Rapid chemical dehydration of biologic samples for scanning electron microscopy using 2,2-dimethoxypropane.

Acidified 2,2-dimethoxypropane (DMP) has been used as a rapid chemical dehydrating agent en route to plastic embedding and ulthrathin sectioning for conventional electron miscroscopy (J Histochem Cytochem 23:107, 1975). We have used DMP to dehydrate biologic specimens prior to critical point drying and metal coating for scanning electron microscopy. There is no difference in either the gross architecture or the fine surface structure of mouse small intestine and trachea, rat trachea and kidney, and cultured fibroblasts, between samples dehydrated in DMP for 5 min to 30 days and those conventionally dehydrated in ethanol or acetone. DMP dehydration is advantageous in speed, economy and apparent completeness.     

Efficacy of Metarhizium anisopliae microsclerotial granules

The entomopathogenic fungus Metarhizium anisopliae has very recently been shown to produce microsclerotia (MS) – compact, heavily melanised, hyphal aggregates – in liquid media. Soil incorporation bioassays of dried MS preparations of three isolates of M. anisopliae were conducted using third instar Tetanops myopaeformis (sugarbeet root maggot) in clay and/or clay loam field soils as a model system to demonstrate efficacy. At rates as low as 23 mg MS granules/100 g dry soil, the biocontrol efficacy of MS granules of M. anisopliae Strain F52 produced in liquid media with a high carbon concentration (36 g/L) and high C:N ratios (30:1, 50:1) were superior to MS preparations produced in low carbon (8 g carbon/L) media and a high carbon medium with a 10:1 C:N ratio. Bioassays using MS formulations of M. anisopliae strains MA1200 and TM109 produced in high carbon and high C:N ratio media were superior in efficacy to the other MS production media tested. MS preparations of M. anisopliae F52 showed superior efficacy against the sugarbeet root maggot in comparison with more conventional, conidia-covered nutritive (corn grit) granules in a clay and clay soil. The MS granules were also highly efficacious against the sugarbeet root maggot at soil moisture levels as low as 0.983 A _w (?2.33 MPa). Granular preparations incorporating Metarhizium MS can serve as a viable formulation for the use of this fungus against soil insects.     

Discovery of Fusarium solani as a naturally occurring pathogen of sugarbeet root maggot (Diptera: Ulidiidae) pupae: prevalence and baseline susceptibility

The fungus Fusarium solani (Mart.) Sacc. was discovered as a native entomopathogen of the sugarbeet root maggot, Tetanops myopaeformis (R?der), in the Red River Valley of North Dakota during the 2004 sugarbeet production season. This is the first report of a native pathogen affecting the pupal stage of T. myopaeformis. Forty-four percent of larvae collected from a field site near St. Thomas (Pembina Co.) in northeastern North Dakota during May and June of 2004 were infected with the entomopathogen. The mean LC(50) of F. solani, assessed by multiple-dose bioassays with laboratory-reared pupae, was 1.8x10(6)conidia/ml. After isolation and confirmation of pathogenicity, a pure isolate of the fungus was deposited in the ARS Entomopathogenic Fungal Collection (ARSEF, Ithaca, NY) as ARSEF 7382. Symptoms of F. solani infection included rapid pupal tissue atrophy and failure of adults to emerge. Transverse dissections of infected pupae revealed dense hyphal growth inside puparia, thus suggesting fungal penetration and pathogenicity. Mycelia emerged from pupae after host tissues were depleted. Exposure of older pupae to lethal concentrations caused rapid mortality of developing adults inside puparia. A second, more extensive field survey was conducted during the 2005 cropping season, and F. solani infection was observed in root maggots at most locations, although at lower levels (1-10%) of prevalence than in 2004. Aberrant timing or amounts of rainfall received could have caused asynchrony between pathogen and host during the second year of the experiment.     

Chemical Dehydration of Specimens with 2,2-Dimethoxypropane (DMP) for Paraffin Processing of Animal Tissues: Practical and Economic Advantages over Dehydration in Ethanol

Chemical dehydration can be accomplished using 2,2-dimethoxy-propane (DMP). In the presence of an acid catalyst, this liquid reacts with water generating methanol and acetone as products. Although DMP is more expensive per milliliter than ethanol and other solvents used for dehydration, it is an economical alternative because a much smaller volume is needed. Slow penetration of DMP was previously thought to restrict its use to tiny specimens, but we now show that pieces of tissue as thick as 2 cm are dehydrated by overnight immersion in acidified DMP. We also show that dehydration in acidified DMP does not impair the staining of UNA or other basophilic components of animal tissues. The temperature and concentrations of methanol and H⁺ in the chemical dehydrating agent are too low to produce histochemicaUy detectable methylation or nucleic acid extraction.     

