Insect Biochemistry最新文献

Using ethanol-chloroform fractionation in conjunction with standard column chromatography techniques catalase has been purified to electrophoretic homogeneity from mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni. The specific activity of purified catalase was 2.2 × 10⁵ units (IU = 1 μmol H₂O₂ decomposed mg protein⁻¹ min⁻¹). The purified enzyme's native molecular weight was in the 247,000–259,000 Da range and was tetrameric with an apparent molecular weight of 63,000 Da for each subunit. In addition, biochemical properties of the enzyme were studied with emphasis on substrate specificity, kinetics, and the mechanism of inactivation by the irreversible inhibitor 3-amino-1,2,4-triazole (AT). The apparent K_m of the purified catalase for H₂O₂ was 54.2 mM and 50% of the maximal rate occurred at 16 mM H₂O₂. Purified catalase was ineffective in metabolizing organic hydroperoxides and, unlike other catalases, lacked peroxidase activity. Lastly, AT in the presence and absence of H₂O₂ was an effective inhibitor of catalase activity (I₅₀ = 100 mM) suggesting that a portion of the purified catalase was complexed with hydrogen peroxide in a compound 1 configuration.

A unique pattern of antioxidant enzymes exists in phytophagous insects for defense against endogenous and exogenous sources of toxic forms of oxygen, and data presented herein describe a profile of these enzymes in many tissues of larvae of the cabbage looper moth, Trichplusia ni. The specific activities of the antioxidant enzymes were high in tissues of high metabolic activities, i.e. Malpighian tubules, hindgut, muscles and gonads. A unique finding was the high constitutive activity of a superoxide dismutase (SOD) in hemocytes, probably consisting predominantly of the CuZn-SOD, which is analogous to the exclusive presence of this form of SOD in vertebrate erythrocytes and leukocytes. In all other tissues, the activity of Mn-SOD was higher than that of the CuZn-SOD which is converse to the pattern in vertebrate tissues. The glutathione peroxidase (GPOX) activity, present in all tissues and with highest levels in the gonads, did not seem to be the selenoprotein typical of the mammalian GPOX. $\text{Glutathione}\text{-S-}\text{transferase}$ (GST) activity paralleled that of its glutathione peroxidase activity (GSTPX). The high GSTPX activity suggests that GSTPX and not GPOX, forms a sequential team with glutathione reductase (GR) to reduce deleterious lipid hydroperoxides and to reduce the oxidized glutathione, GSSG, back to GSH. Catalase (CAT) which decomposes H₂O₂ has very high activity apparently correlated with the low GPOX activity. Finally, the integumental epithelium, and the gut (combined sections) possessed higher amounts of antioxidant enzymes than other tissues. Thus, a physiological relationship may occur between the antioxidant enzyme levels in tissues of T. ni with particularly high metabolic activity and associated endogenous oxidative stress. In addition, another physiological role of these enzymes may be to protect from exogenous oxidative stress exerted by dietary redox-active pro-oxidants in the gut, and to the potential of photodynamically mediated oxygen toxicity in peripheral organs such as the integument.

Lipophorin in Hyalophora cecropia changes from a high density form in diapausing pupal hemolymph (1.143 g/ml in males and 1.139 g/ml in females) to a predominantly low density form in resting adults (1.043 g/ml). The transition takes place primarily during adult eclosion. One week before eclosion, lipophorin undergoes changes in females that are correlated with yolk formation. It becomes 50% more concentrated than in males, its density drops to 1.124 g/ml (compared with 1.134 g/ml in males), it binds apolipophorin III with a stoichiometry of 1:1, and it is joined by small amounts of an adult-like low density lipophorin. Following ovariectomy, lipophorin differed only marginally from this, suggesting that a feedback mechanism prevents it from being encumbered with lipids beyond the level in females that are making yolk. During adsorptive endocytosis by the oocyte, 94% of the lipophorin that is deposited in the yolk is converted to a very high density form (1.232 g/ml), presumably due to the lipid unloading demonstrated earlier in two other lepidopterans. Apolipophorin III and some additional antigenic epitopes of the hemolymph lipophorins were undetectable in the unloaded yolk protein. Yolk contains in addition small amounts of free apoliphorin III and of a high density lipophorin similar to that of the hemolymph; both of these components occur at levels expected for proteins accumulated as solutes in the fluid phase of the oocyte's endocytotic vesicles. Several models are suggested to explain how the apolipophorin III that is bound to lipophorin in the hemolymph of vitellogenic females escapes deposition in the yolk.

