Jack W. Dillwith

Correlation of housefly sex pheromone production with ovarian development

Abstract Pheromone production in the housefly was monitored during oogenesis and in ovariectomized insects by gas-liquid chromatography (GLC) and radio-GLC. The presence of vitellogenic ovaries was required for the initiation of (Z)-9-tricosene (muscalure), (Z)-9,10-epoxytricosane and (Z)-14-tricosen-10-one synthesis. Methylalkane synthesis was enhanced by developing ovaries. Insects ovariectomized within 12 hr after emergence produced no detectable amounts of (Z)-9-tricosene, C 23 epoxide nor C 23 ketone and synthesized less methylalkanes than the controls. This effect was reversed by ovary implants. When flies were ovariectomized after oviposition, synthesis of (Z)-9-tricosene, C 23 epoxide and C 23 ketone continued. Thus, initiation of the synthesis of these C 23 pheromone compounds required a vitellogenic ovary, but the ovary was not required to maintain synthesis.

Biosynthesis of the hydrocarbon components of the sex pheromone of the housefly, Musca domestica L.☆

Abstract Biosynthesis of hydrocarbons, including components of the sex pheromone of the housefly Musca domestica L., was investigated. In vitro studies with isolated tissues from adult flies showed that the hydrocarbon components of the pheromone were synthesized primarily by the epidermal cells in abdominal segments two to seven. The incorporation of [3H or 14C]-labelled acetate, palmitate, stearate and oleate into the saturated and unsaturated hydrocarbon components showed that (Z)-9-tricosene (muscalure) was synthesized de novo by female insects and the distribution of label was consistent with a pathway in which oleic acid was elongated and then decarboxylated. A comparison of the incorporation and distribution of labelled acetate, propionate and succinate into hydrocarbons indicated that the mono- and dimethylalkanes were formed by the substitution of a methylmalonyl-CoA for malonyl-CoA during chain elongation. The incorporation of radioactivity from [1-14C]-propionate increased dramatically in female insects two days after adult emergence, which corresponds in time to the production of methyl branched alkanes. In contrast, this substrate was not efficiently incorporated at any time into male insects.

Vitamin B12 levels in selected insects

Abstract Vitamin B 12 concentrations were determined by radioassay in the housefly, five species of termites, and 17 other phylogenetically diverse insect species. Vitamin B 12 was not detected in the housefly Musca domestica , which apparently cannot interconvert propionate and succinate. In contrast, the termite Zootermopsis angusticollis readily interconverts succinate and methylmalonate, and contains high amounts of vitamin B 12 (940 pg/mg dry tissue), as do four other species of termites. Experiments involving selective elimination of either gut tract protozoa or bacteria in Coptotermes formosanus indicate that intestinal bacteria are the major source of vitamin B 12 in this termite. The other insect species examined have undetectable to moderate amounts of vitamin B 12 .

Biosynthesis of myristate in an aphid: Involvement of a specific acylthioesterase

Abstract Approximately 70% of the total fatty acids of the pea aphid, Acyrthosiphon pisum , is myristate. The fatty acids of the phospholipid fraction consist of over 80% 18-carbon saturated and unsaturated fatty acids, whereas myristate accounts for almost 90% of the fatty acids of the triacylglycerol fraction. In vivo studies showed that [1- 14 C]acetate was incorporated into both saturated (primarily myristate) and monounsaturated fatty acids, with the 14-carbon fatty acid incorporated almost exclusively into the triacylglycerol fraction and the 16- and 18-carbon fatty acids preferentially incorporated into the phospholipid fraction. A 105,000 g particle-free supernatant preparation incorporated radiolabel from [ 3 H]acetyl-coenyzme A (CoA) primarily into myristate at low malonyl-CoA concentrations, and primarily into 16- and 18-carbon fatty acids at high malonyl-CoA concentrations. Using low malonyl-CoA concentrations, [ 3 H]acetyl-CoA was preferentially incorporated into myristate at low ionic stengths, and into 16- and 18-carbon fatty acids at high ionic strengths. Sepharose 6B column chromatography was used to partially purify the fatty acid synthetase (FAS). The partially purified FAS produced 16- and 18-carbon fatty acids exclusively, regardless of malonyl-CoA concentrations or ionic strengths. However, when a separate heat-labile fraction from the Sepharose 6B column was added to the partially purified FAS, it restored the ability of the FAS to produce myristate. Adding larger amounts of this fraction to the partially purified FAS resulted in the formation of a higher percentage of myristate. This fraction contained thioesterase activity, and was partially resolved into two thioesterase activities by Sephadex G-100 column chromatography. The later eluting thioesterase, but not the earlier eluting one, was responsible for chain termination at 14 carbons. This thioesterase readily cleaved 18-, 16-, and 14-carbon acyl-CoAs, but had a much lower specificity for a C 12 CoA. This later eluting thioesterase also restored activity to FAS treated with phenylmethanesulfonyl fluoride. These results suggest the presence of an acylthioesterase in the aphid which releases the growing acyl chain from the FAS when it reaches 14 carbons.