Effects of fall and spring seeding date and other agronomic factors on infestations of root maggots, Delia spp. (Diptera: Anthomyiidae), in canola

Several agronomic benefits can result from fall seeding of canola (Brassica spp.), but extensive research data are lacking on the potential impact of this practice on infestations of root maggots (Delia spp.) (Diptera: Anthomyiidae), which are major pests of the crop in western Canada. Field experiments making up 13 location by year combinations were conducted in central Alberta, Canada, from 1998 to 2001 to determine the effect of fall versus spring seeding of canola on root maggot damage. Depending on the experiment, interactions with seeding rate, seed treatment, timing of weed removal, and canola species (cultivar) also were investigated. Root maggot damage declined with an increase in seeding rate for plots seeded in May but not in fall or April. Susceptibility to infestation was greater for plants of Brassica rapa L. than Brassica napus L., but seed treatment had no effect on damage by these pests. Combined analysis using data from all experiment by location by year combinations indicated that seeding date had no significant effect on root maggot damage. The extended emergence of Delia spp. adults, which spans the appearance of crop stages vulnerable to oviposition regardless of seeding date, prevented reduced root maggot attack. Covariance analysis demonstrated the importance of increasing seeding rate for reducing root maggot infestations, a practice that can be especially beneficial for May-seeded canola when growing conditions limit the ability of plants to compensate for root maggot damage. Results determined with the small plot studies described here should be validated in larger plots or on a commercial field scale, but both the combined and covariance analyses indicate that seeding canola in fall does not predispose plants to greater damage by larval root maggots than seeding in spring.     

Insect feeding-induced differential expression of <Emphasis Type="Italic">Beta vulgaris</Emphasis> root genes and their regulation by defense-associated signals

Root responses to insect pests are an area of plant defense research that lacks much information. We have identified more than 150 sugar beet root ESTs enriched for genes responding to sugar beet root maggot feeding from both moderately resistant, F1016, and susceptible, F1010, genotypes using suppressive subtractive hybridization. The largest number of identified F1016 genes grouped into the defense/stress response (28%) and secondary metabolism (10%) categories with a polyphenol oxidase gene, from F1016, identified most often from the subtractive libraries. The differential expression of the root ESTs was confirmed with RT-PCR. The ESTs were further characterized using macroarray-generated expression profiles from F1016 sugar beet roots following mechanical wounding and treatment of roots with the signaling molecules methyl jasmonate, salicylic acid and ethylene. Of the examined root ESTs, 20, 17 and 11% were regulated by methyl jasmonate, salicylic acid and ethylene, respectively, suggesting these signaling pathways are involved in sugar beet root defense responses to insects. Identification of these sugar beet root ESTs provides knowledge in the field of plant root defense and will lead to the development of novel control strategies for control of the sugar beet root maggot.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users     

Infochemicals mediating the foraging behaviour of Aleochara bilineata Gyllenhal adults: sources of attractants

Aleochara bilineata Gyll. (Coleoptera: Staphylinidae) adults feed on eggs and larvae of cabbage maggot, Delia radicum (L.) (Diptera: Anthomyiidae), and might be used to control this pest. We undertook tests in a Y-tube olfactometer to determine if infochemicals are involved in the food foraging behaviour of A. bilineata adults and to precisely determine the potential sources of stimuli. A. bilineata adults oriented toward the stimuli from both the food-plant of the prey (rutabaga: Brassica napus var. napobrassica (L.) Reichb.) and the prey-plant complex (rutabaga infested by cabbage maggot larvae), but adults significantly preferred the effluvia of infested to uninfested rutabaga in a choice test. The sources of infochemicals in the infested rutabaga were the cabbage maggot larvae themselves and their frass. However, in a choice test adults preferred the effluvia of the larvae to that of the damaged rutabaga from which those larvae were obtained. It is not expected that a generalist predator uses precise herbivore-derived signals in food foraging activity, but the preference of A. bilineata adults for the larval volatiles over frass volatiles may reflect the dependence of this species on dipteran pupae for reproduction.     

