Detection of aerosolized bacterial spores (Bacillus atrophaeus) using free-flying honey bees (Hymenoptera: Apidae) as collectors

An aerosol cloud of Bacillus atrophaeus (previously B. subtilis variety niger) spores, an anthrax surrogate, was created in a large 0.4 ha (1 ac), bee-containing, open-mesh tent. Bees from a B. atrophaeus uncontaminated hive flying through the cloud adsorbed the spores in statistically significant quantities. After removal of the B. atrophaeus contaminated hive and introduction of another B. atrophaeus uncontaminated hive, the bees again were monitored for the next few days for B. atrophaeus spores. B. atrophaeus spores accumulated on the bees bodies following their exposure to the residual B. atrophaeus contamination in the tent. The spore loads on the bees quickly returned to background levels after the hives were removed from the contaminated tent area. It may therefore be practical to use honey bee colonies to monitor foraging areas for disease-causing spores.     

Sub-lethal effects of pesticide residues in brood comb on worker honey bee (Apis mellifera) development and longevity

Background

Numerous surveys reveal high levels of pesticide residue contamination in honey bee comb. We conducted studies to examine possible direct and indirect effects of pesticide exposure from contaminated brood comb on developing worker bees and adult worker lifespan.

Methodology/Principal Findings

Worker bees were reared in brood comb containing high levels of known pesticide residues (treatment) or in relatively uncontaminated brood comb (control). Delayed development was observed in bees reared in treatment combs containing high levels of pesticides particularly in the early stages (day 4 and 8) of worker bee development. Adult longevity was reduced by 4 days in bees exposed to pesticide residues in contaminated brood comb during development. Pesticide residue migration from comb containing high pesticide residues caused contamination of control comb after multiple brood cycles and provided insight on how quickly residues move through wax. Higher brood mortality and delayed adult emergence occurred after multiple brood cycles in contaminated control combs. In contrast, survivability increased in bees reared in treatment comb after multiple brood cycles when pesticide residues had been reduced in treatment combs due to residue migration into uncontaminated control combs, supporting comb replacement efforts. Chemical analysis after the experiment confirmed the migration of pesticide residues from treatment combs into previously uncontaminated control comb.

Conclusions/Significance

This study is the first to demonstrate sub-lethal effects on worker honey bees from pesticide residue exposure from contaminated brood comb. Sub-lethal effects, including delayed larval development and adult emergence or shortened adult longevity, can have indirect effects on the colony such as premature shifts in hive roles and foraging activity. In addition, longer development time for bees may provide a reproductive advantage for parasitic Varroa destructor mites. The impact of delayed development in bees on Varroa mite fecundity should be examined further.     