The amount of glutathione S-transferase activity present in various stages of development and in various tissues of an insecticide susceptible strain of Helicoverpa zea was determined. GSH S-transferase activity toward CDNB (1-chloro-2,4-dinitrobenzene) was detected in the egg, and increased throughout the larval stages and was the highest in the 2 day old fifth instar larvae. GSH S-transferase activity toward DCNB (1,2-dichloro-4-nitrobenzene) could only be detected in the 2 day old fourth instar larvae, and the 2 day old fifth instar larvae. Enzyme activity using CDNB and DCNB as substrates was found in different tissues of the 2 day old fifth instar larvae, i.e. fat body, gut and cuticle. The fat body contained the highest enzyme activity for both substrates. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for CDNB conjugation in different stages of larval development and in the different tissues of the 2 day old fifth instar larvae, was approx. 0.24 mM. The enzyme responsible for CDNB-conjugation was purified 48 fold by GSH-agarose affinity chromatography. SDS gel electrophoresis and chromatofocusing resolved only one isozyme which is a homodimer, consisting of two equal size subunits having 23,900 mol. wt, with a pI value of 8.0.

Females of the German cockroach, Blattella germanica, produce a contact sex pheromone consisting of the methyl ketones 3,11-dimethyl-2-heptacosanone, 3,11-dimethyl-2-nonacosanone, 29-hydroxy- and 29-oxo-3,11-dimethyl-2-nonacosanone. We review evidence in support of the hypothesis that in adult females the hydrocarbon 3,11-dimethylnonacosane is oxidized to the corresponding methyl ketone pheromone. Recent studies on the precursors and directionality of synthesis of the methyl-branched alkane indicate that it is formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation. The hydrocarbon is then hydroxylated and oxidized at the 2-position to form the methyl ketone pheromone. The in vivo synthesis of pheromone and its accumulation on the cuticle are correlated to the synthesis of juvenile hormone (JH) by the corpora allata (CA) in vitro and to oocyte development, suggesting common regulation by JH of pheromone production as well as other reproductive events. The patterns of pheromone and hydrocarbon production in starved, allatectomized and head-ligated females, as well as in females rescued with hormone-replacement therapy, suggest two mechanisms of regulation of pheromone production: a JH-induced conversion of hydrocarbon to pheromone is related to the CA cycle and to oocyte development, while a JH-independent mechanism, which is probably related to feeding, supplies precursors for hydrocarbon biosynthesis.

The transparent accessory reproductive gland of Rhodnius prolixus synthesizes and accumulates a variety of polypeptides. Ouchterlony immunodiffusion demonstrates that the hemolymph contains proteins which react with polyclonal antibodies against extracts of transparent accessory glands. Accessory glands and hemolymph contain a 170 kDa polypeptide with similar mobility on SDS-polyacrylamide gel electrophoresis. This polypeptide reacts with antibodies against extracts of accessory glands. Surgical removal of the accessory glands prevents the appearance of the 170 kDa polypeptide in the hemolymph. In vivo labeling of accessory gland proteins with a mixture of [¹⁴C]amino acids demonstrates that the newly synthesized TARG polypeptide appears in the hemolymph between days 2 and 3 after feeding. It is concluded that a specific polypeptide which is synthesized in the transparent accessory gland is exported to the hemolymph.

Egg-specific protein (ESP) is a yolk protein which is synthesized in developing ovarian follicles of the silkworm, Bombyx mori. We have isolated a gene coding for ESP from a genomic DNA library using cDNA as a probe and sequenced about 2.9 kb including 5′- and 3′-flanking regions. Three closely located sites were identified as transcription initiation sites by primer extension analysis. In the 5′-upstream region, there are several short sequences which are homologous to the regulatory elements of other genes: short sequences homologous to the ecdysteroid response elements of the heat shock protein genes and the glue protein genes, $\text{cis-}\text{acting}$ elements of chorion genes, and a protein factor binding site of the fibroin gene. Southern hybridization analysis revealed the presence of a single copy of the ESP gene per a haploid genome. The amino acid sequence was highly homologous to the human gastric and rat lingual lipases.