Oat cover cropping and soil insecticides in an integrated sugarbeet root maggot (Diptera: Otitidae) management program

Sugarbeet, Beta vulgaris L., producers occasionally establish cereal cover crops to minimize early-season soil erosion, wind abrasion, and mechanical injury of seedlings. We evaluated the use of living oat, Avena sativa L., cover cropping as a cultural tactic to minimize feeding injury from sugarbeet root maggot, Tetanops myopaeformis (R?der), larvae at five field sites during 1996, 1998, and 1999. Sweep-net sampling yielded 4.8-, 11.2-, and 7.2-fold more flies from oat cover-cropped chlorpyrifos, terbufos, and untreated control plots, respectively, than in noncover counterparts. However, larval feeding injury in terbufos-treated plots was reduced when cover-cropped (383 seeds/m2) at St. Thomas in all years. A reduced oat seeding rate (224 seeds/m2) also enhanced root protection in terbufos-treated plots at St. Thomas in 1999. Less root injury was sustained in cover-cropped chlorpyrifos plots than in noncover counterparts at St. Thomas in 2 study yr. Oat cover cropping also frequently resulted in reduced T. myopaeformis feeding injury in the absence of a soil insecticide. Although trends toward increased yields were often evident, significant yield benefits were limited to a 6.8% root yield increase in oat cover plots when compared with noncover treatments overall at St. Thomas in 1996 and an 18.4% sucrose yield increase in terbufos-treated plots at St. Thomas in 1999. These findings suggest that beneficial interactions between planting-time soil insecticides and cereal cover crops are achievable in areas infested by T. myopaeformis. Demonstrated reductions in root feeding injury, combined with additional agronomic benefits, may warrant use of this production practice as part of an integrated management program for this key insect pest of sugarbeet.     

Responses of the specialist biological control agent,Aleochara bilineata,to vegetational diversity in canola agroecosystems

Plant biodiversity is known to affect insect populations, both herbivores and their natural enemies, and as a consequence, habitat management through increased plant species composition and abundance can be exploited for sustainable pest management. In agroecosystems where crop monocultures are the routine production practice, plant biodiversity can be increased by maintaining small populations of weeds, with potential beneficial effects arising from concomitant increases in the abundance of predator and parasitoid populations. We manipulated weed populations in both species of canola, Brassica rapa L. and Brassica napus L., to investigate responses of adults of Aleochara bilineata Gyllenhal (Coleoptera: Staphylinidae), an important natural enemy of root maggots (Delia spp., Diptera: Anthomyiidae). Larval root maggots feed on canola taproots, disrupting the flow of water and nutrients within the plants, causing substantial yield reductions. Aleochara bilineata is a predator–parasitoid that attacks all root maggot pre-imaginal life stages. Activity density of A. bilineata increased as monocotyledonous weed biomass declined. Significant preferences between canola species were observed, with A. bilineata associated most frequently with B. rapa compared with B. napus. Our research suggests that improved management of root maggot infestations in canola through enhancement of populations of the A. bilineata predator–parasitoid could be accomplished by reducing weed infestations; however, such recommendations should consider other predators in the system and the role of weeds in reducing root maggot oviposition and damage.     

Comparative effectiveness of different insecticides for organic management of blueberry maggot (Diptera: Tephritidae)

Laboratory and field assays using insecticides for organic pest management were conducted on the blueberry maggot, Rhagoletis mendax Curran. Topical exposure of flies to spinosad (Entrust), pyrethrum (PyGanic 1.4 EC), azadirachtin (Aza-Direct), and phosmet (Imidan 70-W) resulted in significantly higher mortality compared with the water control after 2 and 24 h. After 24 h, there were no significant differences in fly mortality among treatments of Entrust, PyGanic, or Imidan, whereas fly mortality to Aza-Direct was significantly lower. Another laboratory assay evaluated mortality of flies after residual exposure to these insecticides on leaves, after 24 and 48 h. In this assay, there were no significant differences in fly mortality after 48 h among treatments of PyGanic, Aza-Direct, and the water control, whereas significantly higher fly mortality resulted from exposure to Entrust and Imidan. A repellency assay found no measurable effects of Aza-Direct. Large-scale field trials found no treatment effect for number of adults of the blueberry maggot captured in sticky traps; however, there were significantly lower levels of fruit-infesting larvae in treated plots compared with the untreated control. Spinosad bait (GF-120 NF Naturalyte Fruit Fly Bait), Entrust, and PyGanic were not different from imidacloprid (Provado 1.6 F). However, there was a significantly higher infestation in the plot treated with azadirachtin (Agroneem) compared with Provado. Overall, the insecticides evaluated in these trials showed good ability to control blueberry maggot, suggesting that they can be incorporated in a blueberry maggot management program under organic standards.     