Use of a Foam Spatula for Sampling Surfaces after Bioaerosol Deposition

The present study had three goals: (i) to evaluate the relative quantities of aerosolized Bacillus atrophaeus spores deposited on the vertical, horizontal top, and horizontal bottom surfaces in a chamber; (ii) to assess the relative recoveries of the aerosolized spores from glass and stainless steel surfaces with a polyester swab and a macrofoam sponge wipe; and (iii) to estimate the relative recovery efficiencies of aerosolized B. atrophaeus spores and Pantoea agglomerans using a foam spatula at several different bacterial loads by aerosol distribution on glass surfaces. The majority of spores were collected from the bottom horizontal surface regardless of which swab type and extraction protocol were used. Swabbing with a macrofoam sponge wipe was more efficient in recovering spores from surfaces contaminated with high bioaerosol concentrations than swabbing with a polyester swab. B. atrophaeus spores and P. agglomerans culturable cells were detected on glass surfaces using foam spatulas when the theoretical surface bacterial loads were 2.88 × 10⁴ CFU and 8.09 × 10⁶ CFU per 100-cm² area, respectively. The median recovery efficiency from the surfaces using foam spatulas was equal to 9.9% for B. atrophaeus spores when the recovery was calculated relative to the theoretical surface spore load. Using a foam spatula permits reliable sampling of spores on the bioaerosol-exposed surfaces in a wide measuring range. The culturable P. agglomerans cells were recovered with a median efficiency of 0.001%, but staining the swab extracts with fluorescent dyes allowed us to observe that the viable cell numbers were higher by 1.83 log units than culturable organisms. However, additional work is needed to improve the analysis of the foam extracts in order to decrease the limit of detection of Bacillus spores and Gram-negative bacteria on contaminated surfaces.Surface sampling is performed on a frequent basis in all situations where clean environment monitoring is needed, e.g., in health care facilities and in the pharmaceutical industry and food industry. An anthrax bioterrorist event in the fall of 2001 has emphasized the importance of efficient sampling methods for detection of pathogenic microorganisms on surfaces within intentionally contaminated locations (22). Unfortunately, our knowledge on the most effective sampling methodology as well as the level of confidence we may have in the results obtained by wiping, swabbing, and other sample collection strategies is still limited (1). Moreover, in most of the studies performed so far, bacteria and/or spores were collected from test samples or coupons of various materials, inoculated with a suspension of microorganisms that had been placed and spread over the surface, and then dried (14, 15). This may not mimic the true situation of surface contamination by a pathogen that has been intentionally released. Edmonds et al. (12) recently reported lower swabbing efficiencies of different types of swab materials used for sampling glass, polycarbonate, and vinyl surfaces contaminated with dry aerosol-deposited Bacillus atrophaeus spores compared to the surfaces inoculated by spore suspensions. Solid surface contamination from exposure to aerosolized spores fits the real world better than the previous models.Therefore, in our study we decided to generate aerosols of various concentrations of B. atrophaeus spores as well as the vegetative cells of Pantoea agglomerans inside a chamber where the bioaerosol particles were allowed to gravitationally settle on solid surfaces. The aerosolization of P. agglomerans was performed to verify the recovery of Gram-negative bacteria according to the recommendations of Budowle et al. (5). The main goal of our study was to establish the range of detection when bioaerosol-contaminated surfaces were swabbed using a commercially available foam spatula.     

Inactivation of Bacillus Endospores in Envelopes by Electron Beam Irradiation

The anthrax incidents in the United States in the fall of 2001 led to the use of electron beam (EB) processing to sanitize the mail for the U.S. Postal Service. This method of sanitization has prompted the need to further investigate the effect of EB irradiation on the destruction of Bacillus endospores. In this study, endospores of an anthrax surrogate, B. atrophaeus, were destroyed to demonstrate the efficacy of EB treatment of such biohazard spores. EB exposures were performed to determine (i) the inactivation of varying B. atrophaeus spore concentrations, (ii) a D₁₀ value (dose required to reduce a population by 1 log₁₀) for the B. atrophaeus spores, (iii) the effects of spore survival at the bottom of a standardized paper envelope stack, and (iv) the maximum temperature received by spores. A maximum temperature of 49.2°C was reached at a lethal dose of ~40 kGy, which is a significantly lower temperature than that needed to kill spores by thermal effects alone. AD₁₀ value of 1.53 kGy was determined for the species. A surface EB dose between 25 and 32 kGy produced the appropriate killing dose of EB between 11 and 16 kGy required to inactivate 8 log₁₀ spores, when spore samples were placed at the bottom of a 5.5-cm stack of envelopes.     

DNA extraction and PCR detection of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Summary Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.     

Spore formation in the Actinoplanaceae (Actinomycetales)

Spore development in four genera, Actinoplanes, Dactylosporangium, Planomonospora, and Streptosporangium, was studied by transmission and scanning electron microscopy. Actinoplanes and Streptosporangium formed spores by fragmentation of a hypha within its expanded outer sheath, as do many other actinomycetes. Dactylosporangium and Planomonospora formed spores endogenously by development of wall material within the parent hypha. In this respect, they resembled the genera Actinobifida and Thermoactinomyces. The term sporangium has therefore been used to describe structures which are not homologous. It was suggested that the term should be confined to structures in which endogenous spore formation occurs.     

