[Effect of temperature on post-diapause reproductive development in Listronotus maculicollis (Coleoptera: curculionidae)].

The annual bluegrass weevil Listronotus maculicollis requires chilling exposure to terminate reproductive diapause during overwintering, but the effects of temperature on its post-diapause development in spring remain unclear. To explore this effect, overwintering adults were transferred from cold conditions (6°C/4°C, L:D 10:14) to different warm-up temperatures at L:D 12:12. When weevils were transferred to 7, 14 and 21°C in December and late January, the sizes of male and female reproductive organs were significantly smaller at 7°C than at 14 and 21°C. When weevils were transferred to 7, 9, 11, 13 and 15°C in late January, higher temperatures facilitated the post-diapause development. In both sexes, the sizes of reproductive organs and developmental rate increased with temperature. Reproductive organs did not grow significantly at 7°C in males and at 7-9°C in females, at which the percentage of developing weevils remained low. The time required for 50% of individuals to resume development was 44, 18, 13 and 8 days at 9, 11, 13 and 15°C, respectively, in males and 19, 14 and 8 days at 11, 13 and 15°C, respectively, in females. The threshold temperature for post-diapause development was 7.8°C in males, based on which 61.7 degree-days coincided with 50% of individuals developing. Under field conditions, the percentage of male and female maturity and insemination rate were low until early March, but all reached 100% by late March.

Responses of Poa annua and three bentgrass species (Agrostis spp.) to adult and larval feeding of annual bluegrass weevil, Listronotus maculicollis (Coleoptera: Curculionidae).

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby, is an economically important pest of short-cut turfgrass in Eastern North America. Wide spread insecticide resistance warrants the development of alternative management strategies for this pest. ABW damage typically occurs in areas with a high percentage of annual bluegrass, Poa annua L., the preferred ABW host. Damage to bentgrasses, Agrostis spp., is much rarer and usually less severe. To aid the implementation of host plant resistance as an alternative ABW management strategy we investigated the tolerance of three bentgrass species to ABW feeding. Responses of P. annua, creeping bentgrass, Agrostis stolonifera L., colonial bentgrass, Agrostis capillaris L., and velvet bentgrass, Agrostis canina L., to adult and larval feeding were compared in greenhouse experiments. Grass responses were measured as visual damage, dry weight of the grass stems and leaves, color, density and overall grass quality. To determine possible mechanisms of grass tolerance constitutive fiber and silicon content were also determined. The three bentgrass species tolerated 2-3 times higher numbers of ABW adults and larvae than P. annua before displaying any significant quality decrease. Creeping bentgrass had the lowest damage ratings. ABW infestation caused higher plant yield reduction in P. annua (up to 42%) than in bentgrasses. Observed differences among the grass species in fiber and silicon content in the plant tissue are unlikely to play a role in the resistance of bentgrasses to ABW. Our findings clearly show that A. stolonifera is the best grass species for the implementation of host plant resistance in ABW management.

Developing wax-based granule formulations for mating disruption of oriental beetles (Coleoptera: Scarabaeidae) in turfgrass.

Oriental beetle, Anomala orientalis Waterhouse (Coleoptera: Scarabaeidae), is a pest of turfgrass that may be controlled by applications of synthetic pheromone (Z)-/ (E)-7-tetradecen-2-one to disrupt mating. Laboratory experiments were conducted to determine release profiles of pheromone from experimental wax-based granules, a proprietary wax granule, and rubber septa commonly used in pheromone traps. Rubber septa loaded with 10, 100, and 300 microg per septum provided steady rates of pheromone release (zero-order) over 4 wk of laboratory evaluation (total = 1.1, 9.0, and 26.9 microg/4 wk, respectively). Septa with 1,000 microg per septum had a significant decline in the rate of pheromone release for this 4-wk exposure time (total = 119 microg/4 wk). A large proprietary wax granule (44 mg per granule, 25% wt:wt pheromone) provided a steady rate of pheromone release (total = 2,347 microg/4 wk per granule). Experimental granules (16 mg per granule) made of soywax with higher pheromone loads (10% wt:wt) approached zero-order release (steady state) (total = 69 microg/4 wk per granule), whereas smaller granules (4 mg per granule) with less pheromone (0.1% wt:wt) provided first-order release profiles (decreasing rate with longer exposure time) (total = 0.35 microg/4 wk per granule). A field trial in turfgrass demonstrated the potential of selected granular formulations to provide effective mating disruption for up to 4 wk, as measured by pheromone trap shutdown. Documenting pheromone release profiles for these experimental granules and rubber septa provides valuable information that will support future field evaluations of mating disruption as a control strategy.

