Alison F. Moldenke

Phylogeny of Photorhabdus and Xenorhabdus species and strains as determined by comparison of partial 16S rRNA gene sequences

Partial 16S rRNA gene sequences of 16 strains of the genera Photorhabdus and Xenorhabdus were determined by direct sequencing of PCR products. Aligned sequences were subjected to phylogenetic analysis by maximum-likelihood and maximum-parsimony methods. Distance matrix and phylogenetic analysis did not separate the genera unambiguously. Taxonomic grouping of the bacteria closely paralleled taxonomic grouping of their nematode associates and their geographic origins. We found at least two well-supported taxonomic groups in Photorhabdus species, which suggests that the genus Photorhabdus is coevolving with the nematodes and may be polyspecific.

Cytochrome P-450 in insects: 3. Increase in substrate binding by microsomes from phenobarbital-induced house flies

Abstract Microsomal membranes prepared from the abdomens of two insecticide-susceptible house-fly strains (NAIDM and CSMA) and one insecticide-resistant strain (Rutgers) were examined for evidence of substrate binding by cytochrome P -450. The substrates studied in detail were benzphetamine, nerolidol, and menthone, and to a lesser extent, aldrin and heptachlor. Contrayy to previous reports, type I spectra were obtained with cytochrome P -450 from the susceptible flies. As expected, Rutgers strain P -450 also exhibited type I spectra. The spectra were typical in all cases but one (nerolidol) and were about one-third to one-seventh the size of those obtained with microsomal P -450 from the Rutgers strain. With the exception of those obtained with menthone, substrate-induced spectra were greatly enhanced (about two- to four-fold) with the P -450 from susceptible flies treated with phenobarbital. This treatment did not increase substrate binding by the P -450 from Rutgers flies. In a comparison of the epoxidation of aldrin and heptachlor by induced and noninduced microsomes, it was noted that only the heptachlor epoxidase was increased in all strains. These studies indicate that phenobarbital induction of susceptible flies results in the production of cytochrome P -450 with spectral and enzymatic properties resembling those of resistant flies.

Effects of nitrogen and Douglas-fir allelochemicals on development of the gypsy moth,Lymantria dispar

Two experiments were conducted to examine the influence of foliar nitrogen, terpenes, and phenolics of Douglas-fir on the development of gypsy moth larvae. In the first experiment, foliar concentrations of nitrogen and allelochemicals were manipulated by fertilizing 3-year-old potted seedlings with 0 or 200 ppm nitrogen. Concentrations of foliar nitrogen (0.33–2.38%) were negatively correlated with the phenolics (15.8–24.4 mg/g). Sixth-instar larvae previously reared on current-year Douglas-fir needles were allowed to feed on these seedlings. Pupal weights (312.8–995.6 mg) were positively correlated with levels of foliar nitrogen, negatively correlated with amounts of foliar phenolics, and uncorrelated with terpene concentrations. In the second experiment, terpene and phenolic extracts from Douglas-fir foliage were incorporated at natural levels into artificial diets with high and low levels of protein nitrogen. Neonate larvae grew faster and were larger on the high nitrogen control diet (4.1–4.5%), however, fourth instars performed better on the control diet with low nitrogen levels (2.5–2.7%). Foliar terpenes incorporated into diet had little effect on neonate fitness, but may induce subtle physiological changes in later instar larvae. Phenolics, alone or in combination with terpenes, excessively suppressed growth and survival, with no individuals living through the fourth instar, regardless of the nitrogen level. Incorporating foliar phenolic extracts into artificial diet caused unnatural levels of toxicity and failed to clarify the effects of Douglas-fir phenolics on gypsy moth fitness. Foliar nitrogen is a key factor influencing gypsy moth development on Douglas fir, but may be mitigated to some degree by phenolics.

Cytochrome P-450 in insects. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase, and monooxygenase activities in susceptible and resistant strains of Musca domestica

Abstract Development and phenobarbital (PB) induction of microsomal cytochrome P -450, cytochrome P -450 reductase, two epoxidation, and two O -demethylation activities were examined in chronologically timed populations of insecticide-susceptible (NAIDM) and -resistant (Rutgers) house flies. Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Untreated insects of both strains had comparable reductase levels, whereas cytochrome P -450 and associated monooxygenase activities were 1.5-fold or more higher in Rutgers. Maximum induction, as well as toxicity, occurred at a lower PB concentration in NAIDM than Rutgers. The drug caused consistently higher increases in enzymes and activities within 12 hr of starting treatment in both strains. When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min −1 mg protein −1 ) in all enzymes returned to control values in 24 hr while metabolic capacity (product min −1 insect −1 ) achieved control values within 48 hr. The changes in turnover numbers (pmol product min −1 pmol P -450 −1 ), in conjunction with the differences in the monooxygenation of the four substrates, suggest that PB treatment induced both a quantitative and qualitative change in NAIDM monooxygenation but only a quantitative change in Rutgers monooxygenation.

Cytochrome P-450 in insects: 4. Reconstitution of cytochrome P-450-dependent monooxygenase activity in the house fly

Abstract Two cytochrome P -450-containing fractions were isolated from detergent-solubilized house fly microsomes by hydrophobic chromatography on a tryptamine-Sepharose gel. These fractions (designated P -450-1 and P -450-2) were distinctive in their spectral characteristics and in their profiles following electrophoresis in the presence of sodium dodecyl sulfate. Both fractions exhibited NADPH-dependent epoxidase activity when reconstituted with purified house fly cytochrome P -450 reductase and phospholipid. The aldrin epoxidase activity of fraction P -450-1 was twice that of P -450-2 even though heptachlor epoxidase activity of the fractions was equivalent. O -Demethylase activity with 7-methoxy-4-methylcoumarin was detectable only in the P -450-2 fraction.

