Variation in Soybean Aphid (Hemiptera: Aphididae) Biotypes Within Fields.

Soybean aphid (Aphis glycines Matsumura (Hemiptera: Aphididae)) has been a major pest of soybean in North America since its detection in this continent in 2000 and subsequent spread. Although several aphid resistance genes have been identified, at least four soybean aphid biotypes have been discovered, with three of them being virulent on soybean cultivars with certain soybean aphid resistance genes. These biotypes are known to vary across years and locations, but information on their variation within single fields is limited. An investigation was conducted to study the variation of soybean aphid biotypes within single townships and fields in Minnesota. Screening of 28 soybean aphid isolates collected from seven soybean fields (six soybean fields in Cairo and Wellington Townships of Renville County, MN and one field in Wilmar Township of Kandiyohi County, MN) revealed the existence of multiple known biotypes of soybean aphid within single fields of soybean. We found up to three biotypes of soybean aphid in a single field. Two biotypes were found in five fields while only one field had only a single biotype. Three isolates presented reactions on a panel of resistant and susceptible indicator lines that were different from known biotypes. These results highlight the importance of characterizing soybean aphid biotypes in small geographical areas and utilizing generated knowledge to develop soybean cultivars pyramided with multiple resistance genes. The outcome will be decreased use of insecticides, thereby improving economic and environmental sustainability of soybean production.

Multicolored Asian lady beetle (Coleoptera: Coccinellidae) activity and wine grape phenology: implications for pest management.

We determined the phenology of the multicolored Asian lady beetle, Harmonia axyridis (Pallas), adults in relation to the phenology of wine grapes (Frontenac and Marechal Foch) in Minnesota and Wisconsin vineyards to establish a management window for H. axyridis infestations in wine grapes. In addition, we also assessed the flight activity of H. axyridis in an agricultural landscape. The phenology of berry development and ripening was determined by recording berry size and sugar content of randomly selected berries. The phenology of H. axyridis was determined by tracking its flight activity with yellow sticky cards in vineyards and with a blacklight trap in an agricultural landscape. Berry development and ripening showed three distinct growth periods or phases. The end of growth period I averaged 9 July (Frontenac) and 11 July (Marechal Foch). Veraison, which marks the end of growth period II, averaged 25 July (Frontenac) and 3 August (Marechal Foch). Harvest, the third growth period averaged 18 September for Frontenac and 17 September for Marechal Foch. A major peak of H. axyridis captures occurred between veraison and harvest (i.e., the grape susceptible stage). A similar peak in the summer was observed in the agricultural landscape approximately 10 d before the major peak in the vineyards.

A simple method for in-field sex determination of the multicolored Asian lady beetle Harmonia axyridis.

The multicolored Asian lady beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), has become a popular study organism due to its promise as a biological control agent and its potential adverse, non-target impacts. Behavioral and ecological research on H. axyridis, including examinations of its impacts, could benefit from non-destructive or non-disruptive sexing techniques for this coccinellid. External morphological characters were evaluated for H. axyridis (succinea color form) sex determination in laboratory and field studies. The shape of the distal margin of the fifth visible abdominal sternite accurately predicted H. axyridis sex for all beetles examined. Males consistently had a concave distal margin, while females had a convex distal margin. In addition, pigmentation of the labrum and prosternum were both significantly associated with H. axyridis sex; males had light pigmentation and females had dark pigmentation. Labrum and prosternum pigmentation increased from light to dark with decreasing rearing temperature and increasing time after adult eclosion for females. Male pigmentation was only affected by a decrease in rearing temperature. Validation through in-field collections indicated that these predictors were accurate. However, labrum pigmentation is a more desirable character to use to determine sex, because it is more accurate and easily accessible. Therefore, we recommend using labrum pigmentation for in-field sex determination of H. axyridis. Implications of this diagnostic technique for applied and basic research on this natural enemy are discussed.

Toxicity of commonly used insecticides in sweet corn and soybean to multicolored Asian lady beetle (Coleoptera: Coccinellidae).

