Small-angle x-ray scattering study of insect lipophorin

The structure of lipophorin in insect blood (hemolymph) was investigated by a small-angle x-ray scattering method over the temperature range 0-45 degrees C. The small-angle x-ray scattering profile of lipophorin exhibited a symmetrical sphere with heterogeneous internal electron density. Cockroach and locust lipophorins, which contain hydrocarbons, demonstrated centrosymmetrical distribution of electron density inside the particles. A previous study suggested that the hydrocarbon-rich region is located in the core of lipophorin particle (Katagiri, C., Kimura, J., and Murase, N. (1985) J. Biol. Chem. 260, 13490-13495). Distance distribution functions, P (r), calculated for a simulated three-layer model (electron-rich shell, middle layer, and electron-deficient core) with radial electron density distribution, show good agreement with those observed experimentally for cockroach and locust lipophorins. The dimensions and electron density obtained for the middle layer reveal that this layer is occupied mainly by diacylglycerol and apolipophorin II. Thus, the present study together with previous reports strongly suggest that insect lipophorin is composed of centrosymmetrical three layers; an outer shell with apolipophorin I and phospholipid, a middle layer with diacylglycerol and apolipophorin II, and a core with hydrocarbons.     

Characterization of Colorado potato beetle lipophorin: A hydrocarbon-rich diacylgycerol-poor lipophorin

• 1.1. The lipid composition of lipophorin from the Colorado potato beetle, Leptinotarsa decemlineata Say, was analyzed.
• 2.2. This insect lipophorin contains 44% lipid and is characterized by large amounts of hydrocarbons and small amounts of diacylglycerol.
• 3.3. This is the first observation of a diacylglycerol-poor insect lipophorin in haemolymph.
• 4.4. Since the main energy source for flight in the Colorado potato beetle is proline, the low diacylglycerol content in lipophorin must be related to its peculiar flight metabolism.
• 5.5. This lipophorin, however, can still take up appreciable amounts of diacylglycerol from the locust fat body. Hydrocarbon uptake by this lipophorin was also demonstrated.
• 6.6. The main function of this lipophorin therefore seems to be transport of hydrocarbons from oenocytes to the cuticle.

     

Molecular characteristics of lipophorin,the juvenile hormone-binding protein in the hemolymph of the Colorado potato beetle

Lipophorin, the protein that specifically binds juvenile hormone in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, is a high-density lipoprotein of M_r ～ 574,000. Lipophorin contains 43% lipid and is composed of two apoproteins: apolipophorin I (M_r ～ 251,000) and apolipophorin II (M_r ～ 78,000). Both apoproteins contain mannose residues. Carotenoids make up a substantial part of the lipid fraction. Lipophorin constitutes about 25% of the total hemolymph proteins. Its concentration in the hemolymph (26 μM in 4-day-old long-day and 40 μM in 4-day-old short-day beetles) changes with different physiological conditions concomitant with changes in total protein content. Lipophorin specifically binds 10R-juvenile hormone III with high affinity. The dissociation constant for 10R-juvenile hormone III is 12 ± 2 nM. One lipophorin molecule contains one specific juvenile hormone-binding site. The concentration of binding sites therefore equals that of lipophorin in hemolymph.     

Structural studies of lipophorin in insect blood by differential scanning calorimetry and 13C nuclear magnetic relaxation measurements. Location of hydrocarbons

The possible structure of lipophorin in insect blood (hemolymph) was investigated by differential scanning calorimetry (DSC) and 13C nuclear magnetic relaxation studies. The DSC heating curves of intact lipophorins showed endothermic peaks between -3 and 40 degrees C for lipophorins which contain hydrocarbons, whereas no such peaks were observed for lipophorins which do not contain this lipid. Hydrocarbon fractions isolated from the lipophorins showed endothermic peaks similar to those obtained from intact lipophorin in terms of the transition temperatures, the shapes, and the enthalpy changes. 13C spin lattice relaxation times of the (CH2)n resonance of hydrocarbons of intact lipophorin were measured as a function of temperature and revealed that the motions of hydrocarbon chains changed coincidentally with the onset and offset of phase transition. These data suggest the presence of a hydrocarbon-rich region within the lipophorin particles.     

Insect lipophorin conversions. Compositional analysis of high- and low-density lipophorin of Acherontia atropos and Locusta migratoria.

