Comparative metabolism of isoleucine by corpora allata of nonlepidopteran insects versus lepidopteran insects,in relation to juvenile hormone biosynthesis

We studied the metabolism of [U-¹⁴C]isoleucine by intact and homogenized corpora allata (CA) from various insect species to determine how this substrate is converted to precursors of juvenile hormone (JH). CA homogenates of the lepidopterans Manduca sexta, Hyalophora cecropia, and Samia cynthia metabolize [U-¹⁴C]isoleucine to several products including 2-keto-3-methyl-valerate, 2-methylbutyrate, CO₂, propionate, and acetate. Intact CA of male H. cecropia produce particularly high levels of 2-keto-3-methylvalerate, indicating a highly active branched-chain-amino acid transaminase. In contrast, CA homogenates from the nonlepidopterans Periplaneta americana, Schistocerca nitens, Tenebrio molitor, and Diploptera punctata barely metabolize [U-¹⁴C]isoleucine. However, P. americana CA homogenate metabolizes [U-¹⁴C]2-keto-3-methylvalerate, the transamination product of [U-¹⁴C]isoleucine, more rapidly than does a homogenate of M. sexta CA. Furthermore, intact CA from P. americana incubated with [U-¹⁴C]2-keto-3-methylvalerate incorporate low levels of ¹⁴C into JH III, but do not metabolize this substrate to JH II or JH I. Intact CA from female Diploptera punctata produce very high levels of JH III, but are also unable to incorporate radiolabel from [U-¹⁴C]isoleucine into JH III, which substantiates our findings with other nonlepidopteran CA. The results suggest that CA of nonlepidopteran insects lack an active branched-chain amino acid transaminase and, consequently, are unable to utilize these substrates for JH biosynthesis.     

Pertussis toxin-sensitive G protein that supports vitellogenin uptake by promoting patency

Ovarian follicles of Hyalophora cecropia stopped accumulating [³⁵S]vitellogenin when incubated in pertussis toxin, a G_i protein inactivator. At a cellular level, the responses to pertussis toxin resembled those described earlier to cell-permeant analogs of cyclic AMP. They included accelerated ³⁶Cl^– exchange, ⁸⁶Rb⁺ uptake, and follicle cell swelling, which in turn resulted in a loss of epithelial patency. A 34% rise in follicular cAMP content accompanied these changes. In particulate fractions of follicle homogenates, pertussis toxin catalyzed the ADP-ribosylation of a polypeptide that resolved at 39 kDa in SDS-PAGE; rabbit antibodies to a C-terminal decapeptide common to 39 kDa mammalian G_iα-3 and G_oα were bound in immunoblots at this same location. The findings suggest that a pertussis toxin-sensitive Gα facilitates epithelial patency during vitellogenesis by suppressing cAMP levels. When follicles are released from this restraint, either experimentally with pertussis toxin or by progressing to the next phase in their normal program of development, cAMP levels rise and vitellogenesis terminates. Arch. Insect Biochem. Physiol. 39:36–45, 1998. © 1998 Wiley-Liss, Inc.     

Effects of ionophores on vitellogenin uptake by Hyalophora oocytes

Oocytes of Hyalophora cecropia that were incubated in vitro with [³⁵S]vitellogenin incorporated label within 10 min into an intermediate-density compartment identified by sucrose density gradient centrifugation. During a subsequent 20-min chase this presumptive endosomal label was transferred to a compartment with the higher density of protein yolk spheres. When vitellogenin uptake was inhibited by 10 μM nigericin or monensin, or 50 μM carbonyl cyanide m-cholorophenylhydrazone, a somewhat larger and more focused peak of label accumulated in the endosome region of the gradient, and the transfer of this label to the yolk spheres was blocked. Valinomycin, at concentrations as high as 100 μM, did not inhibit uptake or processing, even though successful insertion into the oocyte membrane could be demonstrated by the effects of this ionophore on the membrane potential and K⁺ permeability of the follicle. Inhibition of processing by nigericin and monensin is consistent with a model of endocytosis in which the ionophores prevent acidification of the endosomes by promoting H⁺-K⁺ exchange with the cytoplasm. Several alternative possibilities were ruled out by physiological analyses entailing the measurement of cytoplasmic pH and membrane potentials.     