The effect of the ectoparasitic mite, Varroa destructor on adult worker honeybee (Apis mellifera) emergence weights, water, protein, carbohydrate, and lipid levels

Wet weight, dry weight and water contents of emerging honeybees (Apis mellifera L. [Hymenoptera: Apidae]) infested with the ectoparasitic mite Varroa destructor (Anderson) (Acari: Varroidae) were all negatively correlated with increasing numbers of mites. It was estimated that for every female mite present during the bees' development, the host would lose three percent of its body water. Parasitised bees also emerged with lower head and abdomen concentrations of protein and with lower abdominal carbohydrate concentrations. Lipid concentrations were not detectably affected by V. destructor infestation. The losses of metabolic reserves were not, however, judged to be serious enough to be directly responsible for the high bee mortality and ultimate colony collapse that are associated with the arrival of Varroa in a hive. Some 8.5% of the emerging bees exhibited morphological deformities and deformity was positively correlated with increasing numbers of mites in brood cells. Deformed bees were, however, found in all categories of parasitosis, suggesting that other factors, such as infectious agents, may be involved. Mites that fed on either live or dead U¹⁴C- labelled bees acquired the label within 24 h and it was calculated that an adult female mite consumes 0.67 l haemolymph 24 h^–1. It was also demonstrated that ¹⁴C was transmitted to a previously non-radio-labelled bee when a mite that had been feeding on a labelled bee changed hosts. The level of transfer was above that which could have arisen through contamination of the mites' mouthparts and supports the suggestion that Varroa is an important vector of pathogens such as viruses.     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Sorophores of<Emphasis Type="Italic">Lygodium</Emphasis> Sw. (Schizaeaceae) from the Late Cretaceous of New Jersey

We report here on a series of specimens of charcoalified sorophores with characteristics of the extant fern genusLygodium (Schizaeaceae) collected from sediments of the Raritan Formation (Late Cretaceous). Each elongate lobed fertile pinnule (sorophore) is flattened and bears alternately arranged sporangia on one surface. Each sporangium is covered by an indusium continuous with the margin of the lamina. Sporangia are oblong in shape, short stalked, and have an apical annulus formed by a single ring of radiating cells that dehisces longitudinally. The sporangial cap or distal face is formed by only one cell. All of these features are characteristic of the extant genusLygodium. Small numbers of trilete, psilate spores are found in the sporangia. Megafossils assignable toLygodium are known from the Upper Cretaceous of North America and Germany with worldwide distribution during the Tertiary. The newLygodium fossils are compared with others previously referred to the genus.     

Acaricide,Fungicide and Drug Interactions in Honey Bees (Apis mellifera)

Background

Chemical analysis shows that honey bees (Apis mellifera) and hive products contain many pesticides derived from various sources. The most abundant pesticides are acaricides applied by beekeepers to control Varroa destructor. Beekeepers also apply antimicrobial drugs to control bacterial and microsporidial diseases. Fungicides may enter the hive when applied to nearby flowering crops. Acaricides, antimicrobial drugs and fungicides are not highly toxic to bees alone, but in combination there is potential for heightened toxicity due to interactive effects.

Methodology/Principal Findings

Laboratory bioassays based on mortality rates in adult worker bees demonstrated interactive effects among acaricides, as well as between acaricides and antimicrobial drugs and between acaricides and fungicides. Toxicity of the acaricide tau-fluvalinate increased in combination with other acaricides and most other compounds tested (15 of 17) while amitraz toxicity was mostly unchanged (1 of 15). The sterol biosynthesis inhibiting (SBI) fungicide prochloraz elevated the toxicity of the acaricides tau-fluvalinate, coumaphos and fenpyroximate, likely through inhibition of detoxicative cytochrome P450 monooxygenase activity. Four other SBI fungicides increased the toxicity of tau-fluvalinate in a dose-dependent manner, although possible evidence of P450 induction was observed at the lowest fungicide doses. Non-transitive interactions between some acaricides were observed. Sublethal amitraz pre-treatment increased the toxicity of the three P450-detoxified acaricides, but amitraz toxicity was not changed by sublethal treatment with the same three acaricides. A two-fold change in the toxicity of tau-fluvalinate was observed between years, suggesting a possible change in the genetic composition of the bees tested.

