the liposomes. In the systems containing only zwitterionic lipids, a small increase in the d-spacing (lamellar stacking spacing) is observed without any disorder effect suggesting a weaker interaction of the peptide and lipid. Circular Dichroism measurements of the peptide, in the presence and absence of the different lipid systems studied, show that the secondary structure of the peptide is modulated by the lipid environment. Considerable amounts of -helix in the presequence is only observed in the systems containing negatively charged lipids. These are the same systems for which the disordering effect is observed with X-ray diffraction. It is proposed that p25 disorders the bilayer stacking by corrugating the membranes. The results are discussed in terms of the relevance of the specific lipid properties (e.g., electric charge and ability to form inverted phases) in determining how the peptide interacts with the lipid and affects its structural organization. It is suggested that the lipid properties relevant for the disordering effect induced by the peptide are the same as those involved in the formation of contact sites between mitochondrial membranes during the import of nuclear coded proteins.     

A neutron diffraction study of the influence of ions on phospholipid membrane interactions.

Neutron diffraction is used to examine the effects of Ca2+ and ClO4- ions on interactions and some structural features of dipalmitoylphosphatidylcholine membranes in both solid and fluid lamellar phases. The results are described within the framework of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory with reference to electrostatic, van der Waals, and hydration components of disjoining pressure. The Hamaker constants are evaluated under equilibrium conditions. Addition of 100 mM CaCl2 to the aqueous phase substantially increases the lamellar repeat spacing (d), which is interpreted in terms of adsorption of Ca2+ ions to bilayers followed by electrostatic repulsion between membranes. The rise of NaClO4 concentration in the presence of 100 mM CaCl2 leads to gradual decrease in d, evidently resulted from the diminution of Ca(2+)-induced positive surface potential by both electrostatic screening and binding of ClO4- ions. In the absence of CaCl2, elevation of NaClO4 concentration to 100-300 mM drastically enhances the repeat spacing and then dramatically decreases d at about 1 M NaClO4. Estimation of the hydration coefficients showed that the pronounced decrease of the repeat spacing at high NaClO4 concentrations was resulted mainly from the (partial) disruption of the structure of intermembrane bound water by chaotropic ClO4- ions and subsequent decrease in hydration repulsive pressure. Moreover, in the case of solid membranes (20 degrees C) high concentrations of ClO4- induced formation of interdigitated phase paralleled with marked reduction in bilayer thickness and corresponding increase in the effective cross-sectional area per lipid molecule.     

Gel phase polymorphism in ether-linked dihexadecylphosphatidylcholine bilayers

The structure and properties of the ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) have been examined as a function of hydration. By differential scanning calorimetry, DHPC exhibits an endothermic (chain melting) transition with the transition temperature (limiting value, 44.2 degrees C) and enthalpy (limiting value, delta H = 8.0 kcal/mol) being hydration dependent. For hydration values greater than 30 wt % water, DHPC exhibits a pretransition at approximately 36 degrees C (delta H = 1.1 kcal/mol) and a subtransition at approximately 5 degrees C (delta H = 0.2 kcal/mol). By X-ray diffraction, at 22 degrees C DHPC exhibits a normal bilayer gel structure with the bilayer periodicity increasing from 58.0 to 62.5 A over the hydration range 9.5-25.4% water. At 30-32% water, two coexisting gel phases are observed with d = 63-64 A and d = 44-45 A; at higher hydration, only the latter phase is present, reaching a limiting d = 47.0 A at 37.5% water. Two different gel phases clearly exist at low and high hydrations. Electron density profiles at low hydration (9.5-25.4%) show a bilayer thickness dp-p = 46 A, whereas at greater than 32% water the bilayer thickness is markedly reduced, dp-p = 30 A. These and other structural parameters indicate a hydration-dependent gel----gel structural transition between a normal bilayer (two chains per polar group) and the chain-interdigitated bilayer (four chains per polar group) arrangement described previously for DHPC [Ruocco, M. J., Siminovitch, D. J., & Griffin, R. G. (1985) Biochemistry 24, 2406-2411].(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray diffraction from paclitaxel-loaded zwitterionic and cationic model membranes

