Localization of phosphorylase kinase subunits at the sarcoplasmic reticulum of rabbit skeletal muscle by monoclonal and polyclonal antibodies

Molecular structures related to phosphorylase kinase have been localized by light and electron microscopy in tissue sections of rabbit skeletal muscle employing polyclonal antibodies directed against the holoenzyme as well as monoclonal antibodies specific for its alpha-, beta- or gamma-subunits. In frozen sections of prefixed muscle fibres both known major regions of glycogen deposition, the intermyofibrillar space and the perinuclear area, are stained predominantly. In sections of unfixed muscle in which cytosolic phosphorylase kinase was removed by extensive washes prior to immunostaining the immunolabel is mainly associated with the sarcoplasmic reticulum (SR). This membrane location is further confirmed by immunoblot analysis of proteins solubilized from isolated SR with Triton X-114. Employing monoclonal antibodies two membrane proteins are identified as the alpha- and beta-subunits of phosphorylase kinase by Western blots. Immunoprecipitates reveal also the gamma-subunit; the delta-subunit, i.e., calmodulin, is enriched with the solubilized enzyme. It proves that a SR membrane associated form of holophosphorylase kinase exists in muscle. Functionally, this kinase might be involved in phosphorylation of phosphatidylinositol present on the SR Ca2+ transport ATPase and thereby might play a role in regulation of Ca2+ transport.     

Evaluation of a granular formulation containing Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) microsclerotia in controlling eggs of Aedes aegypti (Diptera: Culicidae)

We evaluated the potential of a granular formulation of Metarhizium brunneum F52 containing microsclerotia (MbMSc granules) for control of Aedes aegypti by targeting eggs. MbMSc granules produced infective conidia within 14 days after application to 2.5?g moist potting soil, producing 5.9?×?10⁵, 2.08?×?10⁶ and 6.85?×?10⁶ conidia from 1, 5 and 25?mg MbMSc granules, respectively. Application of MbMSc triggered premature eclosion of eggs (EC_50?=?12?mg) with percentages as high as 31?±?2.9% and 67?±?4.3% of the eggs treated with 5 and 25?mg MbMSc granules, respectively, after 14 days on moist filter paper. Premature eclosion of eggs started at 3 days subsequent to MbMSc granule application and survival of larvae was significantly reduced for granule treated eggs (74?±?2.2%, 39?±?2.0% and 23?±?4.9% larvae survived for 1, 5 and 25?mg granule treatments, respectively, EC₅₀?=?4.9?mg). When MbMSc granules were applied in moist potting soil with mosquito eggs, rates of 1, 5 and 25?mg of MbMSc granules significantly reduced adult emergence with only 81?±?2.1%, 47?±?1.9%, and 34?±?2.1% emergence, respectively (EC₅₀?=?7?mg). Eggs treated with increasing concentrations of fungal conidia enhanced premature eclosion of eggs with an EC₅₀?=?1.6?×?10⁶ conidia/mL. Our results demonstrate that MbMSc granules are a promising candidate for control of A. aegypti and that fermentative production of Mb F52 microsclerotia as the active propagule has the potential for use for mosquito control.     

Formulation of entomopathogenic nematode-infected cadavers

Entomopathogenic nematodes are commercially applied in aqueous suspension. These biocontrol agents may also be applied in nematode-infected insect cadavers, but this approach may entail problems in storage and ease of handling. We determined the feasibility of formulating nematode-infected insect cadavers to overcome these hindrances. All experiments were conducted with Heterorhabditis bacteriophora Poinar and Galleria mellonella (L.). Nonformulated cadavers were used as controls. Of 19 formulations tested (including combinations of starches, flours, clays, etc.) 1 (starch-clay combination) was found to adhere to the cadaver and to have no significant deleterious effects on nematode reproduction and infectivity; other formulations exhibited poor adhesion or reduced nematode reproduction. Two formulations enabled cadavers to be partially desiccated without affecting reproduction; other formulations and nonformulated cadavers exhibited reduced reproduction upon desiccation. Four-day-old cadavers were more amenable to desiccation than 8-day-old cadavers. Formulated cadavers were more resistant to rupturing and sticking together during agitation than nonformulated cadavers.     