The formation of low-density lipophorin (LDLp) in insect hemolymph, resulting from association of high-density lipophorin (HDLp) with both lipid and apolipophorin III, is considered to provide a reutilizable lipid shuttle for flight muscle energy supply. The changes in lipid and apolipoprotein composition of LDLp, isolated after flight activity, compared to that of HDLp in the hemolymph at rest, were studied in two evolutionary divergent insects, the hawkmoth Acherontia atropos and the migratory locust, Locusta migratoria. Using FPLC on Superose 6 prep grade as a novel technique to separate the apolipophorins of HDLp and LDLp, the ratio of apolipoprotein I, II, and III in HDLp of both species was demonstrated to be 1:1:1, whereas flight activity resulted in a ratio of 1:1:10 in LDLp. Injection of adipokinetic hormone into resting moths showed that, depending on the dose, the number of apolipophorin III molecules in LDLp can exceed that recovered after the physiological condition of flight. Analysis of the lipophorin lipids demonstrated that in addition to the considerable increase in diacylglycerol in the LDLp particle, which is consistent with the role LDLp in energy supply, particularly the hydrocarbons were increased compared to HDLp, rendering the mechanism of LDLp formation from HDLp even more complex.     

Biosynthesis and secretion of insect lipoprotein.

Biosynthesis of high density lipophorin (HDLp) was studied in larvae and adults of the migratory locust, Locusta migratoria. In an in vitro system, fat bodies were incubated in a medium containing a mixture of tritiated amino acids. Using SDS-PAGE and immunoblotting, it was shown that larval and adult fat bodies secreted both HDLp apoproteins, apolipophorin I (apoLp-I) and apolipophorin II (apoLp-II). Radiolabel was recovered in both apoproteins, indicative of de novo synthesis. The density of the fractions containing the apoproteins synthesized and secreted by larval and adult fat bodies was determined by density gradient ultracentrifugation. A radiolabeled protein fraction was found at density 1.12 g/ml. Using an enzyme-linked immunosorbent assay for detecting apoLp-I and apoLp-II, it was demonstrated that both apoproteins were present in this fraction, which had a density identical to that of circulating HDLp in hemolymph. Lipid analysis revealed that it contained phospholipid, diacylglycerol, sterol, and hydrocarbons. From these results it is concluded that the fat body of the locust synthesizes both apoLp-I and apoLp-II, which are combined with lipids to a lipoprotein particle that is released into the medium as HDLp.     

The molecular and metabolic essentials for long-distance flight in insects

Summary The mechanism of long-distance flight in insects was investigated by comparing lipid mobilization and transport in gregarious- and solitary-phase locusts and in the American cockroach. Unlike the gregarious-phase locust, both the American cockroach and the solitary locust were unable to form low-density lipophorin (loaded with increased amount of diacylglycerol) even when injected with adipokinetic hormone (AKH). The cockroach fat body responded to AKH. However, not only does the American cockroach lack apolipophorin-III (apoLp-III) in the haemolymph, but the fat body contains only an extremely small amount of diacylglycerol and a relatively large triacylglycerol pool. By contrast, the solitary-phase locust had apoLp-III in the haemolymph, but the fat body was only one-seventh or less in weight of the fat body of the gregarious locust. Furthermore, the fat body of the solitary locust contains a very small amount of triacylglycerol (1/20 or less of that of the gregarious locust) with only a trace of diacylglycerol. It was concluded that in the American cockroach and the solitary locust, the stores of fuel in the fat body are insufficient to maintain prolonged flight.Abbreviations AKII adipokinetic hormone - apoLp-III apolipophorin III - HDLp high-density lipophorin - LDLp low-density lipophorin - LTP lipid transfer particle - MW molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

The effect of lintnerisation on wheat and potato starch granules

Wheat and potato starches were hydrolysed with 2·2 n hydrochloric acid at 35°C for a period of time up to 15 days. The residues (lintnerised starches) were washed and freeze dried, and studied by differential scanning calorimetry (DSC), wide-angle X-ray scattering (WAXS), small-angle light scattering (SALS), small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). These techniques showed that profound changes took place in the first day of hydrolysis (during which time the extent of hydrolysis was 7·7% for potato starch and 12·5% for wheat starch). In particular, the gelatinisation enthalpy (ΔH) decreased, the X-ray crystallinity increased and the SANS and SAXS peaks (indicative of a regular spacing between crystalline and amorphous regions) virtually disappeared. The reduction in ΔH is surprising and is discussed at length. It was also shown that freeze drying results in a considerable lowering of the gelatinisation temperature of potato starch (and also of ΔH) while that of wheat starch is only slightly affected.     