We studied the incorporation of the hydrophobic anticancer drug paclitaxel (PXL), into a variety of lipid matrices by X-ray diffraction (XRD) measurements. Liposome suspensions from cationic and zwitterionic lipids, containing different molar fractions of paclitaxel were made and deposited on planar glass substrates. After drying at controlled relative humidity, aligned multilayer stacks were obtained. The structure perpendicular to the substrate plane was investigated by X-ray diffraction measurements. Bragg peaks to several orders were detected, indicative of well-ordered multilamellar lipid layers. The drug induced a modification of the bilayer spacing, which was the characteristic for a given type of lipid matrix. With an excess of the drug, Bragg peaks of drug crystals could be observed. The results provide insight into the solubility of paclitaxel in the different lipid membranes. A structural model of the organization of the drug in the membrane was discussed.     

Location and effect of procaine on lecithin/cholesterol membranes using X-ray diffraction methods

X-ray diffraction studies were made on lecithin/cholesterol multilayers with very high water content and containing the local anaesthetic procaine. Narrow-angle diffraction experiments show that the procaine molecules are located with the uncharged aromatic amine group approx. 10 Å from the centre of the bilayer. The polar tertiary amine group of these molecules is almost certainly located in the polar head-group region of the membrane. Wide-angle diffraction experiments show that the incorporation of procaine molecules into such lipid membranes produces an approx. 30% increase in the spread of acyl chain separation, although the average spacing between the chains is slightly reduced.     

Water state and diffusion through lipid bilayers: Effect of hydration degree

The dependences of adsorbed water state (obtained from the variations in ¹H NMR spectra with the angle between the bilayer normal and magnetic field direction) and water diffusion along the bilayer normal (measured using pulsed field gradient ¹H NMR) on hydration degree have been studied in macroscopically oriented bilayers of dioleoylphosphatidylcholine. The angle dependences of the shape of NMR spectrum are qualitatively different only for water concentrations higher and lower than that achieved by hydration from saturated vapors (χ_eq, about 23%). At concentrations lower than χ_eq, all water in the sample either makes the hydration shells of the lipid polar heads or is in fast exchange with the shell water, so the spin-echo signal from water is detected only within a narrow range of angles close to the magic angle, 54.7°. At concentration exceeding χ_eq, the spin-echo signal from water is retained at all orientations, suggesting that a portion of water between bilayers (quasi-free water) slowly exchanges with water bound to the polar heads. There is an inverse dependence of the coefficient of water self-diffusion through the bilayer system on the hydration degree, which is described in the Tanner model with account of water self-diffusion in the hydrophobic part of the bilayer. Bilayer permeability, distribution coefficient of molecules between aqueous and lipid phases, and water self-diffusion coefficient in the hydrophobic region of the bilayer are estimated.     

Bilayer dimensions and hydration of glycolipids

X-ray diffraction measurements are available on a wide range of glycolipid multilamellar assemblies in excess water, but not at the defined water contents that are needed to derive bilayer dimensions. For lamellar crystalline phases and gel phases with untilted chains, or where the tilt angle is known, the cross-sectional area per chain from wide-angle diffraction can be used to determine the area per lipid molecule at the bilayer surface. Using the lipid molecular volume from densitometry, it is then possible to obtain the bilayer thickness and hence, from the lamellar repeat spacing, the water layer thickness and degree of hydration of the lipid polar groups. This is done here by using the available data for bilayer-forming diacyl and dialkyl glycosylglycerols, and for certain glycosphingolipids. The lamellar crystalline phases of these glycolipids are largely anhydrous, and the degree of hydration of the lamellar gel phases is much lower than that of the corresponding phosphoglycerolipid gel phases. A point of current uncertainty is whether the chains in the gel phases of diacyl glycoglycerolipids are appreciably tilted, unlike their dialkyl counterparts.     