Conclusions/Significance

Interactions with acaricides in honey bees are similar to drug interactions in other animals in that P450-mediated detoxication appears to play an important role. Evidence of non-transivity, year-to-year variation and induction of detoxication enzymes indicates that pesticide interactions in bees may be as complex as drug interactions in mammals.     

Gluconobacters from honey bees

Fifty-sixGluconobacter strains and oneAcetobacter strain were isolated from honey bees and their environment in three different regions in Belgium and identified phenotypically. Polyacrylamide gel electrophoresis of the soluble cell proteins showed that two different types exist within theGluconobacter isolates: strains from type A were found in samples of the three regions, whereas strains from type B were only isolated in two of the three regions. Both types could occur in bees from the same region, from several hives of one bee keeper and from one hive. Strains from type A were almost identical with collection strainG. oxydans subsp.suboxydans NCIB 9018, whereas strains from type B consituted a new protein electrophoretic type within the genusGluconobacter. AlthoughGluconobacter is apparently associated with honey bees, it is not known whether it is important or required for the bees or any hive product.     

Persistence and Decontamination of Bacillus atrophaeus subsp. globigii Spores on Corroded Iron in a Model Drinking Water System

Persistence of Bacillus atrophaeus subsp. globigii spores on corroded iron coupons in drinking water was studied using a biofilm annular reactor. Spores were inoculated at 10⁶ CFU/ml in the dechlorinated reactor bulk water. The dechlorination allowed for observation of the effects of hydraulic shear and biofilm sloughing on persistence. Approximately 50% of the spores initially adhered to the corroded iron surface were not detected after 1 month. Addition of a stable 10 mg/liter free chlorine residual after 1 month led to a 2-log₁₀ reduction of adhered B. atrophaeus subsp. globigii, but levels on the coupons quickly stabilized thereafter. Increasing the free chlorine concentration to 25 or 70 mg/liter had no additional effect on inactivation. B. atrophaeus subsp. globigii spores injected in the presence of a typical distribution system chlorine residual (~0.75 mg/liter) resulted in a steady reduction of adhered B. atrophaeus subsp. globigii over 1 month, but levels on the coupons eventually stabilized. Adding elevated chlorine levels (10, 25, and 70 mg/liter) after 1 month had no effect on the rate of inactivation. Decontamination with elevated free chlorine levels immediately after spore injection resulted in a 3-log₁₀ reduction within 2 weeks, but the rate of inactivation leveled off afterward. This indicates that free chlorine did not reach portions of the corroded iron surface where B. atrophaeus subsp. globigii spores had adhered. B. atrophaeus subsp. globigii spores are capable of persisting for an extended time in the presence of high levels of free chlorine.     

Chromium and nickel tolerance of Trema orientalis (Blume) L. in tissue culture

Callus of Trema orientalis derived from both contaminated and uncontaminated sources were tested in vitro for their relative tolerance to chromium and nickel. The calluses derived from contaminated source were metal-tolerant and showed better growth than those obtained from uncontaminated plants. The specificity of metal tolerance shown by the parent material was maintained in the calluses. Compared to the uncontaminated explants, the calli derived from contaminated sources exhibited higher catalase and peroxidase activities but a reduced acid phosphatase activity. Biochemical studies, provided evidence that the contaminated sources were physiologically distinct from the uncontaminated ones. Thus, this study indicated that seeds of Trema orientalis collected from contaminated sites were tolerant to chromium and nickel, and may have the advantage of being used in sustainable revegetation programmes on chromiferous minewastes.     