Sex pheromone of the housefly: Metabolism of (Z)-9-tricosene to (Z)-9,10-epxytricosane and (Z)-14-tricosen-10-one

Abstract Components of the sex pheromone of the female housefly, (Z)-9,10-epoxytricosane and (Z)-14-tricosen-10-one, are absent on the surface of newly emerged insects, first appear on females on day 2, and increase in amount to day 10. All body parts contain these components, with the legs and abdomen containing the largest amounts. The incorporation of [1- 14 C]acetate into the non-hydrocarbon cuticular (NHC) fraction, which includes 9,10-epoxytricosane and (Z)-14-tricosen-10-one is very low in newly emerged and one-day-old female houseflies and then increases dramatically from day 2 to 6. The major labelled components in this fraction are the C 23 epoxide and ketone. The increased amounts and incorporation of [1- 14 C]acetate into the C 23 epoxide and ketone correlate closely with the production of (Z)-9-tricosene. [9,10- 3 H](Z)-9-Tricosene is readily converted to oxygenated components in female insects giving rise to the C 23 epoxide (85.5%), C 23 ketone (13.0%) and more polar components (1.5%). Both female and male insects of all ages metabolize [9,10- 3 H](Z)-9-tricosene to the epoxide and ketone. All major body parts in both males and females metabolized (Z)-9-tricosene when it was applied to the surface of the insect, with the highest rate of metabolism observed by the legs of male insects.

Induction of female sex pheromone production in male houseflies by ovary implants or 20-hydroxyecdysone☆

Abstract Implanting ovaries or injecting 20-hydroxyecdysone into male houseflies induced sex pheromone production, including (Z)-9-tricosene (muscalure), 9,10-epoxytricosane and (Z)-14-tricosen-10-one, which normally occurs only in vitellogenic females. Control males did not produce detectable amounts of these compounds. Injection of 20-hydroxyecdysone (5 μg/insect per day) for 3 days resulted in the accumulation of 1.81 μg/insect of (Z)-9-tricosene, 0.97 μg/insect of 9,10-epoxytricosane and 0.12 μg/insect (Z)-14-tricosen-10-one. Multiple injections of 20-hydroxyecdysone at doses as low as 50 ng resulted in the accumulation of 23:1, C23 epoxide and C23 ketone; shifted the distribution of label within the alkenes from 27:1 to 23:1 and decreased the amount of label in the hydrocarbon fractions as alkenes. Structures of the C23 alkene and epoxide produced by the males were verified by gas chromatography-mass spectrometry. Radioactivity from [1-14C] acetate was incorporated into the C23 alkene, epoxide and ketone in male insects after ovaries were implanted or they were injected with 20-hydroxyecdysone. Synthesis of the C23 pheromone components decreased rapidly within several days after the administration of 20-hydroxyecdysone ceased, indicating that the enzymes involved in sex pheromone production were not permanently induced by hormone treatment. Ecdysone was also effective in initianing pheromone production in males, whereas inokosterone and cholesterol were not effective. Data presented demonstrate that male houseflies possess the metabolic capability to produce the sex pheromone components, and this suggests that 20-hydroxyecdysone alters the production of cuticular hydrocarbons such that the C23 sex pheromone components become major products.

Characterization of the acyl-CoA desaturase in the housefly, Musca domestica L.

