Identification and immunochemical characterization of the human erythrocyte membrane glycoproteins that carry the Xga antigen.

When the membrane components of Xg(a+) erythrocytes were separated by electrophoresis and immunoblotted with an anti-Xga of human origin, two diffuse bands of approximate Mr 22,000-25,000 and 26,500-29,000 were stained. These reactive components were not evident in membranes from proteinase-treated Xg(a+) erythrocytes. Neuraminidase treatment of erythrocytes before immunoblotting resulted in one diffuse Xga-reactive band, the leading edge of which had a slightly increased mobility corresponding to a decrease in Mr of approx. 1500. These results suggest that the membrane components that carry Xga are sialoglycoproteins. A genetic relationship exists between Xga and the antigen recognized by the monoclonal antibody 12E7. An immunochemical comparison of the structures that carry the Xga and 12E7 antigens demonstrated that they differ in Mr and in the way in which they are modified by neuraminidase. This is in accord with evidence that Xga and 12E7 are the products of two separate structural loci.     

Preparation and application of a fluorescein-labeled peptide for determining the affinity constant of a monoclonal antibody-hapten complex by fluorescence polarization.

A simple and rapid method for determining the affinity constant of a monoclonal antibody-peptide complex under equilibrium conditions is presented. A peptide corresponding to sequence 178-185 of meningococcal strain MC50 class 1 outer membrane protein, which is recognized by monoclonal antibody MN12 (mouse IgG2a), was synthesized. After fluorescein was coupled to the peptide, the peptide-fluorescein conjugate was used for binding studies with MN12, employing fluorescence polarization of the fluorescein label to probe the bound fraction of the peptide. Scatchard analysis showed that the affinity constant was pH dependent. Storage of MN12 under alkaline conditions resulted in a loss of antigen-binding sites, but did not alter the affinity constant. Sips plots showed a homogeneity index of unity.     