Lipophorin in the larval and adult stages of Musca domestica

The major Musca domestica hemolymph lipoprotein, lipophorin, was purified from larval and from adult animals. The housefly lipophorin is composed of two apoproteins, apolipophorin I (M_r ∽ 253,000) and apolipophorin II (M_r ∽ 85,000). The lipophorin contains about 3.9% carbohydrates and reacts positively with concanavalin A. The density of larval lipophorin is equal to 1.152 g/ml and of adult lipophorin to 1.106 g/ml. The amount of lipophorin per animal increases during the larval stage, is constant during pupal stage, and suffers a great reduction at the pharate adult stage. The amount of lipophorin remains stable during the whole first gonotrophic cycle of the housefly. Lipophorin is not detected in the eggs of this insect.     

Effect of adipokinetic hormone on the structure and properties of lipophorin in locusts

The reversible association of a low molecular weight hemolymph protein (mol wt 20,000 estimated by SDS-polyacrylamide gel electrophoresis) with lipophorin, following treatment with adipokinetic hormone (AKH), was demonstrated by density gradient ultracentrifugation and by specific precipitation of lipophorin from the hemolymph of resting and AKH-injected locusts. The injection of AKH also stimulated the loading of diacylglycerol from fat body by lipophorin and resulted in a lower density lipophorin ("activated lipophorin"). The activated lipophorin particles (diameter 21.7 +/- 3.0 nm, 15.8 to 33.6 nm) were larger and more heterogeneous in size than those of resting lipophorin (14.5 +/- 1.6 nm, 11.9 to 19.2 nm). A theoretical analysis based on the experimental data (e.g., density gradient profile, electron microscopic observation, and diacylglycerol content) suggests that very large lipophorin particles result from intermolecular fusion of the lipophorin molecules that are activated by AKH. Attempts to demonstrate the effect of AKH on the structure of lipophorin, in vitro, were unsuccessful.     

Juvenile hormone and the control of adult diapause in the Colorado potato beetle,Leptinotarsa decemlineata

One of the major hormonal events preceding environmentally controlled diapause in the Colorado potato beetle, Leptinotarsa decemlineata, is the inhibition of the corpora allata. This paper reviews briefly the data developed by our laboratory on the role of the neuroendocrine system in the control of the corpus allatum. In addition, we demonstrate that during transport in the hemolymph JH is bound to a JH-III specific binding protein. The JH-binding sites of this protein have a high affinity for JH-III and display stereoselectivity. New data are presented showing that, in beetle hemolymph, JH-III specific binding occurs with the lipophorin fraction.     

Genetic Structure of Populations of the Colorado Potato Beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The genetic structure of a Colorado potato beetle population from Kiev oblast was examined by cluster analysis of individual RAPD patterns. The obtained clustering indicates that the population is structured. This may be explained by adaptation to pyrethroid insecticides used for controlling the population size of this pest. Microevolutionary factors affecting the genetic structure of local populations of Colorado potato beetle are discussed.     

Genetic structure of populations of the Colorado potato beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The genetic structure of a Colorado potato beetle population from Kiev oblast was examined by cluster analysis of individual RAPD patterns. The obtained clustering indicates that the population is structured. This may be explained by adaptation to pyrethroid insecticides used for controlling the population size of this pest. Micro-evolutionary factors affecting the genetic structure of local populations of Colorado potato beetle are discussed.     

Manduca sexta lipid transfer particle acts upon a lipoprotein to catalyze lipid and apoprotein disproportionation