Use of insecticides with low toxicity to natural enemies is an important component of conservation biological control. In this study, we evaluated the toxicity of insecticides used in sweet corn, Zea mays L., and soybean, Glycine max (L.) Merr., to the multicolored Asian lady beetle, Harmonia axyridis (Pallas), under laboratory and field conditions. Field experiments conducted in sweet corn in 2003 and 2004 and in soybean in 2003, showed that H. axyridis was the most abundant predator. In sweet corn, densities of H. axyridis larvae in plots treated with spinosad or indoxacarb were generally higher than in plots treated with chlorpyrifos, carbaryl, bifenthrin, and A-cyhalothrin. In soybean, densities of H. axyridis larvae in plots treated with chlorpyrifos were higher than in plots treated with lambda-cyhalothrin. Laboratory experiments were conducted to evaluate the acute toxicity of insecticides to eggs, first and third instars, pupae, and adults. Spinosad, followed by indoxacarb, were the least toxic insecticides for all life stages of H. axyridis. Conventional insecticides showed high toxicity to H. axyridis when applied at field rates under laboratory conditions. Overall, first instars were most susceptible to the insecticides tested, followed by third instars and adults, eggs, and pupae. Our results suggest that spinosad, and to a lesser extent indoxacarb, offer reduced toxicity to H. axyridis and would be beneficial for conservation biological control in agricultural systems where H. axyridis is abundant.

Phytophagous preferences of the multicolored Asian lady beetle (Coleoptera: Coccinellidae) for autumn-ripening fruit.

The objective of this study was to assess the potential pest status of Harmonia axyridis (Pallas) on autumn-ripening fruit. In autumn, H. axyridis has been observed feeding on pumpkins, apples, grapes, and raspberries in Minnesota. To determine whether H. axyridis can inflict primary feeding damage to fruit (i.e., breaking the skin of the fruit), we conducted laboratory feeding experiments with undamaged pumpkins, apples, grapes, and raspberries. The only fruit that H. axyridis was able to damage directly was raspberry. Laboratory choice tests were conducted to determine whether H. axyridis exhibits a preference between damaged and undamaged fruit, between cultivars of fruit, and between sugar water and water alone. For all fruits tested, H. axyridis showed a preference for damaged fruits over undamaged fruits. H. axyridis also exhibited a strong preference for sugar water over water alone. However, few differences were exhibited in preference between cultivars of fruit. In autumn, it seems that H. axyridis is an opportunist, taking advantage of previously damaged fruit, caused by other agents.

Confirmation of bean leaf beetle, Cerotoma trifurcata, feeding on cucurbits.

The objective of these studies was to assess the degree to which bean leaf beetle, Cerotoma trifurcata (Forster), will feed on cucurbits. In 2003, we documented an infestation of C. trifurcata in a commercial pumpkin field near Rosemount, MN, USA. To evaluate C. trifurcata feeding on cucurbits, we conducted laboratory no-choice and choice test feeding studies. In the laboratory, C. trifurcata fed most heavily on cotyledon-stage cucumber plants, followed by pumpkin and squash. With soybean plants present, C. trifurcata still fed on cucumber plants. However, C. trifurcata appeared to prefer soybeans until the quality of the soybean plants was diminished through feeding damage. This is the first known report of C. trifurcata feeding on cucurbits. The pest potential of C. trifurcata in cucurbit cropping systems should be further evaluated.

The multicolored Asian lady beetle, Harmonia axyridis: a review of its biology, uses in biological control, and non-target impacts.

Throughout the last century, the multicolored Asian lady beetle, Harmonia axyridis (Pallas) has been studied quite extensively, with topics ranging from genetics and evolution to population dynamics and applied biological control being covered. Much of the early work on H. axyridis was conducted in the native Asian range. From the 1980's to the present, numerous European and North American studies have added to the body of literature on H. axyridis. H. axyridis has recently gained attention in North America both as a biological control agent and as a pest. This literature review was compiled for two reasons. First, to assist other researchers as a reference, summarizing most of the voluminous body of literature on H. axyridis pertaining to its biology, life history, uses in biological control, and potential non-target impacts. Secondly, to be a case study on the impacts of an exotic generalist predator.

Determination and correlation of in vitro viability for hairless mouse and human neonatal whole skin and stratum corneum/epidermis.

