Insect Biochemistry最新文献

As suggested by their mechanism-derived IUPAC nomenclature, epoxide hydrolases (EH) are characterized by the hydrolysis of epoxides. In this study EH activity from M. sexta was monitored in cell fractions from fat body and integument using juvenile hormone III (JH) as a substrate. Experiments were conducted to identify a suitable detergent and detergent concentration for solubilization of microsomal and mitochondrial juvenile hormone epoxide hydrolase (JHEH) activity. Triton X-100 efficiently released total and specific JHEH activity from membranes. A pH optimum range was determined for EH activity in cell fractions from different tissue sources. Potential inhibitors of JHEH activity were tested. One of the better inhibitors was a glycidol analog of JH. EH activity on JH III was compared to activity on JH III bisepoxide, cis-stilbene oxide, and trans-stilbene oxide. JHEH tolerance to detergents, salt, and elevated temperature was investigated. As observed in a similar study on D. melanogaster, the effect of inhibitors, substrates, detergents, salt and temperature suggests the presence of EH isozymes. Polyethylene glycol and ammonium sulfate were used to precipitate JHEH activity from solubilized microsomes and cytosol.

Larvae of the house fly, Musca domestica were reared aseptically on diets which contained either cholesterol, campesterol or sitosterol as the dietary sterol at a concentration of 0.1% dry wt. Analysis of puraria (24 h post-pupariation) reared on campesterol or sitosterol diets revealed they contained from 2.7 to 4.6% cholesterol, indicating an ability to accumulate this sterol even where it is present in only minute quantities. Purparia on all diets produced the 27-carbon molting hormones, ecdysone and 20-hydroxyecdysone. When the concentration of campesterol was increased to 0.2% dry wt, puparia also contained the 28-carbon ecdysteroid, makisterone A, although it accounted for only 20.7% of the total ecdysteroid produced.

In the seasonally diphenic butterfly Araschnia levana¹⁴C-labelled tryptophan and 3-hydroxykynurenine, the principal precursors of ommochromes, injected into young pupae caused a pattern specific radiolabel of mature red scales. [¹⁴C]glucose and [³⁵S]methionine also labelled red scales but only when injected shortly before or during the time of pigment synthesis in the wing. In developing non-diapause pupae contents of 3-hydroxykynurenine increased until an abrupt decrease when pigments appeared in the wings. In diapausing pupae 3-hydroxykynurenine remained low but increased after injection of 20-hydroxyecdysone which induced pupal-adult development. Supply of wing scale cells with ommochrome precursors via the haemolymph was analysed after injection of [³H]tryptophan. In developing pupae haemolymph contents of [³H]tryptophan and [³H]3-hydroxykynurenine increased at the time of wing pigment formation and decreased shortly before adult emergence. In diapausing pupae haemolymph contents of [³H]tryptophan and [³H]3-hydroxykynurenine were low compared to non-diapause pupae but increased at the time of wing pigment formation after injection of 20-hydroxyecdysone. Isolated wings incubated in Grace's medium containing [¹⁴C]tryptophan and [¹⁴C]3-hydroxykynurenine incorporated radiolabel specifically into red portions of the wing colour pattern due to labelling of ommatin. Incorporation into red wing areas occurred specifically depending on different colour patterns of the spring- and the summer-morph.

The results demonstrate that both tryptophan as well as 3-hydroxykynurenine are delivered via the haemolymph, and both can serve as precursors of ommatin formation in the scale cells. Therefore, the complete set of enzymes for the tryptophan-ommatin pathway is present in scale-forming cells.

The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum, was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of (RS)-methoprene or JH II had a juvenilizing effect on parasitized T. ni, and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.

Musca domestica larval midgut display in cells and luminal contents a proteolytic activity with a pH optimum of 3.0–3.5. This activity is abolished by pepstatin and is insensitive to soybean trypsin inhibitor and to sulfhydryl proteinase inhibitors. The acid proteinase occurs in multiple forms with M_r values in the range 40,000–80,000 and with pI values of about 5.5. The proteinase inactivates at 60°C according to apparent first-order kinetics and Lineweaver-Burk plots of its activity against albumin concentration are rectilinear, suggesting that the multiple forms have similar properties. The proteinase reacts slowly with diazoacetylnorleucine plus CuSO₄, is stable in alkaline media, is inhibited by dithiothreitol, hydrolyses hemoglobin better than albumin and is virtually not active upon synthetic substrates for pepsin. These properties are similar to those of cathepsin D. The specific activity of the acid proteinase determined by titration with pepstatin is 680 units/mg of proteinase and the K_D of the pepstatin-proteinase complex is 1.5 nM at 30°C. The acid proteinase occurs mainly in midgut subcellular fractions characterized by a high specific activity of molybdate-inhibited acid phosphatase and a large number of secretory-like vesicles. It is proposed that the M. domestica midgut acid proteinase is a cathepsin D-like proteinase evolved to function in luminal contents. The lack of ATP activation of the midgut enzyme supports this hypothesis, since ATP is thought to regulate cathepsin D-proteolysis inside lysosomes.

Brush border membrane from Heliothis virescens catalyzed delta-endotoxin-induced leakage of ⁸⁶Rb⁺-K⁺ and H₂O from phospholipid vesicles. Activated delta-endotoxin [CrylA(c)-55 kDa] from Bacillus thuringiensis kurstaki strain EG2244 producing a single CrylA(c) toxin, when incorporated into phospholipid vesicles, made these vesicles more leaky to ⁸⁶Rb⁺-K⁺ than phospholipid vesicles without toxin. This effect was assayed by following the movement of ⁸⁶Rb⁺ into the vesicles in response to a KCl gradient. When toxin was added to the outside of phospholipid vesicles, ⁸⁶Rb⁺ uptake was impeded. Vesicles prepared with H. virescens brush border membrane catalyzed the association of the toxin with the vesicle, and stimulated KCl gradient-induced ⁸⁶Rb⁺ uptake. Toxin did not catalyze the leakage of ³⁶Cl⁻, suggesting that the toxin created a cation-selective leak. Toxin enhanced the permeability of phospholipid vesicles to H₂O, demonstrated by the enhanced rate of vesicle shrinking under increased osmotic pressure. This was analyzed spectrophotometrically by following the rate of vesicle shrinking in response to a 10 mM KCl gradient. In the presence of concentrated phosphatidylcholine vesicles, toxin spontaneously associated with the vesicles so as to enhance the rate of vesicle shrinking in an osmotic gradient. The rate of vesicle shrinking the presence of KCl and toxin was catalyzed by the presence of brush border reconstituted into the vesicles, reducing the effective toxin concentration 1000-fold.

The following hydrocarbon classes have been identified in insect cuticular lipids: A, $\text{n-}\text{alkanes}$; B, olefins, comprising B1, alkenes, B2, alkadienes and B3, alkatrienes; and C, methylalkanes, comprising monomethyl-, dimethyl-, trimethyl- and tetramethylakanes. Biosynthetic pathways have been described for most of the main classes and hydrocarbon composition may be considered a product of an insect's genotype and thus available for taxonomic use. Olefins and methylalkanes usually occur as isomeric mixtures and hydrocarbon composition can be complex. This provides the chemotaxonomist with many potential characters. Preliminary investigations show that hydrocarbon composition not only separates species but also reflects links between species and between higher taxa. A hydrocarbon chemotaxonomy which also takes into account the elongation-decarboxylation pathways used by insects to synthesise their hydrocarbons could provide information on the evolution of hydrocarbon biosynthesis in the insects.