A novel reaction, catalyzed by Manduca sexta lipid transfer particle (LTP), transforms low density lipophorin (LDLp) into two distinct lipoprotein species. A population of LDLp particles serves as lipid donor or acceptor in LTP-catalyzed production of a very low density lipophorin (VLDLp) and a high density lipophorin (HDLp) product. The products result from facilitated net transfer of lipid mass from donor LDLp particles to acceptor LDLp particles. Transfer of apolipophorin III (apoLp-III) from donor to acceptor lipoprotein occurs during the reaction to produce a lipid- and apoLp-III-enriched VLDLp species and lipid- and apoLp-III-depleted HDLp species. The VLDLp produced in this in vitro reaction contains more lipid and apoLp-III than any previous lipophorin species reported and further demonstrates the scope of the lipid binding capacity of lipophorin. Lipid analysis and radiolabeling studies confirmed that unidirectional net transfer of lipid mass and apoLp-III from donor to acceptor occurs. When 3H-lipid-LDLp was used as substrate in the LTP-catalyzed disproportionation reaction the density distribution of radioactivity and protein provided evidence of vectorial transfer of diacylglycerol, phospholipid, and free fatty acids. Electron micrographs of the original LDLp population and of the LTP-induced product lipoprotein population provided further support for the interpretation derived from biochemical studies. This LTP-catalyzed disproportionation was observed only with apoLp-III-rich LDLp suggesting that the presence of increased amounts of this apoprotein dramatically affects the properties of the particle and appears to be directly related to the capacity of the lipoprotein to bind lipid.     

基于GIS的马铃薯甲虫扩散与河流关系研究——以新疆沙湾县为例

马铃薯甲虫是一种重要的外来入侵害虫,自1993年传入我国新疆北部地区以来,迅速扩散传播,对我国马铃薯等茄科作物生产造成严重威胁。为明确马铃薯甲虫传播扩散与河流等生态因子之间的关系,结合马铃薯甲虫虫情调查,利用地理信息系统(GIS)的空间分析功能,对新疆沙湾县马铃薯甲虫发生与河流的关系进行研究。结果表明:在沙湾县内,马铃薯甲虫扩散方向与河流流向基本一致,均呈由南向北方向;不同缓冲区内,马铃薯甲虫危害级别差异极显著,缓冲区危害级别从大到小依次:缓冲区2(1-2 km)>缓冲区4(3-4 km)>缓冲区5(4-5 km)>缓冲区1(0-1 km)>缓冲区3(2-3 km);不同河流流域内马铃薯甲虫危害级别也差异极显著,危害级别排序依次是:金沟河>安集海河>玛纳斯河>西岸大渠。     

Effect of crop rotation distance on populations of Colorado potato beetle (Coleoptera: Chrysomelidae): development of areawide Colorado potato beetle pest management strategies

The Colorado potato beetle, Leptinotarsa decemlineata (Say), overwinters adjacent to field edges and infests nearby fields in the spring, primarily by walking. Crop rotation is known to be an effective cultural control against Colorado potato beetle populations limiting spring infestations. Spatial separation is an important consideration in optimizing the rotational effect because long-distance rotations have been shown to reduce Colorado potato beetle infestations. To determine the effect of long-distance rotations on Colorado potato beetle populations in commercial Wisconsin potato production, a geographic area of >18,200 ha (>45,000) acres in southern Portage County, Wisconsin, was selected as an experimental area for areawide pest management of the Colorado potato beetle. From 1997 to 1999, beetle populations at edges within each potato field in this region were determined by spring and fall field sampling. The rotational distance between current and previous potato fields was measured and analysis was run between the distance and Colorado potato beetle populations. Long-distance rotations of >400 m were an effective cultural control management strategy to limit adult beetle infestations in the spring. This strategy can be optimized when collaborating growers are able to maximize their rotational distances by coordinating their rotational schemes within large areawide, geographic locations. Deploying long-distance rotations within a geographic area over many years would limit Colorado potato beetle populations and could result in a significantly reduced Colorado potato beetle populations entering fields in the spring.     

Flight take-off performance of Colorado potato beetle in relation to potato phenology

The flight take-off frequency of adult Colorado potato beetles, Leptinotarsa decemlineata (Say), from potato plants, Solanum tuberosum L. 'Red Pontiac' at the bloom stage of development was 2.2-2.5-fold that of Colorado potato beetle from plants at the vegetative stage. Tests were conducted in a flight chamber over a period of 3 h. Prefeeding Colorado potato beetles for 48 h on potato plants at the bloom or at the vegetative stage before placing them into the flight chamber resulted in the same significantly higher flight take-off frequency from potato plants at the bloom stage than from plants at the vegetative stage. These results demonstrate that the factor in potato plants in bloom that stimulates the flight take-off of the Colorado potato beetle is independent of the feeding history of the beetles and begins acting only when the beetles are in the presence of the plant. According to these results, the dispersal of adult Colorado potato beetles from potato fields in bloom to younger potato fields with plants at the vegetative stage, previously reported in the literature, is at least partly explained by the effect of plant phenology on the frequency of flight take-off. Results confirm the value of planting potato fields of similar phenology over as wide an area as possible to reduce Colorado potato beetle dispersal between fields. Results also imply that staggering the planting dates of conventional potato refuge areas near Colorado potato beetle transgenic or conventionally resistant potato fields is a sound management practice, because it promotes the movement of wild beetles over to the adjacent younger resistant crops.     