Decontamination Options for Bacillus anthracis-Contaminated Drinking Water Determined from Spore Surrogate Studies

Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (10² and 10⁶ spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor''s desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA''s efficacy standard for a biocide, although they were found to be as effective for concentrations of 10² spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.Developing a decontamination approach that can be safely and effectively applied to civilian water resources and facilities following a terrorist or catastrophic release of Bacillus anthracis spores poses many challenges. For example, if a municipal drinking water system were contaminated directly or indirectly during or after such an incident, it would be essential to assess the potential health risks posed by water consumption or other water uses (e.g., recreational and bathing) and then to apply one or more proven technologies, if deemed necessary, to decontaminate the water supply quickly and cost-effectively. Treatment of drinking water implies the use of a decontamination approach that would not pose adverse health risks to humans or result in unacceptable damage to the environment. A major obstacle in killing spores of Bacillus spp. on or in virtually any matrix is their high level of resistance to treatments such as harsh chemicals, heat, desiccation, and UV light (14, 20). Because of the substantial and widely reported resistance of Bacillus spores to inactivation, a decontaminant proven to be efficacious in killing such spores for site-specific applications is likely to be effective against all other biological warfare agents as well.Whereas nearly all biological warfare agents are intended for aerosol application, many have strong potential as waterborne threats and could inflict heavy casualties when ingested (2). B. anthracis in particular has been identified as a “probable” (12) or an actual (24) water threat. Even though the principal risk associated with the consumption of water containing B. anthracis spores would likely arise from an ingestion hazard, water used for bathing, showering, or recreational purposes might also pose cutaneous as well as aerosol exposure hazards. There is controversy regarding the long-term viability of B. anthracis in water, and experimental evidence is limited. However, according to a review of nonkinetic studies on survival of virulent strains in the environment (21), B. anthracis spores can survive from 2 to 18 years in pond water and 20 months in seawater or distilled water. B. anthracis spores have been reported by others to be stable in water for 2 years (24).Various decontamination approaches have been evaluated for efficacy against biological warfare agents, including Bacillus spores, on hard, nonporous surfaces. Recommendations by the U.S. Environmental Protection Agency (EPA) include the use of sodium hypochlorite (1:9 dilution of bleach to 5,250 to 6,000 ppm, corrected to pH 7, with a 60-min contact time at 20°C [6, 17]), and liquid chlorine dioxide with a 30-min wet contact time at 20°C (7). Liquid hydrogen peroxide/peroxyacetic acid (known as peroxy compounds and marketed as ready-to-use solutions), generally with a 15- to 20-min wet contact time and concentration as specified by the manufacturer, has also been recommended (13). Other products, such as hydrogen peroxide solution (3 to 25%) and potassium peroxymonosulfate, have been evaluated for efficacy against Bacillus spores as well (27). Although disinfectants at various concentrations have been tested previously against the spores of B. anthracis and their surrogates, wide variations in test protocols make meaningful comparisons among studies virtually impossible (9, 11, 17).In contrast to surface cleanup of spores, fewer assessments of efficacy utilizing suspension tests with the aforementioned chemicals or other methods have been reported for the decontamination of Bacillus species spores in water, and much of the published work has assessed only relatively high concentrations of spores in water. For example, one previous investigation commenced evaluations with 0.2-ml suspensions of approximately 10⁹ spores/ml of various Bacillus spp. to which 20 ml of aqueous ozone or 20 ml of hydrogen peroxide solution was added to assess sporicidal action (10), and others have reported mechanisms of deactivating B. subtilis spores prepared in concentrations of up to approximately 10⁸ spores/ml (26) and approximately 10⁹ spores/ml (17). Inactivation by chlorination of various Bacillus spp. with initial concentrations of approximately 1 × 10⁴ CFU/ml has also been tested (16). However, relatively low spore concentrations would be expected to result from dilutions following contamination of a large public water system. Therefore, it is reasonable to evaluate the effectiveness of decontaminants or other methods against even lower spore concentrations in water than have been previously assessed. In addition to assessing the parameter of Bacillus spore concentration in water, it is essential to identify the most effective commercially available chemical that will kill all the spores or minimize population growth, while considering the effects of the chemical on the environment and in humans.Several objectives served to focus our investigation. First, five potential candidate decontaminants were selected because of their relative safety and ultimate degradation in the environment without substantive adverse consequences. The five chemicals were also chosen as a way of comparing the effectiveness of available free chlorine content, pH, and oxidation potential on spore inactivation. From an evaluation of those chemical parameters, we sought to determine the most effective option for inactivating Bacillus spore surrogates suspended in water. As a second objective, we attempted to identify the lowest concentration of the selected chemicals necessary to achieve the EPA''s biocide standard of a >6-log kill. As a third objective, we wanted to assess the effect of reduced spore concentration on chemical biocide efficacy. As an important step in ascertaining an efficient, safe, and cost-effective water treatment method that could potentially provide safe water to the general population in the event of B. anthracis contamination—and limit the potential risk of contracting gastrointestinal or cutaneous anthrax as well—the following parameters were evaluated: chemical decontaminant type, chemical decontaminant concentration (0.01% to 5%), contact time of spores with chemical decontaminant (10 min to 1 h), spore type (Bacillus atrophaeus or Bacillus thuringiensis), and low versus relatively high spore concentrations (approximately 10² and 10⁶ spores/ml, respectively).Use of B. atrophaeus and B. thuringiensis spores as surrogates for B. anthracis is widely reported in the literature. For example, Szabo et al. (23) used B. atrophaeus subsp. globigii spores as a surrogate for B. anthracis to investigate the persistence and decontamination of those surrogates on corroded iron in a model drinking water system, and Rice et al. (16) used spores of B. thuringiensis as an “appropriate surrogate for spores of B. anthracis” for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Furthermore, the EPA (5) concluded that “B. globigii can serve as a conservative surrogate for B. anthracis during studies of inactivation by chlorination.”     