Microbial Mineral Weathering for Nutrient Acquisition Releases Arsenic

Tens of millions of people in Southeast Asia drink groundwater contaminated with naturally occurring arsenic. How arsenic is released from the sediment into the water remains poorly understood. Here, we show in laboratory experiments that phosphate-limited cells of Burkholderia fungorum mobilize ancillary arsenic from apatite. We hypothesize that arsenic mobilization is a by-product of mineral weathering for nutrient acquisition. The released arsenic does not undergo a redox transformation but appears to be solubilized from the apatite mineral lattice during weathering. Analysis of apatite from the source area in the Himalayan basin indicates the presence of elevated levels of arsenic, with an average concentration of 210 mg/kg. The rate of arsenic release is independent of the initial dissolved arsenic concentration and occurs at phosphate levels observed in Bangladesh aquifers. We also demonstrate the presence of the microbial phenotype that releases arsenic from apatite in Bangladesh aquifer sediments and groundwater. These results suggest that microbial mineral weathering for nutrient acquisition could be an important mechanism for arsenic mobilization.Tens of millions of people in Southeast Asia are drinking groundwater containing elevated levels of arsenic that is naturally released from the sediment into the groundwater (22, 49). Multiple mechanisms have been proposed to explain this phenomenon, including arsenic mobilization during reduction of Fe(III) (hydr)oxides (14, 22, 27), arsenate reduction and mobilization (25, 28, 51), and redox cycling and release from arsenic-rich sulfides (30). Each mechanism probably plays a role under certain conditions but cannot fully explain the prevalence of arsenic or all the features of its distribution in groundwater (39, 42).It is well known that bacteria can obtain nutrients from solid-phase minerals (1, 4, 5, 36, 47). Subsurface environments are oligotrophic, and bacteria have been shown to preferentially weather minerals that contain phosphate and ammonia, both required for growth (36, 37). Microbial production of organic acids, ligands, and extracellular polysaccharides could all promote dissolution of minerals (35, 44-46). This is different than microbes using solid-phase minerals in redox processes such as Fe(III) reduction, resulting in the release of toxic metals (2, 24, 28). During redox reactions, microbes use the solid-phase minerals for energy. During nutrient acquisition, microbes access trace nutrients from the minerals for cellular growth (5), and redox alterations may not be occurring. Despite the importance of bacterially promoted mineral weathering for nutrient acquisition, the release of potentially dangerous ancillary elements such as arsenic from minerals to groundwater has not been investigated.We propose a novel mechanism for arsenic mobilization in aquifer systems: microorganisms weather and dissolve minerals to obtain nutrients and, during this process, cause an ancillary release of arsenic into the groundwater. Microcosm experiments were conducted with Burkholderia fungorum, phosphate-free artificial groundwater (AGW), and natural apatite. Apatite from the Himalayas was also analyzed for arsenic. Finally, bacteria extracted from Bangladesh sediments and water samples were incubated on mineral phosphate-solubilizing (MPS) plates to determine if this phenotype is present in arsenic-rich deltaic environments. Results indicate that ancillary arsenic release may mobilize arsenic in aquifer systems.The generalized formula for apatite is Ca₅(PO₄)₃(F,Cl,OH). Purified apatite was obtained from the Himalayas (18) and was digested with hot nitric acid. Digestions were performed to determine the total amount of arsenic associated with the apatite. Approximately 0.5 g of apatite was digested in 5 ml of nitric acid at 90°C for 1 h and then brought to 20 ml with deionized water (8). The total amount of arsenic was measured by inductively coupled plasma mass spectrometry. For microcosm experiments, apatite was purchased from Ward''s Scientific and ground with mortar and pestle to a fine powder. It contained 470 mg/kg arsenic. The percentages of As(V) and As(III) in the solid-phase arsenic from the Ward''s Scientific apatite and one Himalayan sample were determined by X-ray absorption near-edge structure spectroscopic analyses at the Stanford Synchrotron Radiation Laboratory on beam line 11-2 (13, 31, 34).Burkholderia fungorum was utilized because of its ability to weather rocks for nutrient acquisition. Specifically, experiments with basalt and granite have shown that it can weather these rocks and release phosphate into the environment (47, 48). Members of the Burkholderia genus have been isolated from granitic soils (48) and have been shown to be efficient at mineral weathering (40). Unfortunately, little work has been done to characterize microbial populations in the aquifers and soils of Southeast Asia, and isolates were not available (9, 19, 20). The metabolic function of Burkholderia fungorum is indistinguishable from those of other heterotrophic bacteria and therefore was chosen as a model organism (12).Burkholderia fungorum (ATCC BAA-463) was grown overnight at 30°C while being shaken in tryptic soy broth, washed, and resuspended in P-limited AGW. Approximately 5 × 10⁵ cells/ml were added to each microcosm. In abiotic controls, cells were killed by being autoclaved and then added to the microcosm. The P-limited AGW is based on the recipe of Wu et al. (47) and is similar to low-ionic-strength groundwater; per liter of solution, the major components are glucose (0.2 g), NH₄Cl (0.04 g), KCl (5.0 × 10⁻⁴ g), and MgSO₄ (5.0 × 10⁻⁴ g) (added as 1 × 10⁻³ g MgSO₄ · 7H₂O). Glucose was added as the carbon source. Each 500-ml Wheaton bottle contained 200 ml of P-limited AGW and 2 g of apatite. Bottles were shaken at 30°C throughout the course of the experiment. The microcosm experiments were performed under oxic conditions. Experiments included addition of (i) 2 ml of live B. fungorum cells, (ii) 2 ml of killed B. fungorum cells, (iii) a no-cell control, (iv) 2 ml of live B. fungorum cells and an arsenate spike, and (v) 2 ml of live B. fungorum cells with a pH 7 phosphate spike. Samples were spiked with arsenic because recent field studies have shown that arsenic release is independent of the initial arsenic concentration (39). Therefore, a field-relevant method should indicate release in the presence of increasing initial arsenic concentrations. A phosphate spike was included to determine if release occurred under field-relevant phosphate