BACKGROUND AND DESIGN: Viable tissue is essential to assess the rate and extent of biotransformation during percutaneous absorption in vitro. We assessed the viability of hairless mouse whole skin (WS) and stratum corneum/epidermis (SCE) and human neonatal SCE following separation from the dermis by EDTA phosphate-buffered saline (EDTA-PBS) incubation or by heat treatment by measuring the conversion of dextrose to lactate. Lactate concentrations in receptor fluid samples were determined using a Sigma diagnostic lactate determination kit. A standard curve was prepared and samples assayed spectrophotometrically at 340 nm using a lambda 2 beta spectrophotometer. Standard curves were prepared for each experiment and correlation coefficient values (r) were calculated. RESULTS: Our results showed that hairless mouse SCE was associated with glucose conversion to lactic acid at an increased rate if incubated in EDTA-PBS for 4 h and used immediately. Lactate production was greater with the dermis present (EDTA-PBS WS). The rate of glucose to lactate conversion in hairless mouse SCE was 20-25% of that found in WS. Compared with Dulbecco's modified PBS (DMPBS)-treated WS controls, the rate of lactate production in EDTA-PBS-treated WS was nearly a 50% less. Heat treatment in water at 60 degrees C to separate SCE from hairless mouse WS appeared to eliminate viability. Viability of hairless mouse SCE, as measured by glucose conversion to lactate, was comparable to human neonatal SCE. CONCLUSIONS: These results suggest that the dermis is a significant contributor to glucose metabolism and that incubation in EDTA-PBS is a contributing factor to the overall decrease in metabolic capacity of the tissue. As a result of these findings, hairless mouse SCE appears to be useful as a model for human neonatal SCE in percutaneous absorption studies.

Molecular cloning and functional analysis of the promoter of the human squalene synthase gene.

We have cloned and characterized the 5'-flanking region of the gene encoding human squalene synthase. We report here the promoter activity of successively 5'-truncated sections of a 1 kilobase of this region by fusing it to the coding region of a luciferase reporter gene. DNA segments of 200 base pairs (bp) 5' to the transcription start site, as determined by primer extension analysis, show a strong promoter effect on the expression of the luciferase chimeric gene and a high response to the presence of sterols when transiently transfected into the human hepatoma cell line HepG2 or to the hamster-derived CHO-K1 cells. An approximately 50-fold induction of luciferase activity, in the absence of sterols, was observed in transiently transfected HepG2 cells for fusion constructs containing sections of 200, 459, and 934 bp of the putative human squalene synthase promoter. Loss of promoter activity and response to sterols was localized to a 69-bp section located 131 nucleotides 5' to the transcription start site. Sequence analysis of this region showed that it contained a sterol regulatory element 1 (SRE-1) previously identified in other sterol regulated genes (Smith, J. R., Osborne, T. F., Brown, M. S., Goldstein, J. L., and Gil, G. (1988). J. Biol. Chem. 263, 18480-18487) and two potential NF-1 binding sites. Additional CCAAT box, SRE-1 element, and two Sp1 sites were identified 3' to this section. Sequences within this 69-bp DNA, including the SRE-1 cis-acting element, show strong binding to the purified nuclear transcription factor ADD1 (Tonzonoz, P., Kim, J. B., Graves, R. A., and Spiegelman B. M. (1993) Mol. Cell Biol. 13, 4753-4759) by mobility shift assay and footprinting analyses.

Percutaneous absorption of flurbiprofen in the hairless rat measured in vivo using 19F magnetic resonance spectroscopy.

The objective of this investigation was to develop a new methodology using 19F-magnetic resonance spectroscopy (MRS) to measure the in vivo percutaneous absorption of flurbiprofen through hairless rat skin. A 2% W/V flurbiprofen gel (Klucel HF, hydroxypropyl cellulose 1.5% to 2% W/V) containing isopropyl alcohol, water, and propylene glycol (55:35:10 v/v/v) was prepared. A 2-mg dose (100 mg of gel) was applied to the skin of the lower back of an anesthetized hairless rat, contained with a rubber o-ring, and occluded with a lexan plastic cover slip. The animal was placed on an MR surface coil (3.5-cm diameter tuned to 19F) and measurements taken continuously over approximately 3 h in 10-min intervals with a 2-tesla GE CSI nuclear magnetic resonance (NMR) spectrometer. One measures the disappearance of MR signal intensity per interval, which directly relates to the percent of drug disappearance over time, which in turn was converted to a flux value. The flux of flurbiprofen in vivo was found to be 95 +/- 22 micrograms/cm2/h. This is approximately four times greater than the flux of flurbiprofen through excised human skin reported by Akhter and Barry (22 +/- 14 micrograms/cm2/h). This new in vivo method measures drug disappearance and can be readily transferred to man. This method may be adapted to study other fluorine compounds or other nuclei with magnetic properties. It avoids exposure of a patient or animal to the radiation used in x-ray fluorescence methods or to 14C- or 3H-radiolabeled drugs.