Abstract Long chain acyl-CoA desaturase activity has been characterized in a microsomal fraction from the adult housefly, Musca domestica. NADPH was a four-fold better electron donor than NADH in 1.5–2.0-day-old males and three-fold better in 1.5–2.0-day-old females. The optimal pH for the desaturase was between 6.8 and 7.2 and most of the desaturase activity was in the microsomal fraction. The substrate specificity for both sexes was: 18:0-CoA > 16:0-CoA ≥ 14:0-CoA . Freezing and thawing decreased desaturase activity. Cyanide inhibited the desaturase, while CO had no effect. Ozonolysis of the isolated product followed by radio-GLC of the ozonides from both [14C]-18:0-CoA and [14C]-16:0-CoA showed the desaturase was specific for the Δ9 position. In males, desaturase activity increased about 26-fold from emergence until the insects were between three and four days old. In females, the activity increased about 12-fold from emergence until day two; then it sharply decreased on day three maintaining a 6–8-fold increase for the next four to five days. By day 11, the activity had decreased to the value observed at emergence for both sexes. NADH cytochrome c reductase activity and cytochrome b 5 concentrations showed cyclic behaviour with maximal activity or concentration occurring at days one and six in both sexes. The NADPH cytochrome c reductase had only one maximum which occurred at day one in both sexes. In males where large amounts of alkenes were being made, cuticular alkene synthesis closely paralleled desaturase activity. Females did not synthesize large amounts of alkene after day two, and the correlation with desaturase activity was not as strong.

Occurrence and metabolism of arachidonic acid in the housefly, Musca domestica (L.)

Gas-liquid chromatography (GLC) on polar, non-polar and capillary columns and chemical ionization-gas chromatography-mass spectrometry (CI-GC-MS) were used to verify the presence of arachidonic acid (20:4) and eicosatrienoic acid (20:3, n-6) at low levels in adult male and female houseflies. The phospholipid (PL) fraction contained the highest amounts of these fatty acids. An analysis of the distribution of radiolabelled 20:4, 20:3 (n-6), oleic acid (18:1) and palmitic acid (16:0) when injected into the insect showed that a large proportion of the radioactivity from 20:4 was incorporated into the PL fraction, whereas the radioactivity from the other fatty acids was recovered at higher levels in the free fatty acid and triacylglycerol fractions. When [3H]20:4 was injected into the haemolymph it was rapidly sequestered into the PL fraction. Over 80% of the recovered radiolabel from 20:4 was found in PL, 10 min after injection. Approximately one-half of the [3H]20:4 in the PL was recovered in phosphatidylethanolamine (PE), the major PL of the housefly. Liberation of the acyl moiety from the 2 position by phospholipase A2 showed that over 80% of the sequestered 20:4 was in the 2-position. Radio-GLC showed that the injected [3H]20:4 was incorporated intact into PL. When large amounts of unlabelled 20:4 were injected into the insect or were included in the diet of adult insects prior to administration of [3H]20:4, then larger amounts of radioactivity were recovered in the TG and FFA fractions. These data document the presence of 20:4 in the housefly and demonstrate that injected [3H]20:4 was readily incorporated into PL specifically into the 2-position.

Inhibition of DNA polymerase by captan

Captan inhibits DNA polymerases of both eukaryotic and prokaryotic sources. When polymerases were employed in assays with various nucleotides as template-primer, no specificity in the base sequence of polynucleotide was required for inhibition. Sucrose gradient centrifugation and preincubation studies showed the inhibition was caused by an irreversible alteration of the polymerase. Captan and DNA compete for the same site on the polymerase, thus DNA can serve a protective role in the elimination of captans action. The pyrophosphate exchange activity associated with the polymerase is not inhibited by captan and the fidelity with which DNa polymerase I copies the DNA template also is not altered by captan treatment.

Inhibition of DNA polymerase β activity in isolated bovine liver nuclei by captan and related compounds

Abstract The effects on DNA synthesis of the fungicide captan and several structurally related compounds were investigated in isolated bovine liver nuclei. Captan, folpet, captafol, and trichloromethanesulfenyl chloride inhibited DNA synthesis to the same degree with ID 50 values of approximately 50 μ M in a 40-min assay. The inhibition is concentration dependent and the degree of inhibition increases with time. Studies with structural analogs of captan indicated that inhibition of DNA synthesis by captan is mediated through the trichloromethylthio moiety of the captan molecule. In addition, the data indicate thiophosgene is probably not the toxic species involved in the inhibition of DNA synthesis. The isolated nuclei used in this study were shown to exhibit only a single DNA polymerase activity which was determined to be of the β or low-molecular-weight type. In addition to its inhibition in intact nuclei, captan inhibited the activity of the β polymerase in nuclear extracts as well as in partially purified enzyme preparations. These results indicate that captan inhibits DNA synthesis in our preparation of isolated nuclei by acting directly on the DNA polymerase-catalyzed reaction rather than by causing a nonspecific or indirect effect in the nuclear system such as alterations in the nuclear membrane or aggregation of the nuclei. The site of captans inhibitory action is the DNA polymerase molecule. The interaction of captan with the polymerase results in irreversible inhibition of the enzyme. Interaction of captan with the template, if it occurs, does not appear to be involved in mediating the inhibition.