Effect of light energy on alkali-released virions from Anagrapha falcifera nucleopolyhedrovirus

We compared the insecticidal activities of occluded and nonoccluded AfMNPV baculovirus obtained by dissolving the occlusion bodies (OB) with sodium carbonate. Droplet feeding and cotton leaf feeding bioassay techniques were used to determine the dose response against neonate Trichoplusia ni (Hübner) and loss of insecticidal activity when the virus was exposed to simulated sunlight from a xenon light source. Using droplet bioassays to determine a dose response, nonoccluded virus (NOV) was 20 times more active (LC(50) = 4.8 x 10(3) OB/ml, dissolved) than occluded virus (LC(50) = 9.6 x 10(4) OB/ml) when the samples remained wet. However, NOV lost activity when air dried before being tested by droplet (LC(50) > 1.0 x 10(6) OB/ml) or leaf feeding (LC(50) > 3.0 x 10(6) OB/ml) bioassays. Adding sucrose to NOV prevented the loss of insecticidal activity when samples were dried. The activity of NOV with 2% sucrose was similar to that of occluded virus samples, with or without sucrose, in both droplet feeding and leaf feeding assays. These results indicate that the OB protected the insecticidal activity of virions from the detrimental effects of drying. The OB also provided some protection from the detrimental effects of simulated sunlight (xenon) exposure. NOV samples exposed to xenon light had significantly greater loss of insecticidal activity than did similar samples of occluded virus. Without advancement in technologies, such as formulations, possible benefits of increased insecticidal activity from the use of nonoccluded virus is probably not sufficient to offset the rapid loss of activity due to drying or light exposure.     

Sunlight persistence and rainfastness of spray-dried formulations of baculovirus isolated from Anagrapha falcifera (Lepidoptera: Noctuidae)

Nuclear polyhedrosis viruses such as the one isolated from the celery looper, Anagrapha falcifera (Kirby) (AfMNPV), have the potential to be successful bioinsecticides if improved formulations can prevent rapid loss of insecticidal activity from environmental conditions such as sunlight and rainfall. We tested 16 spray-dried formulations of AfMNPV to determine the effect of different ingredients (e.g., lignin, corn flour, and so on) on insecticidal activity after simulated rain and simulated sunlight (at Peoria, IL) and natural sunlight exposures (at Tifton, GA). The most effective formulation contained pregelatinized corn flour and potassium lignate, which retained more than half of its original activity after 5 cm of simulated rain, and almost full activity after 8 h of simulated sunlight. In Georgia, formulations made with and without lignin were compared for persistence of insecticidal activity when exposed to natural sunlight. In addition, the effect of fluorescent brighteners as formulation components and spray tank additives was tested. Results showed that the formulations with lignin had more insecticidal activity remaining after sunlight exposure than formulations without lignin. The inclusion of brighteners in the formulation did not improve initial activity or virus persistence. However, a 1% tank mix significantly enhanced activity and improved persistence. Scanning electron micrographs revealed discreet particles, and transmission electron micrographs showed virus embedded within microgranules. Results demonstrated that formulations made with natural ingredients could improve persistence of virus-based biopesticides.     