Substrate selection of adenylation domains for nonribosomal peptide synthetase (NRPS) in bacillamide C biosynthesis by marine <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis> C89

Nonribosomal peptide synthetases (NRPSs) are multi-modular enzymes involved in the biosynthesis of natural products. Bacillamide C was synthesized by Bacillus atrophaeus C89. A nonribosomal peptide synthetase (NRPS) cluster found in the genome of B. atrophaeus C89 was hypothesized to be responsible for the biosynthesis of bacillamide C using alanine and cysteine as substrates. Here, the structure analysis of adenylation domains based on homologous proteins with known crystal structures indicated locations of the substrate-binding pockets. Molecular docking suggested alanine and cysteine as the potential substrates for the two adenylation domains in the NRPS cluster. Furthermore, biochemical characterization of the purified recombinant adenylation domains proved that alanine and cysteine were the optimum substrates for the two adenylation domains. The results provided the in vitro evidence for the hypothesis that the two adenylation domains in the NRPS of B. atrophaeus C89 preferentially select alanine and cysteine, respectively, as a substrate to synthesize bacillamide C. Furthermore, this study on substrates selectivity of adenylation domains provided basis for rational design of bacillamide analogs.     

Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m²) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ± 5.8 CFU/m² for wet sampling and 100.5 ± 10.2 CFU/m² for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.     

Stingless bees (<Emphasis Type="Italic">Scaptotrigona pectoralis</Emphasis>) learn foreign trail pheromones and use them to find food

Foragers of several species of stingless bees (Hymenoptera, Apidae and Meliponini) deposit pheromone marks in the vegetation to guide nestmates to new food sources. These pheromones are produced in the labial glands and are nest and species specific. Thus, an important question is how recruited foragers recognize their nestmates’ pheromone in the field. We tested whether naïve workers learn a specific trail pheromone composition while being recruited by nestmates inside the hive in the species Scaptotrigona pectoralis. We installed artificial scent trails branching off from trails deposited by recruiting foragers and registered whether newly recruited bees follow these trails. The artificial trails were baited with trail pheromones of workers collected from foreign S. pectoralis colonies. When the same foreign trail pheromone was presented inside the experimental hives while recruitment took place a significant higher number of bees followed the artificial trails than in experiments without intranidal presentation. Our results demonstrate that recruits of S. pectoralis can learn the composition of specific trail pheromone bouquets inside the nest and subsequently follow this pheromone in the field. We, therefore, suggest that trail pheromone recognition in S. pectoralis is based on a flexible learning process rather than being a genetically fixed behaviour.     