conditions. Abiotic experiments were performed with gluconic acid and the Ward''s Scientific apatite. Eight incubations with 0.2 g of apatite and 20 ml of gluconic acid were performed at concentrations ranging from 314 nM to 3.14 M. Samples were shaken at 200 rpm for 24 h at 37°C.Aliquots were removed from each microcosm at the sampling points for analysis of pH, gluconic acid, glucose, cations, and trace metals. pH was analyzed immediately with an Accumet pH electrode (Fisher Scientific). Glucose and gluconic acid samples were filtered with a Millex-GP 0.22-μm filter (Millipore) and frozen. Glucose was analyzed by the Amplex red glucose kit (Invitrogen) using a Stratagene Mx3005P (La Jolla, CA) system in plate reader mode. Gluconic acid was measured by ion chromatography on a Dionex ICS-2000 (Sunnyvale, CA) with an AS11HC column in gradient mode. Trace metal and cation samples were filtered with a Millex-GP 0.22-μm filter (Millipore) and stored in 1% hydrochloric acid at 4°C. Samples were analyzed by inductively coupled plasma mass spectrometry with a high-resolution Axiom single collector instrument (21). Arsenic species, i.e., As(III) and As(V), were determined by hydride generation atomic fluorescence spectrometry (32).The amount of phosphate remaining in the apatite was not examined because only a small percentage of the apatite was solubilized and it would not be possible to detect these small variations. Previous microcosm experiments with phosphate-limited AGW and basalt or granite have shown that little to no cell growth occurs in the absence of a mineral phosphate source and that apatite was most likely the source of phosphate (47, 48). Plating and flow cytometer analyses during the initial experiments indicated that without phosphate or apatite, significantly less growth occurred. However, the fine-grained apatite in the microcosms made flow cytometer counting and plate counting difficult. Therefore, the mineral solids at the end of one round of incubation were examined by scanning electron microscopy (SEM) to better understand microbe-mineral interactions (7). Samples were prepared inside an anaerobic glove bag. To prepare samples for SEM observations, cell-mineral suspensions were fixed in 2.5% glutaraldehyde in a 0.05 M Na cacodylate buffer solution of pH 7. One droplet of fixed cell-mineral gel was placed on the surface of a glass coverslip that was cleaned with 1 mg/ml polylysine solution prior to use. The sample was allowed to settle down onto the coverslip for 20 min. The sample-coated coverslips were sequentially dehydrated using various proportions of ethanol and distilled water, followed by critical point drying (7). The coverslips were mounted onto SEM stubs and Au coated for observations with a Zeiss Supra 35 VP FEG scanning electron microscope. The only time that the samples were exposed to air was during critical point drying and Au coating. The scanning electron microscope was operated at the accelerating voltage of 15 kV. Short working distance (6 to 10 mm) and low beam current (30 to 40 mA) were used to achieve maximum image resolution.MPS phenotype plates were prepared in two layers (10). The bottom layer consisted of 10 ml of 1% purified agar. The top layer consisted of a nutrient medium with 2% purified agar, 1% glucose, 0.1% MgSO₄ (0.21% MgSO₄·7H₂O), 0.1% NaCl, and 0.5% NH₄Cl. After being autoclaved, 0.5 g sterile hydroxyapatite (Sigma), vitamins, and minerals were added to the top layer of media. The hydroxyapatite was washed 10 times in deionized water to remove sorbed phosphate. The vitamin and mineral mix was altered (23), and no phosphate was present in the MPS phenotype plates except for in the solid-phase hydroxyapatite. Plates were inoculated with cells and incubated aerobically at 30°C. When poured, the plate contents are cloudy because of the presence of solid-phase hydroxyapatite in the agar. The only mechanism causing the plates to become clear is the solubilization of hydroxyapatite. Previous studies have shown that clearings on the plates occur only in the presence of the MPS phenotype (10).Four sediment samples and six water samples from Bangladesh were tested for the presence of the MPS phenotype. All water and sediment samples were from Araihazar Upazila, located 30 km east of Dhaka in a transition zone between the uplifted, low-arsenic Pleistocene Madhupur terrace to the northwest and a high-arsenic region on the opposite bank of the Meghna River to the southeast (50). Groundwater arsenic concentrations in the region range from less than 5 μg/liter to 900 μg/liter (43). Sediments were extracted by combining 10 g of sediment with 20 ml of AGW augmented with 1% Tween 80 and shaken overnight at 30°C (3). The sediment was allowed to settle by gravity, two volumes were plated, 50 μl and 100 μl, and the plates were sealed with Parafilm and incubated at 30°C. Collecting microbially pristine sediment samples from Bangladesh aquifers is difficult and requires novel techniques (33, 41) compared to those traditionally utilized (16, 29). Traditional drilling techniques are not available, and extensive work has been undertaken with the local drillers to obtain pristine samples that are not contaminated by drilling fluid (33, 41). Briefly, during drilling a core or evacuated cylinder is lowered through the drill casing to collect sediment from below the casing. Sediment is therefore collected below the casing from undisturbed sediment. The sediment was stored anaerobically at room temperature until use. These sediments are considered to be pristine with unaltered microbial populations and are our best method for assessing in situ microbial processes (42).Groundwater samples obtained by sterile techniques were also examined. Groundwater from six wells was concentrated by ultrafiltration (15), and the concentrated retentate was utilized to inoculate plates. Briefly, 100 liters of water was concentrated to 250 ml using a Rexeed 25S dialysis filter (Dial Medical Supply, Chester Springs, PA), and 100 μl of this retentate was used to inoculate the plates. The plates were then stored at room temperature in the dark, and growth was observed over the first week. This method was performed with a sterile technique at the wellhead in Bangladesh and greatly decreased the chance of having problems with contamination or long-term storage. Standard MPS plates plus three controls were included in the experiment. The four-plate procedure included (i) standard MPS plates, (ii) MPS plates with no phosphate and no hydroxyapatite, (iii) MPS plates with phosphate but no hydroxyapatite, and (iv) MPS plates with phosphate and hydroxyapatite. The goal of using the