Northern hybridization analysis of RNA prepared from various tissues at different developmental stages showed that the ESP gene is expressed only in follicles undergoing vitellogenesis. Injection of 20-hydroxyecdysone into the isolated pupal abdomen stimulated the expression of the ESP gene with a lag period of 2 days.

It now appears that arthropods produce and release a wider variety of juvenile hormones (JH) and related compounds than previously thought. For instance, in the adult crayfish, Procambarus clarkii, the mandibular organs, the homologous structure to insect corpora allata (CA), release both farnesoic acid (FA) and methyl farnesoate (MF), the immediate precursors of JH III, but not JH III itself. In larvae of the cockroach Diploptera punctata, JH III production ceases during the last half of the 4th stadium, but the CA continue to produce and release FA throughout this period. The embryos of the same species also release JH III and a product that coelutes with MF on HPLC. In adult blowfly, Calliphora vomitoria, the CA release JH III bisepoxide and possibly the 6,7-epoxide, in addition to JH III. In the lepidopteran species Pseudaletia unipuncta, male CA produce and release JH acids I, II, and III as well as a product which we have tentatively identified as homo-(and/or) dihomo-FA. In the females, CA produce and release the three common JH homologues and a product that we believe is the esterified version of the male compound, homo/dihomo-MF. Although the release of JH precursors from their sites of synthesis might result in their conversion to the active hormone in peripheral tissues, there is only limited evidence for such a process. Studies on biological activities of these compounds and on the developmental changes in biosynthesis and its regulation should provide information necessary for the defining of these compounds as hormones or otherwise and should improve our understanding of the evolution of the JH biosynthetic pathway in the phylum Arthropoda.

The appearance of chitinolytic enzymes, chitinase and β-N-acetylglucosaminidase, in integuments of fifth-larval instars of the silkworm, Bombyx mori, was investigated by injection of 20-hydroxyecdysone into the hemolymph of the ligated larvae, and by topical application of an imidazole compound (KK-42, 1-benzyl-5[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole) along the dorsal vessel of the larvae at the beginning of spinning behavior. 20-Hydroxyecdysone induced both enzyme activities. However, the induction patterns were different between two types of chitinolytic enzymes. Chitinase was rapidly induced only by high hormone levels (30 μg/insect, 7.5 μg/g live wt) and soon decreased, while β-N-acetylglucosaminidase was gradually induced even by low hormone levels (6 μg/insect, 1.5 μg/g live wt). KK-42 suppressed both the larval-pupal transformation and appearance of chitinolytic enzymes. Application of KK-42 (50 μg/insect) caused 1-day delay in β-N-acetylglucosaminidase and 2-day delay in chitinase. It was shown by immunoblotting and activity staining that the appearance of the enzyme activities was associated with that of the respective enzyme molecules. The molecular species of β-N-acetylglucosaminidase appeared was mainly the 67.5 kDa subunit. In the case of chitinase, several molecular species including active forms (88 and 65 kDa) and zymogenic form (about 215 kDa) were observed. These results suggest that β-N-acetylglucosaminidase is induced in an active form by relatively low ecdysteroid levels, whereas chitinase is induced through activation of the zymogen by higher levels of hormone.

The voltage-sensitive sodium channel is the site of action of two important classes of insecticides, DDT and pyrethroids. We recently used the polymerase chain reaction (PCR) to amplify sodium channel gene sequences in the house fly genome and showed the direct use of the amplification product as a conspecific hybridization probe. This report describes the use of this method to isolate sodium channel gene sequences from seven insect species (representing four orders) and an arachnid, thereby demonstrating its general utility for quickly and efficaciously isolating homologous sequences from distantly related species. DNA sequence analysis of the amplified products revealed that all but a few were homologous to the IS5-6 region of the para gene of Drosophila melanogaster, the region upon which the design of the target primers was based. Although unique nucleotide sequences were obtained for each species (with some species having more than one sequence variant), the inferred amino acid sequences of the 15 residue stretch between the amino acid target sequences were found to be completely conserved or to contain a single conservative replacement of serine with threonine. We suggest that this methodology now permits specific knowledge obtained from molecular genetic analysis of D. melanogaster to be applied straightforwardly to the characterization of many genes and the primary products of their expression in other insect specs.