Thermal behavior of round and wagtail dancing honeybees

The thermal behavior of round and wagtail dancing honeybees (Apis mellifera carnica) gathering sucrose solutions of concentrations between 0.5 and 2 mol·l^-1 was investigated under field conditions by infrared thermography (30–506 m flight distance). During the stay inside the hive thoracic surface temperature ranged from 31.4 to 43.9 °C. In both round and wagtail dancing honeybees the concentration of sucrose in the food influenced dancing temperature in a non-linear way. Average dancing temperature was 37.9 °C in foragers gathering a 0.5 mol·l^-1 sucrose solution, 40.1°C with a 1 mol·l^-1, 40.6°C with a 1.5 mol·l^-1 and 40.7°C with a 2 mol·l^-1 solution. The variability of thoracic temperature was highest with the 0.5 mol·l^-1 and lowest with the 1.5 and 2 mol·l^-1 concentrations. Thoracic temperatures during trophallactic contact with hive bees were similar to dancing temperature at 1.5 mol·l^-1 but lower at the other concentrations. During periods of distribution of food to hive bees (trophallactic contact >2.5s) the dancers' thorax cooled down by more than 0.5°C considerably more frequently with the 0.5 mol·l^-1 solution (65% of cases) than with the 1.5 mol·l^-1 solution (26%). By contrast, heating the thorax up by more than 0.5°C was infrequent with the 0.5 mol·l^-1 solution (2%) but occurred at a maximum rate of 26% with the 1.5 mol·l^-1 solution. Bees gathering the 1 or 2 mol·l^-1 solutions showed intermediate behavior. Linear model analysis showed that at higher concentrations the dancers compensated better for variations of hive air temperature: per 1 °C increase of hive temperature dancing temperature increased by 0.34, 0.22, 0.12, and 0.13 °C with 0.5, 1, 1.5, and 2 mol·l^-1 sucrose solutions, respectively. The results furnish evidence that dancing honeybees follow a strategy of selective heterothermy by tuning their thermal behavior to the needs of the behavior performed at the moment. Thoracic temperature is regulated to a high level and more accurately when fast exploitation of profitable food sources is recommended. Thoracic temperature is lowered when the ratio of gain to costs of foraging becomes more unfavorable.Abbreviations SD standard deviation - SD _reg SD around regression line - H _rel relative humidity at feeding station - T _a air temperature at feeding station - T _i air temperature near the dancers - T _d Thoracic surface temperatures - T _d dancing - T _tr trophallactic contact (distribution of food) - T _w walking - T _stay mean temperature of total stay in the hive     

The pollination biology ofHibbertia stricta (Dilleniaceae)

The exines of pollen grains ofHibbertia stricta (DC.)R. Br. exF. Muell. (Sect.Pleurandra) wear an oily, yellow pollen coat that stains positively for lipids. The pollen is collected by asocial bees, exclusively. The most common floral foragers are members of the genusLasioglossum (subgenusChilalictus;Halictidae) and they harvest pollen via thoracic vibration. As these bees cling to the inflated anthers their pollen smeared bodies come in contact with either of the two wet, nonpapillate stigmas. The stigmas respond positively to cytochemical tests for the presence of esterase immediately following expansion of the corolla, indicating the effective pollination period. The foraging patterns of the bees are narrowly to broadly polylectic. AsH. stricta flowers are nectarless, it is not surprising that bees bearing mixed pollen loads always carry the pollen of at least one nectariferous, coblooming plant. The pollination biology ofH. stricta is compared with otherHibbertia spp. and with pollen flowers in general.