additional plates was to better understand under what conditions hydroxyapatite is mobilized and further constrain the phenotype in Bangladesh. Good photographs of the plates could not be obtained at the field clinic, but consistent patterns were observed and reported.Apatite, Ca₅(PO₄)₃(F,Cl,OH), is an abundant source of phosphate and is present in the aquifers and rivers of Bangladesh (6, 22). Dissolution of 13 purified apatite samples from the Himalayas resulted in an average arsenic concentration of 210 mg/kg (all values are 1, 3, 10, 11, 15, 24, 51, 78, 110, 200, 220, 250, and 1,800 mg/kg). These samples are not from Bangladesh but were upgradient within the drainage basin and may represent source material for the delta. These apatite samples were not used in the incubations, as we used the standard mineral, but indicate that apatite is a potential source of phosphate and also arsenic for the shallow aquifers in Bangladesh. Arsenic X-ray absorption near-edge structure analysis on beam line 11-2 at the Stanford Synchrotron Radiation Laboratory of the Ward''s Scientific apatite used in the microcosms and the 51-mg/kg sample from the Himalayas indicated that both samples had approximately 84% ± 6% arsenate and 16% ± 6% arsenite. We assume that the arsenic is replacing the phosphorus in the mineral structure and not just sorbed on the surface, since washes with water do not release the arsenic from the mineral.Aqueous samples were collected from a series of microcosm incubations over time and analyzed. Control incubations with killed cells and without cells did not release phosphorus, calcium, or arsenic, and glucose levels remained unchanged (Fig. ​(Fig.1).1). In live incubations, phosphorus and calcium concentrations increased over time, indicating dissolution of apatite, and glucose levels decreased, indicating microbial respiration (Fig. ​(Fig.1).1). More phosphorus is released than can be consumed by the bacteria, and the concentrations increase over time. More importantly, arsenic levels systematically increased with calcium and phosphorus from 4 to 35 μg/liter arsenic. The increase was consistent in duplicate bottles and experiments (Fig. ​(Fig.1).1). The arsenic was released as oxidized arsenate in all incubations tested, with an average of 87% ± 5% As(V) and 13% ± 5% As(III) in the aqueous phases of the microcosms (Fig. ​(Fig.1E).1E). The percentages of As(V) and As(III) in the aqueous phase were nearly identical to those in the mineral. Therefore, no arsenic redox chemistry was occurring. This indicates that microbes catalyzed the release of arsenic from the apatite. We hypothesize that microbes preferentially weather minerals for nutrient acquisition, and during this process, arsenic contained in the mineral will be released into the water.Open in a separate windowFIG. 1.Change in concentrations over time during microcosm experiments examining arsenic release from apatite. Results are shown for two duplicate microcosms and for the no-cell control. The killed control is shown in Fig. ​Fig.4,4, and the killed control and no-cell control were always nearly identical. (A) Change in calcium concentrations over time. (B) Change in pH over time. (C) Change in glucose concentrations over time. (D) Change in phosphorus concentrations over time. (E) Change in arsenic concentrations over time. The proportion of arsenate As(V) relative to As(V) plus As(III) is indicated at the fourth time point and is presented as a percentage. The arsenic was released as oxidized arsenate in all incubations tested, with an average of 87% ± 5% As(V) and 13% ± 5% As(III) in the aqueous phases of the microcosms. (F) Change in gluconic acid concentrations over time.SEM was performed to better understand mineral dissolution and potential microbe-mineral interactions. In the killed control (Fig. ​(Fig.2A),2A), apatite grains are observed with no cells and no dissolution features. In the live incubations with cells, glucose, and apatite, there is an intimate relationship between the apatite minerals and the cells (Fig. ​(Fig.2B).2B). Cells are observed on the surfaces of the apatite, and dissolution textures are present on the surfaces of the apatite. These observations are consistent with the idea that apatite was weathered by B. fungorum and that during this weathering process, arsenic was released.Open in a separate windowFIG. 2.Secondary electron images from microcosm sediments at the end of 24 h of incubation. (A) Image of apatite grains from the killed control. Microbes were not observed in association with the minerals, and the minerals appear unaltered. (B) Image of apatite grains from a live microcosm amended with B. fungorum and glucose. Rod-shaped cells are observed in close contact with the apatite minerals, and dissolution features are present on the minerals.Further measurements and experiments were conducted to better understand the mechanisms of apatite dissolution and arsenic release (Fig. ​(Fig.3).3). Control incubations with killed cells and without cells did not show a change in pH over the course of the incubations (Fig. ​(Fig.11 and ​and4).4). In live incubations, the pH level systematically decreased (Fig. ​(Fig.1).1). In the no-cell control, gluconic acid levels were constant, whereas in the live incubations, gluconic acid levels increased (Fig. ​(Fig.1E).1E). The production of gluconic acid from glucose during respiration has previously been observed with Burkholderia fungorum (11, 46, 48). Gluconic acid is typically further oxidized by bacteria but can accumulate under stress (12). In order to compare microbial metabolism to abiotic controls, apatite was shaken for 24 h at 30°C in increasing strengths of gluconic acid (Fig. ​(Fig.3).3). The amount of arsenic release was approximately linear on a log-log scale of gluconic acid and arsenic concentrations. In addition, the amount of arsenic released during the live microcosms due to microbial production of gluconic acid was consistent with the abiotic experiments (Fig. ​(Fig.3).3). This indicates that microbial oxidative degradation of glucose to gluconic acid by microbes in intimate contact with apatite is a likely cause of arsenic release from the mineral structure into the water.Open in a separate windowFIG. 3.Gluconic acid concentration versus arsenic concentration from abiotic experiments, along with the average gluconic acid and arsenic concentrations from the live microcosms in Fig. ​Fig.1.1. A log-log relationship was observed between gluconic acid concentrations and the amount of arsenic released from the apatite. In addition, the amount of arsenic released during microcosm experiments was consistent with that of the abiotic experiments.Open in a separate windowFIG. 