Developmental temperature effects on five geographic isolates of the entomopathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae).

The development of five geographic isolates of Steinernema feltiae at 5, 8, 10, 15, 20, 25, and 28 degrees C in wax moth, Galleria mellonella, larvae was examined. The isolates were from Mediterranean (Sinop from Turkey, SN from France, and Monterey from California), subtropical (Rafaela from Argentina), and tropical (MG-14 from Hawaii) regions. All isolates caused 100% mortality of wax moth larvae and developed and produced progeny between 8 and 25 degrees C. At 28 degrees C, mortality was 100%, but no progeny was observed. The highest infective juvenile production was observed at 15 degrees C for all isolates. In general, the tropical isolate, MG-14, had the lowest production of infective juveniles. The time of emergence of the infective juveniles from the host cadaver showed some differences among isolates, with the Sinop isolate having the earliest emergence time from cadavers at 15 degrees C (10 days) and 20 degrees C (8 days). At 25 degrees C, the infective juveniles of the Sinop, SN, and Rafaela isolates emerged from the cadavers from 5 to 7 days. Time of host death by all isolates showed no differences at 8, 10, 15, 20, and 28 degrees C. At 25 degrees C for all isolates (except the MG-14), shorter times to host death were observed. Host death occurred at 12 days at 8 degrees C, 9 to 11 days at 10 degrees C, 4 to 5 days at 15 degrees C, 3 days at 20 degrees C, and 2 days at 25 and 28 degrees C. For penetration efficiency, the Sinop, SN, and Rafaela isolates penetrated their hosts at 5, 8, and 10 degrees C. Penetration of the infective juveniles was consistently high for all isolates at 15, 20, 25, and 28 degrees C, but it was significantly lower for the MG-14 isolate at 15, 25, and 28 degrees C. No progeny production occurred at 28 degrees C, but nematode penetration did occur with the MG-14 isolate having significantly lower penetration than the other isolates. When nematodes were produced at 8, 15, and 23 degrees C in wax moth larvae, all isolates had infective juveniles with longer body lengths at 8 degrees C followed by 15 and 23 degrees C. To further verify body length at the different temperatures, beet armyworm, Spodoptera exigua, larvae and dog-food agar medium were used, respectively, for in vivo and in vitro culture of the Sinop isolate. Infective juvenile body length showed the same trends, with the longest being at 8 degrees C and decreasing in length from 15 to 23 degrees C. The data suggest that quality of food for the nematode and temperature (that is, developmental time) influence the body length of the infective juvenile.

Intramolecular electron transfer from c heme to d1 heme in bacterial cytochrome cd1 nitrite reductase occurs over the same distances at very different rates depending on the source of the enzyme.

Intramolecular electron transfer over 12 A from heme c to heme d(1) was investigated in cytochrome cd(1) nitrite reductase from Pseudomonas aeruginosa, following reduction of the c heme by pulse radiolysis. The rate constant for the transfer is relatively slow, k = 3 s(-1). The present observations contrast with a corresponding rate of electron transfer, 1.4 x 10(3) s(-1), measured for cytochrome cd(1) from Paracoccus pantotrophus, though the relative positions of the two heme groups are the same in both enzymes. The rate of intramolecular electron transfer within the enzyme from P. aeruginosa was accelerated 10(4)-fold (1.4 x 10(4) s(-1)) by the binding of cyanide to the d(1) heme. A coordination change at the d(1) heme upon its reduction is suggested to be a major factor in determining the slow rate of electron transfer in the P. aeruginosa enzyme in the absence of cyanide.

Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior.

Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center. Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1. Reproducible hysteresis is demonstrated in the redox titrations. In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV. However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2. A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated. In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1). The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes.

Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus.

Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction). Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration. Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron. Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen. On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed. Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically. These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme. At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme. Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen. The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species. This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical. Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH(-) for completion of a catalytic cycle.

Biological control agents for white grubs (Coleoptera: Scarabaeidae) in anticipation of the establishment of the Japanese beetle in California.