The anaerobic metabolism of verapamil in rat cecal contents forms nor-verapamil and thiocyanate.

The anaerobic metabolism of verapamil was studied to determine the role the intestinal flora may have on the disposition of verapamil. Metabolites produced by the flora could be the source of adverse reactions reported only with the p.o. controlled release formulations but not with immediate release formulations. Verapamil was found to be metabolized to thiocyanate, nor-verapamil and several more polar metabolites that were detected by either a specific assay for cyanide and thiocyanate or a high-performance liquid chromatography (HPLC) assay, respectively. The rate of thiocyanate formation with an initial substrate concentration of 2 mM verapamil was found to be 0.008 +/- 0.004 microgram/hr/ml of 1:10 cecal suspension. The N-demethylated metabolite, nor-verapamil, was detected in the cecal suspensions but it also disappeared with time. The rate of verapamil disappearance was dependent on the concentration of bacteria in the incubation mixture; the rate being most rapid with the highest concentration of bacteria. Acetonitrile and butyronitrile were also found to be metabolized by the cecal flora. Cyanide as well as thiocyanate were produced from both organo-nitriles. These results suggest that the cyano group of verapamil, acetonitrile and butyronitrile were all cleaved to form cyanide and/or thiocyanate. With verapamil, cleavage of the cyano group would form new chemical entities that could be pharmacologically active and serve as a source of some of the adverse reactions or side-effects reported.

Diffusion of [2-14C]diazepam across isolated hairless mouse stratum corneum/epidermal tissues.

The objective of this study was to develop a method of preparing mouse stratum corneum/epidermal (SCE) tissue without the dermis for use in drug diffusion studies. The diffusion of radiolabeled diazepam across this new preparation has been studied and the effect of the dermis on diffusion evaluated. Incubation of large pieces of mouse skin in a 20mM EDTA, 15 mM sodium phosphate buffer, pH 7.2, in normal saline for 3-4 h at 37 degrees C resulted in a tissue which easily separated at the epidermal-dermal junction. The resulting tissue contains stratum corneum and epidermis, which are the same layers used in studies with human skin in vitro. The EDTA treatment did not effect diffusion of [2-14C]diazepam across whole mouse skin (SCE and dermis) used as controls. The rate of drug diffusion was greater across SCE than SCE and dermis, however, 0.48-1.12 micrograms/cm2/h versus 0.11-0.52 microgram/cm/h, respectively. The permeability coefficients for mouse SCE ranged from 1.92-4.48 X 10(-2) cm/h. The lag times and diffusion coefficients were 0.36-0.91 h and 0.1-0.6 X 10(-6) cm2/h, respectively. The presence of the dermis decreased the diffusion rate or flux of diazepam. The dermis appears to accumulate drug until it is saturated and then the drug diffuses into the receiving chamber.

Diffusion of [2-14C]diazepam across hairless mouse skin and human skin.