4.(A) Release of arsenic over time at various initial arsenic concentrations. These experiments were conducted to determine if the release of arsenic occurs at all initial arsenic concentrations, as observed in the field. Microcosms were spiked with arsenate. The killed control is shown for reference, which did not release arsenic. (B) Change in pH over time for the arsenic spike experiments. (C) Change in concentration of arsenic over time at different initial phosphate concentrations. All microcosms contained B. fungorum, and the phosphate was spiked at pH 7. (D) pH changes during the phosphate spike experiments. (E) Release of arsenic as a function of initial phosphate concentrations. The gray bars indicate the distribution of detectable phosphate levels observed in Bangladesh aquifers, as determined by the British Geological Survey (BGS) (22).Additional incubations were conducted to examine the effect of increasing initial arsenic and phosphate concentrations and to better constrain field observations. The amount of arsenic released as a function of initial arsenic concentration was examined because field observations indicate that the release of arsenic occurs at a constant rate and is independent of arsenic concentration (39). Results indicate that during ancillary arsenic release, the amount of arsenic released is independent of the initial arsenic concentration (Fig. ​(Fig.4A).4A). In addition, the pH decreases consistently at all arsenic levels consistent with the mechanism of gluconic acid production (Fig. ​(Fig.4B).4B). We examined the amount of arsenic released as a function of phosphate concentrations to determine if release occurs under field-relevant conditions. The amount of arsenic released decreases as the initial aqueous phosphate concentration increases above 0.1 mM (Fig. 4C and E). The arsenic is released when the pH decreases but remains steady when 10 and 100 mM phosphate are present. The limiting phosphate concentrations are higher than concentrations observed in Bangladesh groundwater (Fig. ​(Fig.4E).4E). Microbially mediated arsenic release from a commonly occurring mineral was documented in the presence of increasing arsenic concentrations and at in situ phosphate levels, indicating that the mechanism is consistent with field observations.In separate experiments, we checked for the presence of the MPS phenotype in bacteria collected from sediments of shallow Bangladesh aquifers. Cells extracted from Bangladesh sediment were incubated on MPS phenotype plates that contained only solid-phase phosphate. Each sample gave rise to colonies surrounded by easily observed clearings (Fig. ​(Fig.5).5). These clearings can be present only if the hydroxyapatite is solubilized. Plates had, on average, about 5 to 10 colonies with clearings, which corresponds to about 100 to 200 colonies per gram. After performing initial sediment incubations, we returned to Bangladesh and incubated MPS plates with groundwater that was concentrated by ultrafiltration using sterile techniques. Four types of plates were incubated as follows: (i) standard MPS plates, (ii) MPS plates with no phosphate and no hydroxyapatite, (iii) MPS plates with phosphate but no hydroxyapatite, and (iv) MPS plates with phosphate and hydroxyapatite. A consistent pattern emerged with the plates (data not shown). The standard MPS plates had colonies and abundant clearings (∼100 colonies). The MPS plates with no phosphate and no hydroxyapatite had colonies but significantly fewer colonies (∼20), indicating that phosphate may have been limited. The MPS plates with phosphate but no hydroxyapatite had significant colonies (∼100). The MPS plates with phosphate and hydroxyapatite had colonies (∼100) but no clearings, indicating that cells were using the soluble phosphate. When phosphate was present in either the solid or aqueous phase, significantly more colonies were observed. This would correspond to about 3 colonies per ml of groundwater. When only solid-phase phosphate as hydroxyapatite was present, clearings were observed. This was corroborating evidence that the MPS phenotype is present in Bangladesh, and under low phosphate conditions, hydroxyapatite is solubilized. This indicates that bacteria capable of solubilizing phosphate from solid-phase hydroxyapatite are present in the shallow aquifers of Bangladesh.Open in a separate windowFIG. 5.MPS phenotype plate containing only solid-phase phosphate as hydroxyapatite was incubated with cells extracted from shallow Bangladesh sediment. The clearings are highlighted by arrows and indicate that phosphate-solubilizing bacteria were extracted from the sediment and are present in the shallow aquifers of Bangladesh. Small clearings are observed around many of the other colonies but do not appear in the photographs.We hypothesize that the required conditions for ancillary arsenic release during microbial mineral weathering appear to include a nutrient limitation, the presence of poorly weathered minerals such as apatite, a supply of organic carbon, and an active microbial population. Each of these conditions is present for Bangladesh aquifers. The MPS phenotype is present in Bangladesh, and the in situ phosphate levels should promote its expression and, hence, mineral weathering with the associated arsenic release. Bangladesh aquifers are organic carbon rich, and organic carbon has been correlated with arsenic (26). Ancillary arsenic release may also occur during weathering of feldspars, biotite, and hornblende. These minerals are common in Bangladesh aquifers (6, 31, 38), and they have been shown to be potential sites of microbial weathering (5, 36, 37, 40).We postulate that nutrient acquisition and ancillary arsenic release are independent of redox conditions and will also occur during fermentation, Fe(III) reduction, and methanogenesis. Importantly, we believe that ancillary arsenic release has been obscured by the overprint of iron cycling. Laboratory and field experiments are showing that Fe and As release are decoupled (17, 33, 42) and that arsenic release occurs at a constant rate across various depositional environments (39). These observations are consistent with our novel mechanism that can occur under all redox conditions. Therefore, we consider ancillary arsenic release to potentially be a more important factor than Fe(III) reduction for controlling arsenic release from the minerals into the groundwater of Bangladesh. This novel mechanism may clarify some of the unexplained geochemical patterns related to the presence of arsenic in groundwater and its worldwide impact on human health.     