Evaluation of spray-dried lignin-based formulations and adjuvants as solar protectants for the granulovirus of the codling moth, Cydia pomonella (L)

Commercial formulations of the codling moth, Cydia pomonella L., granulovirus (CpGV) are limited by their short residual activity under orchard conditions in the Pacific Northwest. We evaluated spray-dried lignin-encapsulated formulations of CpGV for improved solar stability based on laboratory bioassays with a solar simulator and in field tests in an infested apple orchard. In laboratory tests, aqueous lignin formulations containing a high dosage of 3 x 10(10) occlusion bodies (OB)/L, with and without the additives titanium dioxide (TiO(2)) and sugar, provided significant solar protection of virus, i.e., mortality of codling moth exposed to lignin formulations that had been irradiated with 9.36 x 10(6) joules/m(2) was 92-94%, compared with 66-67% from a glycerin-stabilized product (Cyd-X) or suspension of pure unformulated virus at the same rates. By comparison, a lower dosage of the lignin formulation (3 x 10(8)OB/L) did not provide significant solar protection. Equivalent dosage-dependent patterns in solar protection were observed in further tests with the lignin formulation, when an intermediate (3 x 10(9)OB/L) as well as the low dosage provided no solar protection. Equivalent rates of a blank lignin formulation (containing no virus) did not affect larval mortality, suggesting a protective effect of the lignin on the virus at the high rate. The use of several spray adjuvants, 'NuFilm-17' and 'Organic Biolink' (sticker-spreaders at 0.06% v/v), 'Raynox' (sunburn protectant at 5% v/v), and 'Trilogy'(neem oil at 1% v/v) did not provide solar protection of a commercial CpGV preparation in laboratory tests. In season long orchard tests (Golden Delicious), the lignin formulation of CpGV applied at 6.57 x 10(12)OB/ha did not significantly improve control of codling moth or protection of fruit compared with Cyd-X at equivalent rates. Our studies show that lignin-based CpGV formulations provided solar protection at relatively high virus dosages. The testing of lignin formulations containing reduced virus concentrations may allow virus solar protection to be achieved at more economical rates.     

Nepetalactones from essential oil of Nepeta cataria represent a stable fly feeding and oviposition repellent

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is one of the most serious pests to livestock. It feeds mainly on cattle and causes significant economic losses in the cattle industry. Standard stable fly control involving insecticides and sanitation is usually costly and often has limited effectiveness. As we continue to evaluate and develop safer fly control strategies, the present study reports on the effectiveness of catnip (Nepeta cataria L.) oil and its constituent compounds, nepetalactones, as stable fly repellents. The essential oil of catnip reduced the feeding of stable flies by >96% in an in vitro bioassay system, compared with other sesquiterpene-rich plant oils (e.g. amyris and sandalwood). Catnip oil demonstrated strong repellency against stable flies relative to other chemicals for repelling biting insects, including isolongifolenone, 2-methylpiperidinyl-3-cyclohexen-1-carboxamide and (1S,2'S)-2-methylpiperidinyl-3-cyclohexen-1-carboxamide. The repellency against stable flies of the most commonly used mosquito repellent, DEET, was relatively low. In field trials, two formulations of catnip oil provided >95% protection and were effective for up to 6 h when tested on cattle. Catnip oil also acted as a strong oviposition repellent and reduced gravid stable fly oviposition by 98%.     

Examining Cuphea as a potential host for western corn rootworm (Coleoptera: Chrysomelidae): larval development

In previous crop rotation research, adult emergence traps placed in plots planted to Cuphea PSR-23 (a selected cross of Cuphea viscosissma Jacq. and Cuphea lanceolata Ait.) caught high numbers of adult western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), suggesting that larvae may have completed development on this broadleaf plant. Because of this observation, a series of greenhouse and field experiments were conducted to test the hypothesis that Cuphea could serve as a host for larval development. Greenhouse-grown plants infested with neonates of a colonized nondiapausing strain of the beetle showed no survival of larvae on Cuphea, although larvae did survive on the positive control (corn, Zea mays L.) and negative control [sorghum, Sorghum bicolor (L.) Moench] plants. Soil samples collected 20 June, 7 July, and 29 July 2005 from field plots planted to Cuphea did not contain rootworm larvae compared with means of 1.28, 0.22, and 0.00 rootworms kg(-1) soil, respectively, for samples collected from plots planted to corn. Emergence traps captured a peak of eight beetles trap(-1) day(-1) from corn plots on 8 July compared with a peak of 0.5 beetle trap(-1) day(-1) on 4 August from Cuphea plots. Even though a few adult beetles were again captured in the emergence traps placed in the Cuphea plots, it is not thought to be the result of successful larval development on Cuphea roots. All the direct evidence reported here supports the conventional belief that rootworm larvae do not survive on broadleaf plants, including Cuphea.     

