Abstract
Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56% lipids and 9 and 7% carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.
Highlights
Cowpea (Vigna unguiculata) is a leguminous crop cultivated in most tropical regions, where its seeds contribute to human and animal diets as an important source of carbohydrates and protein
The soluble protein fraction from homogenates of larvae and adults of C. maculatus was subjected to a KBr gradient ultracentrifugation and the fractions were analyzed by SDS-PAGE (Figure 1)
Lipophorin labeling Purified Lp was labeled with fluorescent fatty acid (BODIPY® FL C16) or phospholipids (TRITC-DHPE; Molecular Probes Inc., Eugene, OR, USA) using the method described by Martin-Nizard et al [22]
Summary
Cowpea (Vigna unguiculata) is a leguminous crop cultivated in most tropical regions, where its seeds contribute to human and animal diets as an important source of carbohydrates and protein. Insects from the order Coleoptera (family Bruchidae) cause serious losses of cowpea and other seeds in storage. One of the most important insect pests of the cowpea is the larva of Callosobruchus maculatus (Coleoptera: Bruchidae), which avidly attacks the seeds during storage. Studies of Coleoptera in general and bruchids in particular are underrepresented in insect biochemistry, despite the fact that Coleoptera is the largest insect order. Most biochemical research on beetles has concentrated on the description of digestive enzymes [1]. A detailed description of the lipid metabolism of these organisms is lacking
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