Impact of nC24 agricultural mineral oil deposits on the searching efficiency and predation rate of the predatory mite Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae)

Walking activity, walking straightness, walking speed and searching efficiency of the predatory mite Phytoseiulus persimilis Athias-Henriot were measured on French bean leaf discs that were sprayed with either distilled water, or one of 0.25%, 0.50% and 1.00% w/w aqueous emulsions of an n C24 agricultural mineral oil (AMO). There was no significant difference in percentage of time that mites spent walking in the control (water-sprayed) conditions and in any of the oil treatments. Walking paths were significantly straighter in the oil treatments than in the control, but differences among the oil treatments did not differ significantly. Walking speeds in the oil treatments were significantly slower than in the control and decreased with increasing oil concentration. Deposits of oil at all concentrations significantly suppressed searching efficiency in comparison with control, and searching efficiency in the 1.00% oil treatment was significantly lower than in the 0.25% oil treatment. First predation of P. persimilis on AMO-contaminated eggs of two-spotted mite ( Tetranychus urticae Koch) on unsprayed leaf discs was significantly delayed in all oil treatments in comparison with the control. However there was no significant effect on the overall predation rate. In the tests of P. persimilis predation on AMO-contaminated T. urticae eggs on sprayed leaf discs, the number of first predation occurrences in the first hour was significantly lower in 0.50% and 1.00% oil treatments than in the control. Overall predation rates were significantly reduced by oil but they did not differ significantly among the oil treatments.     