Distribution of cuticular protein mRNAs in silk moth integument and imaginal discs

The distributions of mRNAs for two cuticular proteins of Hyalophora cecropia were examined with RT-PCR and in situ hybridization. For major regions of larval and pupal cuticle, there was a strong correspondence between the type of cuticle and the predominant cuticular protein message found. Epidermal cells underlying soft cuticle had mRNA for HCCP12, with a RR-1 consensus attributed to soft cuticle, while the epidermal cells associated with hard cuticle had predominantly mRNA for HCCP66, a protein with the RR-2 consensus attributed to hard cuticle. Both messages were found in all areas of the pupal fore- and hind-wings, with modest area-specific difference in concentration being much less than differences in the relative abundance of these cuticular proteins.

mRNA for HCCP12 was present in imaginal discs of feeding larvae of H cecropia. Data from Bombyx mori available at SilkBase (http://www.ab.a.u-tokyo.ac.jp/silkbase/) revealed that imaginal discs from feeding larvae had abundant mRNA for RR-1 cuticular proteins, representing six distinct gene products. Only discs from spinning larvae had mRNAs that coded for RR-2 proteins arising from 10 distinct genes. Thus, lepidopteran wing imaginal discs can no longer be regarded as inactive in larval cuticle production.     

Cuticular proteins: The neglected component

This paper emphasizes the importance of the protein component of cuticles. Correlation of electrophoretic charge distribution of individual cuticular proteins and physical properties of the cuticles from which they were extracted, as well as interpopulation and interspecies conservation of electrophoretic patterns, are used to argue that individual proteins play precise roles in the cuticle. Glycosylation of cuticular proteins is described, but no function for these modifications is yet known. Analogy is drawn to analyses of chorion proteins and the case is made that analysis of amino acid sequence data is likely to provide insights into how cuticular proteins and chitin interact to construct the diverse types of cuticles.     

Cordyceps cateniannulata,a novel entomopathogenic fungus to control Stenoma impressella Busck (Lepidoptera: Elachistidae) in Colombia

Stenoma impressella is one of the most important defoliator pests in oil palm plantations in Colombia. To identify an alternative method for its control was characterized biologically and molecularly two strains of Cordyceps cateniannulata (CPIsp1201 and IPIsp1201) and three strains of Beauveria bassiana (CPBb0502; CPBb0411; CPBb0404) against S. impressella larvae. Virulence was evaluated under laboratory conditions. In an oil palm leaflet, individual larvae obtained from the insect colony were inoculated with 5 μl of a conidial suspension containing 1 × 10⁷ conidia/ml. The five strains were pathogenic against S. impressella larvae. CPIsp1201 and IPIsp1201 strains resulted in the highest mortality and were subsequently evaluated in two bioassays using a dose of 1 × 10¹³ conidia/ha. In the first bioassay, performed under shaded conditions, leaves of oil palms were infested with 75 larvae from the breeding/treatment. The second bioassay was performed in the field using natural populations. No differences were found between strains in both bioassays and the different dosages (5 × 10¹², 1 × 10¹³, and 1.5 × 10¹³ conidia/ha). Finally, the two strains were evaluated under oil palm plantation conditions at a dose of 1 × 10¹³ conidia/ha in 126 naturally infested palms. Larval mortality caused by the strains IPIsp1201 and CPIsp1201 (79.5% and 70.5%, respectively) was higher than the natural mortality registered in the control (37.3%). Cordyceps cateniannulata used at 1 × 10¹³ conidia/ha was effective at controlling S. impressella.     

Adsorptive endocytosis of vitellogenin,lipophorin, and microvitellogenin during yolk formation in Hyalophora

Yolk in Hyalophora cecropia is a mixture of proteins that are derived from the extracellular medium. We have measured for five of these proteins the number of moles deposited in each egg, the molarity of their precursors in the hemolymph at a midpoint in vitellogenesis (day 18 of adult development), and the degree to which they are concentrated by the oocyte, relative to inulin. The proteins were isolated by gel permeation and ion exchange chromatography and used to generate antibodies in rabbits. Preliminary studies established that yolk proteins are essentially quantitatively extractable in media suitable for measuring antigen concentrations by precipitation with antibodies and that yolk and hemolymph forms of the five proteins have, effectively, the same antibody-binding specificities as the isolated standards. Content per egg was about 900 pmol for vitellogenin, 600 pmol for microvitellogenin, and 300 pmol for lipophorin. By contrast, two hemolymph storage hexamers, arylphorin and a flavoprotein, occurred at less than 3 pmol per egg. In principle, yolk precursors are taken in both as solutes in the fluid phase of the endocytotic vesicles and as ligands adsorbed to vesicle membranes. Measurements of inulin uptake indicated that fluid phase endocytosis could account for only 4% of vitellogenin, 1% of microvitellogenin, and 15% of lipophorin in the yolk, when hemolymph precursors are at their day 18 concentrations. By the same comparison, arylphorin and flavoprotein appear to be excluded from the yolk, relative to inulin.