NADPH-cytochrome P-450 reductase from the house fly, Musca domestica: Improved methods for purification, and reconstitution of aldrin epoxidase activity

NADPH-cytochrome P-450 reductase was purified from microsomes prepared from adult phenobarbital-induced house fly abdomens. The reductase was purified 360-fold from a detergent solubilizate of these microsomes by affinity chromatography techniques. The homogeneous reductase has an apparent molecular weight of 74,000, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its specific activity ranged from 32 to 40 mol cytochrome c reduced/min per mg protein, and the enzyme was active in reducing house fly cytochrome P-450 in a reconstituted aldrin epoxidase assay. An important factor in the purification procedure was the inhibition of membrane-bound proteases by chymostatin and leupeptin. Attempts to purify the reductase in the absence of these inhibitors yielded a proteolytically processed form of the reductase that was active with artificial substrates, but did not function with cytochrome P-450 in a reconstituted mixed function oxidase (MFO) assay.

Cytochrome P-450 in insects—V.: Monoterpene induction of cytochrome P-450 and associated monooxygenase activities in the larva of the variegated cutworm Peridroma saucia (Hübner)☆

Abstract 1. The induction of microsomal cytochrome P-450 and associated polysubstrate monoxygenase activities by natural levels (0.01–0.1%) of the naturally occurring monoterpenes pulegone, menthone and menthol was examined in sixth-instar larvae of the variegated cutworm ( Peridroma saucia Hubner). 2. The monoterpenes increased cytochrome P-450 levels, aldrin and heptachlor epoxidase activity in microsomes from midgut and other tissues of the midsection. 3. NADPH-dependent cytochrome c reductase levels generally were not increased in midgut microsomes and were increased in carcass microsomes by higher doses of the inducers. 4. Induction by any of the monoterpenes increased the size of the Type I (substrate) binding spectra obtained with pulegone, menthone and nerolidol, but not those obtained with menthol. 5. The induction of polysubstrate monooxygenases by these compounds may increase the resistance of the larvae to both host plant allelochemicals and to insecticides in the field.

White alder and Douglas-fir foliage quality and interegg-mass influences on larval development of gypsy moth,Lymantria dispar.

Individual families of gypsy moth collected from a single population exhibited different degrees of fitness when fed diets of white alder, a suitable broadleaf host, and Douglas-fir, an unsuitable conifer host. Members of families on diets of Douglas-fir had significantly lower survival, longer larval periods, lower pupal weights, and shorter pupal periods than members of the same families fed alder. Foliar nutritional quality, including nitrogen level and allelochemical composition (terpenes and phenols), was considered the key factor responsible for these differences. Growth parameters differed significantly for families within diet treatments, indicating that the genetic resources of a family did affect performance somewhat. The influence of a familys genetic resources on larval survival was most notable when larvae were under the greatest nutritional stress.

Cytochrome P-450 in insects. 7. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase, and monooxygenase activities in the black blow fly (Phormia regina, Meigen)

Abstract Development and phenobarbital (PB) induction of microsomal cytochrome P -450, NADPH-cytochrome c ( P -450) reductase, two epoxidation, and two O -demethylation activities were examined in chronologically timed populations of female black blow flies ( Phormia regina , Meigen). Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Induction occurred in all enzymes, even at 0.005% PB, and was maximum at 0.15%. Dramatic induction of the O -demethylation of 7-methoxy-4-methylcoumarin was observed in flies dosed with the maximum concentration of the drug. This monooxygenase activity increased to nearly 1400 times the level in control flies, whereas the other O -demethylation (methoxyresorufin) and the two epoxidation reactions exhibited considerably less change. Induction of the structural enzymes of this enzyme system were 10-fold for cytochrome P -450 and 5-fold for NADPH-cytochrome c ( P -450) reductase. These data suggest that PB induces several P -450s in the blow fly, particularly one bearing a high degree of specificity for 7-methoxy-4-methycoumarin.

Toxicity of acephate to larvae of gypsy moth as a function of host plant and bioassay method

We examined toxicity of acephate to third‐instar gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), under different conditions of administration method, availability of food to larvae during bioassay, host plant, and activity of detoxifying enzymes. Larvae that had been fed field‐collected foliage of white alder (Alnus rhombifolia Nutt.) were less susceptible 48 h after treatment with topically applied acephate if they were allowed to continue feeding on foliage during the bioassay period (LD50= 60.6 μg/g larva) than if they were not (LD50= 13.5 μg/g larva). All surviving larvae were replaced on their original food plant after the 48‐h bioassay; of these, 14.4% of the larvae not fed during treatment died before pupation, compared with 1.3% of the larvae fed alder during treatment. The LD50 obtained for topically treated larvae reared and treated on Douglas‐fir, Pseudotsuga menziesii (Mirb.) Franco, (51.1 μg/g larva) was comparable to that obtained for larvae fed alder (60.0 μg/g larva) throughout treatment. Larvae treated orally with acephate, however, were slightly more susceptible when reared on Douglas‐fir (LC50, 20.3 ppm) than when reared on alder (LC50, 27.0 ppm). Post‐treatment mortality in orally treated larvae was 10.3% in those fed alder and 9.5% in those fed Douglas‐fir. Higher cytochrome P‐450 activities in larvae reared on Douglas‐fir apparently did not enhance tolerance to acephate. Both sexes of orally treated larvae took significantly longer to pupate than did controls on both foliage types, as did topically treated males fed Douglas‐fir. Pupal weight generally was slightly, but not always significantly, higher in treated than untreated larvae under all dietary and treatment regimes.