Relative toxicity of nC24 agricultural mineral oil to Tetranychus urticae Koch (Acari: Tetranychidae) and Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) and its possible relationship to egg ultrastructure

The relative toxicity (LC₅₀ values based on µg oil/cm²) is evaluated of aqueous n C24 agricultural mineral oil (AMO) emulsions to the egg, six-legged nymph (larva), eight-legged protonymph and adult stages of two-spotted mite ( Tetranychus urticae ) and its predator, Phytoseiulus persimilis , on French bean leaf discs, using a Potter spray tower to apply of the oil. The egg of P. persimilis was the least susceptible stage (LC₅₀ 444.84) and its LC₅₀ was significantly higher than all other stages tested of either P. persimilis or T. urticae . The LC₅₀ for adult female T. urticae (LC₅₀ 63.89) was significantly lower than the larva (LC₅₀ 93.86); however, there was no significant difference in response between the protonymph (LC₅₀ 70.44) and the larva, which were both higher than T. urticae eggs (LC₅₀ 17.55). LC₅₀s for P. persimilis larva (LC₅₀ 43.87), protonymph (LC₅₀ 41.55) and adult female (LC₅₀ 53.34) were similar. Scanning electron microscopy showed that the egg surface of T. urticae is usually well covered with fine silk that may trap more oil and increase AMO efficacy. Other possible differences in AMO efficacy between T. urticae and P. persimilis may be due to differences in egg size, egg incubation period, egg surface structure and the presence of vulnerable respiratory cones in T. urticae eggs. Dose of 0.2–0.3% (w/w) is considered to be the most appropriate for n C24 AMOs use against T. urticae in combination with P. persimilis in integrated pest management programs.     

Genetic heterogeneity of skin microvasculature

Angiogenesis, the formation of new blood vessels from existing vasculature, is a complex process that is essential for normal embryonic development. Current models for experimental evaluation of angiogenesis often use tissue from large vessels like the aorta and umbilical vein, which are phenotypically distinct from microvasculature. We demonstrate that the utilization of skin to measure microvascular angiogenesis in embryonic and adult tissues is an efficient way to quantify microvasculature angiogenesis. We validate this approach and demonstrate its added value by showing significant differences in angiogenesis in monogenic and polygenic mouse models. We discovered that the pattern of angiogenic response among inbred mouse strains in this ex vivo assay differs from the strain distributions of previous in vivo angiogenesis assays. The difference between the ex vivo and in vivo assays may be related to systemic factors present in whole animals. Expression analysis of cultured skin biopsies from strains of mice with opposing angiogenic response was performed to identify pathways that contribute to differential angiogenic response. Increased expression of negative regulators of angiogenesis in C57Bl/6J mice was associated with lower growth rates.     

Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

Background

Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis.

Methodology/Principal Findings

Mice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice.

Conclusions/Significance

Tumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.