Lipophorin structure analyzed by in vitro treatment with lipases.

Adult Manduca sexta high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-I, -II, and -III) and 52% lipid. The flight-specific low density lipophorin (LDLp) contains 62% lipid and is associated with several additional molecules of apoLp-III. The amount of phospholipid remains constant in lipophorin (140 mol/mol of lipophorin), while the diacylglycerol content varies between different lipophorin species (310 mol/mol HDLp up to 1160 mol/mol LDLp). Both lipophorin particles were enzymatically depleted of phospholipid or diacylglycerol by in vitro incubation with either phospholipase A2 or triacylglycerol lipase. Albumin was used to remove free fatty acids generated during the reaction. Treatment with phospholipase A2 removed all phospholipids (except sphingomyelin) and the resulting particles were stable. Triacylglycerol lipase hydrolyzed large fractions of diacylglycerol. The resulting particles were smaller in size, higher in density, and devoid of apoLp-III. The particles retained apoLp-I and -II and the other lipid components, including a substantial amount of diacylglycerol. Structural integrity of diacylglycerol-depleted lipophorin was confirmed by electron microscopical analysis. When treated with both phospholipase A2 and triacylglycerol lipase, lipophorin precipitated. From these results we conclude that: 1) all phospholipid and apoLp-III are located at the surface of lipophorin, whereas diacylglycerol is partitioned between the sublayers and the surface of the particle; 2) both diacylglycerol and phospholipid play a role in stabilizing lipophorin in the aqueous medium; and 3) lipophorin can be extensively unloaded and still retain its basic structure, a necessary feature for its function as a reusable lipid shuttle.     

Chemical and immunological properties of lipophorins from seven insect orders

The major insect hemolymph lipoprotein, lipophorin, was isolated from adults of eight insect species representing seven insect orders. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to compare their respective apoprotein components. In all species examined lipophorin was composed of at least two apoproteins, apolipophorin I (M_r ～ 250,000) and apolipophorin II (M_r ～ 78,000), and two species had a third apoprotein, apolipophorin III (M_r ～ 17,000). The density of each isolated lipophorin was determined from the refractive index of KBr following density gradient centrifugation. Immunoblotting with anti-larval Manduca sexta apolipophorin I and II of the apoproteins separated by SDS-PACE indicated cross reactivity between anti-M sexta apoLp-ll and apoLp-ll in all species tested. Anti-M sexta apoLp-l exhibited no cross reactivity for any species tested. Fluorescent lectin staining of the apoproteins separated on SDS-PAGE gels revealed the presence of covalently bound carbohydrate residues.     

Pymetrozine causes a nontarget pest, the Colorado potato beetle (Coleoptera: Chrysomelidae), to leave potato plants

Pymetrozine is a selective insecticide that targets aphids. Published assessments of the effects of pymetrozine on nontarget organisms focus mainly on predatory insects, and they rarely indicate toxicity. In a laboratory bioassay, survival of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), larvae was not affected by pymetrozine exposure. We subsequently used pymetrozine to implement low-aphid-density treatments in a field experiment that involved separate manipulations of Colorado potato beetle density. Unexpectedly, the addition of Colorado potato beetle adults and eggs did not increase the densities of Colorado potato beetle larvae in plots that were sprayed with pymetrozine (applied with water and an adjuvant). In control plots sprayed with water and adjuvant (without pymetrozine), addition of Colorado potato beetles increased densities of their larvae. Data collected on a smaller scale suggest that a behavioral mechanism underlies the population-level pattern: Colorado potato beetle larvae become more active and are less likely to remain on a host plant after exposure to pymetrozine. Thus, potato, Solanum tuberosum L., growers who use pymetrozine against aphids also might benefit in terms of Colorado potato beetle control.