Evaluation of lignins and particle films as solar protectants for the granulovirus of the codling moth,Cydia pomonella

The identification of effective solar protectants for field application of the codling moth, Cydia pomonella (L.), granulovirus (CpGV) is of interest to improve its efficacy and commercial viability as a biological pesticide. We evaluated several materials as potential adjuvants to protect CpGV from ultraviolet degradation. In laboratory tests with a solar simulator (9.36×10⁶ J/m²), the addition of kaolin clay (Surround® WP at 3 and 6%, w/v), a paraffin wax-based emulsion (SPLAT? at 5%, v/v), and a bark extract trans-cinnamaldehyde combined with a film agent (both at 1%, v/v) did not significantly reduce larval entries or increase larval mortality in irradiated apples that were treated with a commercial CpGV product (Cyd-X). In semi-field tests in an apple orchard, a spray-dried lignin formulation containing CpGV (6.57×10¹² OBs/ha) and a lignin-based adjuvant used with Cyd-X (both applied at 4.7–5.6 kg lignin/ha) significantly improved residual activity of CpGV compared with Cyd-X alone applied at the same rate. However, the benefits were short lived and could not be detected after 7 days. In orchard tests, we evaluated two additional refined lignin-products (Lignosulfonate and Vanisperse? CB at 5.61 kg/ha) and two particle film materials (kaolin clay, ‘Cocoon?’, and calcium with boron ‘Eclipse?’) as adjuvants for UV protection of Cyd-X (6.57×10¹² OBs/ha) in tests against a dense codling moth infestation. Although all virus treatments were highly effective (causing ≥90% larval mortality), no significant effects of the adjuvant treatments could be detected. In these latter tests, the use of a silicone based wetting agent at 0.025% (v/v) may have been beneficial at increasing mortality among older larvae inside the fruit.     

Comparing formulations for a mixed-microbial biopesticide with Bacillus thuringiensis var. kurstaki and Beauveria bassiana blastospores

Developing a dual agent biopesticide formulation made with blastospores of Beauveria bassiana (Balsamo) Vuillemin strain GHA (Bb) at the ratio of 1:1 mixed with traditional fermentation production of Bacillus thuringiensis kurstaki (Btk) spores and crystals were studied. Twelve potential formulation ingredients were screened for their impact on insecticidal activity, microbe viability, UV protection and product suspensibility of a spray dried Bt/Bb (1:1) mixture applications to cabbage plants against cabbage looper, Trichoplusia ni. Based on the results, Spray-dried mixtures of Bt/Bb made with biochar, chitosan and skim milk powder formulations gave the greatest efficacy and higher solar stability when exposed to simulated sunlight. They therefore can be used as a formulation that combines Bt with Bb in one product. However, the formulation made with aitkin component had significantly more colony forming units (CFUs) of Bt, whereas, formulations made with albumin, sodium carbonate and pharmamedia had more Bb CFUs than other formulations. Albumin, pharmamedia, skim milk powder and biochar had significantly better suspension in water (less settling) than others. These results support the concept of formulating the two biocontrol agents, B. thuringiensis and B. bassiana blastospores for greater efficacy, more viability and longer field persistence as an environmentally friendly sustainable pest control tool.

LSID:urn:lsid:zoobank.org:pub:568D6FE6-E876-417B-BE39-C51F7CBAD6AB