We tested biological control agents for the control of 3rd-instar scarab turfgrass pests, both for the masked chafer Cyclocephala hirta LeConte and the Japanese beetle, Popillia japonica Newman. The former species is endemic in California whereas the latter, although not yet established, constitutes a permanent serious threat to agriculture and horticulture in California. We conducted experiments using C. hirta in California and P. japonica in New Jersey. A field trial conducted in 2 different California turfgrass sites compared the field persistence in the absence of hosts of Bacillus thuringiensis Berliner subspecies japonensis Buibui strain, the milky disease bacterium, Paenibacillus (=Bacillus) popilliae (Dutky), and the entomopathogenic nematodes Steinernema kushidai Mamiya and Heterorhabditis bacteriophora Poinar to that of the organophosphate diazinon. Soil samples taken 0-70 d after applications were bio-assayed with P. japonica. Only diazinon and the entomopathogenic nematode S. kushidai caused substantial mortality and S. kushidai activity persisted significantly longer than diazinon activity. In greenhouse experiments, combinations of entomopathogenic nematode species usually resulted in additive mortality of scarab larvae. Combinations of S. kushidai and diazinon also resulted in additive mortality. In field trials, the efficacy of H. bacteriophora and especially S. kushidai and S. glaseri, was comparable to that of diazinon over 14-18 d. However, it is likely that at least S. kushidai would have outperformed diazinon over an extended period because of its longer persistence and potential for recycling in the hosts. S. kushidai, should it become commercially available, deserves further examination as an alternative to chemical white grub control especially as a highly compatible component of sustainable turfgrass management.

Are there temporarily non-infectious dauer stages in entomopathogenic nematode populations: a test of the phased infectivity hypothesis.

Many studies of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) have reported that only a small proportion (typically < 40%) of infective stages (dauers), even under apparently ideal conditions, actually infect a host. The 'phased infectivity hypothesis' is most frequently invoked to explain this pattern of low infection with entomopathogenic nematodes. It proposes that at a given point in time not all individuals are infectious i.e. infectiousness is delayed in some individuals. We tested experimentally several predictions based on this hypothesis. Specifically, if phased infectivity occurs, we should be able to expose dauers to increasing numbers of potential hosts until dauers no longer infect and still be able to recover viable dauers. These recovered dauers which did not infect should be infectious at some later point in time. However, our results do not support the phased infectivity hypothesis for 3 species of Steinernema: most dauers could be recovered in one sampling round when provided with sufficient suitable hosts. In contrast, Heterorhabditis bacteriophora frequently did not infect all available hosts, and infectious dauers were recovered in subsequent sampling rounds. This result is more consistent with the phased infectivity hypotheses, but further research is needed before we can be more confident in the hypothesis. For all species tested, the number of available hosts influenced population levels of nematode infectivity. This suggests that the infection status of hosts can influence whether a dauer infects. Our results indicate that phased infectivity is not a common phenomenon in entomopathogenic nematode dauers, despite the widespread acceptance of this hypothesis.

Pulse radiolysis studies on cytochrome cd1 nitrite reductase from Thiosphaera pantotropha: evidence for a fast intramolecular electron transfer from c-heme to d1-heme.

Electron transfer within cytochrome cd1 from Thiosphaera pantotropha was investigated by the technique of pulse radiolysis. The reduction of the heme centers in this nitrite reductase occurred in two phases as judged from kinetic difference spectra. In the faster phase, radiolytically generated N-methylnicotinamide (NMA) radicals selectively reduced the c-heme of the enzyme. From the absorbance increase at 420 nm, a characteristic of formation of the ferrousc-heme, the second-order rate constant for this electron transfer process was estimated to be 3.8 x 10(9) M-1 s-1 at pH 7.0. In the slower phase, a decrease of absorption around 420 and 550 nm, corresponding to a reoxidation of the c-heme, was accompanied by an increase of absorption around 460 and 640 nm, characteristic of formation of the reduced d1-heme. This indicated that an intramolecular electron transfer from the c-heme to the d1-heme occurred. The first-order rate constant of this process was calculated to be 1.4 x 10(3) s-1 at pH 7.0 and was independent of the enzyme concentration. In the presence of nitrite the interheme electron transfer rate was not affected, but on a time scale of seconds a new species associated with the d1-heme, having an absorption maximum at 640 nm, was detected and is proposed to reflect ligand binding to this heme. These results suggest the role of the c-heme as the electron acceptor site in cytochrome cd1 and in mediating the electron transfer to the catalytic site of the enzyme. Moreover, the fast interheme electron transfer rate argues against this process being the rate determining step in catalysis.

Effect of nematode-trapping fungi on an entomopathogenic nematode originating from the same field site in California.