Water uptake and distribution in germinating lupine seeds studied by magnetic resonance imaging and NMR spectroscopy

Magnetic resonance imaging (MRI) was used to study temporal and spatial water uptake and distribution in germinating lupine ( Lupinus luteus L.) seeds. During 24 h of imbibition, water was unevenly distributed within the seed and some anatomical parts were more hydrated than others. Water entered the seed through the hilum and micropyle. The embryonic axis was the first to show hydration followed by seed coat and later cotyledons. The changes in water status were characterized by NMR spectroscopy. Analyses of T₂ relaxation times revealed a three-component water proton system (structural, intracellular and extracellular water) in germinating lupine seeds. The data on the components of transverse relaxation time studies indicated the complex exchange processes taking place between water components inside lupine seed over first 2.5 h of hydration, with a distinguished increase in structural water and decrease in other components. This speaks in favor of the high water-absorbing capacity of lupine seeds as related to high protein content. Germination was accompanied by swelling of protein bodies and changes in the organization of stored reserves with gradual disappearance of protein from the cells.     

Phase equilibria of membrane lipids from Acholeplasma laidlawii: importance of a single lipid forming nonlamellar phases

A basis for the reorganization of the bilayer structure in biological membranes is the different aggregate structures formed by lipids in water. The phase equilibria of all individual lipids and several in vivo polar lipid mixtures from acyl chain modified membranes of Acholeplasma laidlawii were investigated with different NMR techniques. All dioleoyl (DO) polar lipids, except monoglucosyldiglyceride (MGDG), form lamellar liquid crystalline (L alpha) phases only. The phase diagram of DOMGDG reveals reversed cubic (III), reversed hexagonal (HII), and L alpha phases. In mixtures of DOMGDG and dioleoyldiglycosyldiglyceride (DODGDG), the formation of an III (or HII) phase is enhanced by DOMGDG and low hydration or high temperatures. For in vivo mixtures of all polar DO lipids, a transition from an L alpha to an III phase is promoted by low hydration or high temperatures (50 degrees C). The phospholipids are incorporated in this III phase. Likewise, III and HII phases are formed at similar temperatures in a series of in vivo mixtures with different extents of acyl chain unsaturation. However, their melting temperatures (Tm) vary in an expected manner. All cubic and hexagonal phases, except the III phase with DOMGDG, exist in equilibrium with excess water. The maximum hydration of MGDG and DGDG is similar and increases with acyl chain unsaturation but is substantially lower than that for, e.g., phosphatidylcholine. The translational diffusion of the lipids in the cubic phases is rapid, implying bicontinuous structures. However, their appearances in freeze-fracture electron microscope pictures are different. The III phase of DOMGDG belongs to the Ia3d space group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of systemic insecticides on lipid biosynthesis in seeds of Indian mustard (Brassica juncea L.)

The systemic insecticides namely phorate (Thimet 10 G) oxydemeton methyl (metasystox 25 EC) and dimethoate (Rogor 30 EC) decreased oil content in the developing seeds of Indian mustard (Brassica juncea L.) but showed an increase in the mature seeds. The inhibitory effect in the developing seed was accompanied by an increase in soluble sugars and a corresponding decrease in malate dehydrogenase and G6P dehydrogenase activity. The relative proportion of triacylglycerols and glycolipids decreased significantly while that of phospholipids and free fatty acids increased in the developing seeds. In the mature seeds, the proportion of triacylglycerols did not change appreciably from that in control. The erucic acid synthesis which was less at 10 and 20 days after fertilization (DAF) increased at 30 DAF with oxydemeton methyl and dimethoate; phorate was ineffective. In mature seeds, the proportion of erucic acid increased at the cost of linoleic and linolenic acids. All the insecticides appreciably decreased the rate of [1-¹⁴C]acetate incorporation into lipids both in vivo and in vitro experiments. In the in vivo experiment, the synthesis of polar lipids was enhanced at 10 and 20 DAF, and the higher doses of oxydemeton methyl and dimethoate at 30 DAF. On the other hand, the ¹⁴C-incorporation into triacylglycerols showed an opposite trend to that of polar lipids. In the in vitro experiment, oxydemeton methyl and dimethoate enhanced the synthesis of polar lipids at 10 and 20 DAF while these inhibited it at 30 DAF. The synthesis of triacylglycerols was inhibited by the use of these insecticides.