The objectives of this study were to investigate the absorption of diazepam applied topically to the hairless mouse in vivo and to determine the diffusion of diazepam across isolated hairless mouse skin and human skin. [14C]Diazepam was readily absorbed after topical administration to the intact hairless mouse, a total of 75.8% of the 14C-label applied being recovered in urine and feces. Diazepam was found to diffuse across human and hairless mouse skin unchanged in experiments with twin-chambered diffusion cells. The variation in diffusion rate or the flux for both human and mouse tissues was greater among specimens than between duplicate or triplicate trials for a single specimen. Fluxes for mouse skin (stratum corneum, epidermis, and dermis) were greater than for human skin (stratum corneum and epidermis): 0.35-0.61 microgram/cm2/h for mouse skin vs 0.24-0.42 microgram/cm2/h for human skin. The permeability coefficients for mouse skin ranged from 1.4-2.4 X 10(-2)cm/h compared with 0.8-1.4 X 10(-2)cm/h for human skin. Although human stratum corneum is almost twice the thickness of that of the hairless mouse, the diffusion coefficients for human skin were 3-12 times greater (0.76-3.31 X 10(-6) cm2/h for human skin vs 0.12-0.27 X 10(-6) cm2/h for hairless mouse) because of a shorter lag time for diffusion across human skin. These differences between the diffusion coefficients and diffusion rates (or permeability coefficients) suggest that the presence of the dermis may present some barrier properties. In vitro the dermis may require complete saturation before the diazepam can be detected in the receiving chamber. [14C]Diazepam was not detected in the receiving chamber of the Franz cell apparatus in experiments with human skin. This indicated that the rate of diffusion was less than 0.09 microgram/cm2/h. Since this diffusion technique more closely resembles topical administration to humans, these results appear to indicate that achieving therapeutic concentrations in humans may be difficult. In addition, the hairless mouse may not be a suitable model for predicting percutaneous absorption of diazepam in humans.

Reduction of nitroheterocyclic compounds by mammalian tissues in vivo.

To determine whether nitro group reduction occurs in mammalian tissues, metronidazole (0.021, 0.064 and 10 mg/kg), misonidazole (0.015 mg/kg) and nitrofurazone (0.13 mg/kg), respectively, were administered to germfree rats. A reduced metabolite [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-ol] and two of its hydrolysis products, urea and (2-hydroxy-3-methoxypropyl)-guanidine, were found in the urines of germfree rats that received misonidazole. When nitrofurazone was administered, a reduced metabolite, 4-cyano-2-oxobutyraldehyde semicarbazone, was detected in the urines. However, acetamide and N-(2-hydroxyethyl)oxamic acid, fragmentation products from the reduction of metronidazole, were not found in significant concentrations in the urine when germfree rats received metronidazole. Apparently metronidazole is reduced so much more slowly than misonidazole and nitrofurazone in the tissues of germfree rats that its reductive metabolites are not detectable. This observation may be explained by the one-electron reduction potentials (E1 7) of these drugs, that of metronidazole (E1 7 = -486 mV) being lower than those of either misonidazole (E1 7 = -389 mV) or nitrofurazone (E1 7 = -257 mV). Under these circumstances, metronidazole reduction is not detected, either because its radical anion forms more slowly than that of the other nitroheterocyclic compounds or because its radical anion interacts more rapidly with oxygen to restore the parent compound.

Comparative misonidazole metabolism in anaerobic bacteria and hypoxic Chinese hamster lung fibroblast (V-79-473) cells.

The metabolism of the radiation sensitizer misonidazole was similar in anaerobic cecal contents and hypoxic Chinese hamster lung fibroblasts (V-79-473). Both systems formed the amino derivative of misonidazole, [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-ol] (AIM), and urea, as well as a metabolite, (2-hydroxy-3-methoxypropyl)-guanidine (G), which has not been described previously. It appears that the nitro group of misonidazole was reduced to form AIM and that this compound was then hydrolyzed to yield either urea or G, the latter in yields of 25% (tissue culture) to 55% (cecal contents). When tested with the Ames tester strain, both G and AIM were slightly mutagenic only for strain TA 98 and then only in the presence of the system for microsomal activation.

Interaction of metronidazole with resistant and susceptible Bacteroides fragilis.

The kinetics of the lethal action of metronidazole and the formation of acetamide have been studied in a strain of Bacteroides fragilis which is relatively resistant to metronidazole. As with a susceptible strain of B. fragilis, the data are consistent with a model in which a labile intermediate in metronidazole metabolism interacts either with water to form acetamide or with a bacterium to cause its death. Although the relatively resistant strain grows more slowly than the susceptible one and is killed less rapidly by metronidazole, the resistant strain displays the same relationship between the lethal action of metronidazole and metronidazole metabolism to acetamide. The relatively resistant strain, like the susceptible one, has an enhanced lethal response to metronidazole in the presence of a strain of Escherichia coli. The results suggest that the proposed labile reactive intermediate of metronidazole forms more slowly in the resistant strains.