We determined whether nematode-trapping fungi may influence the dynamics of a coastal shrub community. The food chain interactions in the shrub community involve the dominant plant species, its major insect herbivore, and an entomopathogenic nematode, Heterorhabditis hepialus. Of the 12 nematode-trapping fungi previously isolated from soils at the study site, 5 were selected for this study. Arthrobotrys oligospora, Geniculifera paucispora, Monacrosporium eudermatum, and Monacrosporium cionopagum efficiently trapped and colonized H. hepialus on agar; in contrast Nematoctonus concurrens trapped but did not infect or colonize the nematode on agar. To determine whether these fungi can suppress H. hepialus in soil, we added the fungi in the form of fungal-colonized nematodes to pasteurized (2 hr at 62 degrees C) and raw (nontreated) soil from the study site. Suppression was measured by comparing nematode invasion into a wax moth larva in fungus-treated and untreated soil in vials at 20 degrees C. Fungal population density in soil was estimated using dilution plating and most probable number procedures. All fungi suppressed H. hepialus if the wax moth larvae were added 4 days after the nematodes. Suppression ranged between 37 and 54% and did not differ among fungi. Suppression was usually greater in raw than in pasteurized soil. Raw soil contained a constant background of nematode-trapping fungi, and A. oligospora was the most common among these; no background was detected in pasteurized soil. The presence of background fungi in raw soil may explain the higher suppression in raw than in pasteurized soil. Fungal propagule densities in our laboratory experiments were similar to those observed in the field, suggesting that nematode-trapping fungi may influence the dynamics of the plant, insect herbivore, and entomopathogenic nematode in the coastal ecosystem.

Competition between two steinernematid nematode species for an insect host at different soil depths.

We studied interactions between 2 entomopathogenic nematode species, Steinernema carpocapsae, an ambusher forager, and Steinernema glaseri, a cruiser forager, when they were provided wax moth larvae as hosts at 0, 2, or 10 cm soil depth. Populations of infective juvenile nematodes in soil were monitored at 30-day intervals over 120 days using wax moth larvae as baits. After application of S. carpocapsae, S. glaseri, or the combination of both species, hosts were added at 30-day intervals. With hosts at 0 cm depth, each nematode species was negatively affected by the presence of the other species at the 30- and 60-day samples. At 90 and 120 days, S. carpocapsae numbers in the combined treatment were as high as in the single species treatment, whereas only few S. glaseri were recovered. With hosts at 2 or 10 cm depth, the presence of S. glaseri had a strong negative effect on S. carpocapsae, but S. glaseri was not affected by the presence of S. carpocapsae. In another experiment, S. carpocapsae dominated over S. glaseri in hosts located at 0 cm depth as measured by penetration efficiency into hosts and progeny production. In contrast, S. glaseri dominated at 2 cm depth. At 2 cm depth, S. carpocapsae penetrated into hosts too slowly to compete successfully with S. glaseri. Steinernema carpocapsae is superior to S. glaseri when competing for a host on the soil surface; however, below the surface S. glaseri is superior to S. carpocapsae.

Density-dependent effects on Steinernema glaseri (Nematoda: Steinernematidae) within an insect host.

Increasing densities of Steinernema glaseri infective juveniles (IJs) in soil affected penetration efficiency and reproduction of the nematodes in larvae of the greater wax moth Galleria mellonella. The penetration efficiency and the proportion of penetrated IJs developing to adults decreased significantly with increasing numbers of IJs present in the soil and entering the hosts, respectively. The number of progeny produced/host cadaver initially increased, with the highest production being between 20.7 +/- 3.3 and 58.0 +/- 6.6 IJs established/host +/- SE and decreased at higher densities. Above 184.4 +/- 37.7 IJs established/host, no progeny emerged from the cadavers.

31P ENDOR studies of xanthine oxidase: coupling of phosphorus of the pterin cofactor to molybdenum (V).

31P ENDOR spectra are described for three different molybdenum(V) species in reduced xanthine oxidase samples. The spectra were not affected by removing the FAD from the enzyme, implying that this is located at some distance from molybdenum. Furthermore, in confirmation of the work of J. L. Johnson, R. E. London, and K. V. Rajagopalan [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6493-6497], NMR and chemical analysis of the phosphate content of highly purified xanthine oxidase showed there are only three phosphate residues per subunit of the enzyme. It is concluded that the ENDOR features are due to hyperfine coupling of the phosphate group of the pterin cofactor to the molybdenum atom. Evaluation of the dipolar component of the coupling has permitted estimation of the molybdenum-phosphorus distances as 7-12 A. This implies that the cofactor is in an extended conformation in the enzyme molecule. Less detailed 31P ENDOR data on sulfite oxidase are consistent with a similar conformation for the cofactor in this enzyme.