Improvements in dehydration and cement line staining for methacrylate embedded human bone biopsies

Undemineralized methacrylate embedded bone biopsies and other bone specimens can be processed much more rapidly by application of acidified 2,2-dimethoxypropane (DMP) dehydration, which requires two hours, than by traditional graded ethanol dehydration, which requires at least four days. This shortened processing time is valuable when biopsy results are urgently needed to detect osteomalacia or to determine bone features prior to possible parathyroidectomy. We have processed over 200 bone specimens with DMP and have compared DMP dehydration to graded ethanol dehydration in 11 biopsies in which two plugs were available from the same patient. DMP dehydration does not compromise the following: tetracycline retention, Goldner's stain, acid phosphatase localization or histochemical identification of aluminum. Cement lines, which provide a record of past remodelling, are useful in clinical interpretation of bone biopsies. We have adapted two stains, toluidine blue and methylene blue/basic fuchsin, for improved cement line identification. Five-micrometer sections individually demineralized in acetate buffer prior to cement line staining show best results with toluidine blue at pH 5.5 and with methylene blue/basic fuchsin at pH 2.5-3.5.     

Function and immuno-localization of aquaporins in the Antarctic midge Belgica antarctica

Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes. Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin), AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins in water relations and freezing tolerance in B. antarctica.     

Water balance in the sugarbeet root maggot Tetanops myopaeformis,during long‐term low‐temperature storage and after freezing

The sugarbeet root maggot Tetanops myopaeformis Röder (Diptera: Ulidiidae) can be stored in moist sand at 4–6 °C for up to 5 years and is freeze‐tolerant. The majority of stored larvae survive in a state of post‐diapause quiescence and the remainder are in a multi‐year diapause. The present study aims to determine larval water content and water loss rates in diapausing and low‐temperature stored larvae. Body water content ranges from 57% to 70.1%. Two distinct groupings of larvae are revealed based on dry weights. The first group consists of the diapausing larvae and larvae stored for 1 year. This group has significantly higher dry weights than the second grouping, which consists of the larvae stored for 2 and 3 years. There are no significant differences within each group. Larval water losses follow a first‐order kinetic relationship with time. Larvae stored for 2 years lose water at a significantly higher rate than diapausing larvae. Larvae exhibit no active water uptake at storage temperatures. A freezing event does not induce a significant decrease in wet weights, nor does it increase larval water loss rates. These results indicate that metabolic water and the microclimate during storage are key factors enabling the long‐term survival of T. myopaeformis larvae during low‐temperature storage, and may provide insights for maintaining other insect species under similar conditions.     

Size of orchard trees as a factor affecting behavioural control of apple maggot flies (Dipt., Tephritidae) by traps

Over a 3-year period (1997–1999), we examined the influence of tree size on effectiveness of traps for behaviourally controlling apple maggot flies, Rhagoletis pomonella (Walsh), in small blocks of trees in Massachusetts commercial apple orchards. Traps were red spheres coated with Tangletrap and baited with an attractive synthetic host fruit odour (butyl hexanoate). Traps were placed 6 m apart on perimeter apple trees of each block and were designed to intercept apple maggot flies immigrating into blocks from unmanaged host trees. Based on captures of adults by unbaited red spheres placed near the centre of each block to monitor degree of adult penetration into the interior and on percentages of fruit injured by apple maggot, traps surrounding blocks of small trees (1.5 m canopy diameter) planted at high density were more effective in controlling apple maggot flies (relative to control obtained by insecticide sprays used in comparison blocks) than were traps surrounding blocks of large trees (3.7 m canopy diameter) planted at low density, with traps surrounding blocks of medium-size trees (2.5 m canopy diameter) planted at medium density generally providing an intermediate level of control.     

Nitrogen application enhanced the expression of developmental plasticity of root systems triggered by mild drought stress in rice

Background

The promoted root growth under developmental plasticity triggered specifically by mild drought stress (MDS) is known to contribute to maintained water uptake and dry matter production (DMP).

Aims

To examine whether the expression of developmental plasticity of root systems and its contribution to DMP would be affected by the levels of nitrogen (N) application.

Methods

Two genotypes (CSSL50 derived from Nipponbare/Kasalath cross and Nipponbare) were grown under soil moisture gradients with a line source sprinkler system. Three N fertilizer treatments were used; 25 (low), 75 (standard) and 150 kg N ha^?1 (high) in 2009 and 60 (low), 120 (standard) and 180 kg N ha^?1 (high) in 2011.

Results

Across varying N level treatments, there were no significant differences in any of the traits examined between the two genotypes under well-watered and severe drought stress conditions. In contrast, under MDS conditions (15–25 % w/w of soil moisture content (SMC) in 2009 and 17–25 % w/w of SMC in 2011), CSSL50 showed greater DMP than Nipponbare. The difference, however, varied with N level treatments since CSSL50’s greater root system development under MDS, was more pronounced at standard and high N levels than at low N level than it was for Nipponbare.

Conclusions

N application enhanced the expression of plasticity in root system development at standard and high N levels as compared with low N level under MDS conditions, which contributed to the maintenance of DMP.     

Cover crop and conidia delivery system impacts on soil persistence of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) in sugarbeet

The sugarbeet root maggot, Tetanops myopaeformis (Röder), is a major North American pest of sugarbeet, Beta vulgaris L. Previous research suggests that moderate T. myopaeformis control is possible with the entomopathogen Metarhizium anisopliae (Metch.) Sorok. We conducted a three-year (2002–2004) experiment to assess impacts of oat, Avena sativa L. and rye, Secale cereale L., cover crops on persistence of corn grit-based granular or spray formulations of M. anisopliae isolate ATCC 62176 (i.e. MA 1200) applied at 8×10¹² viable conidia/ha in sugarbeet. More colony forming units (CFUs) were detected immediately after application [0 days after treatment (DAT)] in spray plots than granule-treated plots. However, 76–92% declines in CFUs per gram of soil occurred in spray plots within 30 DAT. Substantially (i.e. 83–560%) more rainfall occurred in June 2002 than during June of any other year. Subsequently, 71–670% increases in CFU concentrations occurred by 60 DAT in M. anisopliae granule-treated plots with oat or rye cover crops that year. CFU density increases were higher in cover crops in 2002, but no significant cover crop effects were detected. Conidia persisted for up to 30 DAT in M. anisopliae spray plots and 60 DAT in granule-treated plots in 2002; however, no increases occurred in the years with less June rainfall. Trends suggest that M. anisopliae aqueous sprays result in greater conidia concentrations than granules at sugarbeet plant bases in June during T. myopaeformis oviposition and larval establishment on host plants. Increases are possible when delivering conidia via granules, but high post-application rainfall could be necessary for conidia production.     

Population dynamics of apple maggot (Diptera: Tephritidae) in south central Pennsylvania

The apple maggot, Rhagoletis pomonella (Walsh), was monitored with baited yellow panels and red spheres in commercial orchards, abandoned orchards, and unsprayed backyard apple trees in 1998 and 1999. Apple maggot adults were captured in all apple habitats, but the capture levels in the abandoned orchards and unsprayed backyard trees tended to be higher than in the commercial orchards. Peak capture occurred between mid-July and late August in both years. Emergence cages seeded with infested fruits were used to investigate bivoltinism, which was observed in both years.     

Hydraulic adjustment in jack pine and black spruce seedlings under controlled cycles of dehydration and rehydration

Drought adjustments were compared in black spruce ( Picea mariana [Mill] B.S.P), and jack pine ( Pinus banksiana [Lamb.]) by subjecting seedlings to five cycles of dehydration and rehydration. A computer-controlled root misting chamber system, supplied low (−1.5 MPa), moderate (−2.0 MPa), and severe (−2.5 MPa) dehydration, respectively, in cycles 1, 3 and 5. Although cell water relations failed to adjust to chronic dehydration, there was limited osmotic adjustment in black spruce (cycle 3), and water was re-allocated from the apoplast to the symplast in jack pine (cycles 1 and 3). Dehydration postponement was more important than dehydration tolerance. Jack pine was better able to postpone dehydration than black spruce. Specific conductivity, the hydraulic conductivity per unit stem cross-sectional area, was lower in jack pine and slower to decline during chronic dehydration. When specific conductivity was corrected for the greater leaf area in black spruce, the leaf-specific conductivity did not differ in the two species. There was no increase in needle leakage in jack pine and stomata in jack pine seedlings reopened fully after rehydration. Black spruce was more of a 'water spender', and less water stress (−2.0 MPa, cycle 3) was required to lower specific conductivity, compared to jack pine (−2.5 MPa, cycle 5). Leakage from needle membranes increased in black spruce, and stomata failed to reopen after rewatering (cycles 3 and 5). A greater needle area, smaller root system, and a higher specific conductivity lowered the water stress threshold for cavitation in black spruce, which is confined to moister sites in the boreal forest. Jack pine had a larger root system, smaller needle area and lower specific conductivity than black spruce. Because of these static features, jack pine is more drought tolerant and it is often found on sites that are too hot and dry for black spruce.     

Chemical Dehydration for Rapid Paraffin Embedding

We describe chemical dehydration with 2,2-dimethoxypropane (DMP) for rapid paraffin embedding using a mixture of DMP and mineral oil followed by mineral oil as clearing intermediates. This method is useful for classical histological techniques as well as for histochemistry and immunocytochemistry.