A novel approach combining the Calgary Biofilm Device and Phenotype MicroArray for the characterization of the chemical sensitivity of bacterial biofilms

A rapid method for screening the metabolic susceptibility of biofilms to toxic compounds was developed by combining the Calgary Biofilm Device (MBEC device) and Phenotype MicroArray (PM) technology. The method was developed using Pseudomonas alcaliphila 34, a Cr(VI)-hyper-resistant bacterium, as the test organism. P. alcaliphila produced a robust biofilm after incubation for 16 h, reaching the maximum value after incubation for 24 h (9.4 × 10⁶ ± 3.3 × 10⁶ CFU peg^?1). In order to detect the metabolic activity of cells in the biofilm, dye E (5×) and menadione sodium bisulphate (100 μM) were selected for redox detection chemistry, because they produced a high colorimetric yield in response to bacterial metabolism (340.4 ± 6.9 Omnilog Arbitrary Units). This combined approach, which avoids the limitations of traditional plate counts, was validated by testing the susceptibility of P. alcaliphila biofilm to 22 toxic compounds. For each compound the concentration level that significantly lowered the metabolic activity of the biofilm was identified. Chemical sensitivity analysis of the planktonic culture was also performed, allowing comparison of the metabolic susceptibility patterns of biofilm and planktonic cultures.     

Establishing a laboratory model of dental unit waterlines bacterial biofilms using a CDC biofilm reactor

In this study, a laboratory model to reproduce dental unit waterline (DUWL) biofilms was developed using a CDC biofilm reactor (CBR). Bacteria obtained from DUWLs were filtered and cultured in Reasoner’s 2A (R2A) for 10 days, and were subsequently stored at ?70°C. This stock was cultivated on R2A in batch mode. After culturing for five days, the bacteria were inoculated into the CBR. Biofilms were grown on polyurethane tubing for four days. Biofilm accumulation and thickness was 1.3 × 10⁵ CFU cm^?2 and 10–14 μm respectively, after four days. Bacteria in the biofilms included cocci and rods of short and medium lengths. In addition, 38 bacterial genera were detected in biofilms. In this study, the suitability and reproducibility of the CBR model for DUWL biofilm formation were demonstrated. The model provides a foundation for the development of bacterial control methods for DUWLs.     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

Negative pressure wound therapy reduces the motility of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and enhances wound healing in a rabbit ear biofilm infection model

Pseudomonas aeruginosa motility, virulence factors and biofilms are known to be detrimental to wound healing. The efficacy of negative pressure wound therapy (NPWT) against P. aeruginosa has been little studied, either in vitro or in vivo. The present study evaluated the effect of negative pressure (NP) on P. aeruginosa motility in vitro, and the effect of NPWT on virulence factors and biofilms in vivo. P. aeruginosa motility was quantified under different levels of NP (atmospheric pressure, ? 75, ? 125, ? 200 mmHg) using an in vitro model. Swimming, swarming and twitching motility were significantly inhibited by NP (? 125 and ? 200 mmHg) compared with atmospheric pressure (p = 0.05). Virulence factors and biofilm components were quantified in NPWT and gauze treated groups using a rabbit ear biofilm model. Biofilm structure was studied with fluorescence microscopy and scanning electron microscopy. Additionally, viable bacterial counts and histological wound healing parameters were measured. Compared with the control, NPWT treatment resulted in a significant reduction in expression of all virulence factors assayed including exotoxin A, rhamnolipid and elastase (p = 0.01). A significant reduction of biofilm components (eDNA) (p = 0.01) was also observed in the NPWT group. The reduction of biofilm matrix was verified by fluorescence- and scanning electron-microscopy. NPWT lead to better histologic parameters (p = 0.01) and decreased bacterial counts (p = 0.05) compared with the control. NPWT treatment was demonstrated to be an effective strategy to reduce virulence factors and biofilm components, which may explain the increased wound healing observed.     

Laboratory-scale biofiltration of acrylonitrile by <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> DAP 96622 in a trickling bed bioreactor

Acrylonitrile (ACN), a volatile component of the waste generated during the production of acrylamide, also is often associated with aromatic contaminants such as toluene and styrene. Biofiltration, considered an effective technique for the treatment of volatile hydrocarbons, has not been used to treat volatile nitriles. An experimental laboratory-scale trickling bed bioreactor using cells of Rhodococcus rhodochrous DAP 96622 supported on granular activated carbon (GAC) was developed and evaluated to assess the ability of biofiltration to treat ACN. In addition to following the course of treatability of ACN, kinetics of ACN biodegradation during both recycle batch and open modes of operation by immobilized and free cells were evaluated. For fed-batch mode bioreactor with immobilized cells, almost complete ACN removal (>95%) was achieved at a flow rate of 0.1 μl/min ACN and 0.8 μl/min toluene (TOL) (for comparative purposes this is equivalent to 6.9 mg l^?1 h^?1 ACN and 83.52 mg l^?1 h^?1 TOL). In a single-pass mode bioreactor with immobilized cells, at ACN inlet loads of 100–200 mg l^?1 h^?1 and TOL inlet load of ~400 mg l^?1 h^?1, with empty bed retention time (EBRT) of 8 min, ACN removal efficiency was ~90%. The three-dimensional structure and characteristics of the biofilm were investigated using confocal scanning laser microscopy (CSLM). CLSM images revealed a robust and heterogeneous biofilm, with microcolonies interspersed with voids and channels. Analysis of the precise measurement of biofilm characteristics using COMSTAT^® agreed with the assumption that both biomass and biofilm thickness increased along the carbon column depth.     

Doxycycline interferes with quorum sensing-mediated virulence factors and biofilm formation in Gram-negative bacteria

Inhibition of quorum sensing (QS)-regulated virulence factors including biofilm is a recognized anti-pathogenic drug target. The search for safe and effective anti-QS agents is expected to be useful to combat diseases caused by multidrug-resistant bacteria. In this study, effect of a commonly used antibiotic, doxycycline on QS was evaluated using sensor strains of Chromobacterium violaceum (ATCC 12472 and CVO26) and Pseudomonas aeruginosa PAO1. Sub-MICs of doxycycline reduced QS-controlled violacein production in C. violaceum to a significant degree (70 %) and showed a significant reduction of LasB elastase (67.2 %), pyocyanin (69.1 %), chitinase (69.8 %) and protease (65 %) production and swarming motility (74 %) in P. aeruginosa PAO1 over untreated controls. Similar results were also recorded against a clinical strain of P. aeruginosa (PAF-79). Interestingly, doxycycline at respective sub-MICs (4 and 32 μg ml^?1) significantly reduced the biofilm-forming capability and exopolysaccharide production in both the strains of P. aeruginosa (PAO1 and PAF-79) over untreated controls. The results of this study highlight the multiple actions of doxycycline against QS-linked traits/virulence factors and its potential to attenuate virulence of P. aeruginosa.     

Paenibacillus beijingensis sp. nov., a nitrogen-fixing species isolated from wheat rhizosphere soil

A novel nitrogen-fixing bacterium, BJ-18^T, was isolated from wheat rhizosphere soil. Strain BJ-18^T was observed to be Gram-positive, facultatively anaerobic, motile and rod-shaped (0.4–0.9 μm × 2.0–2.9 μm). Phylogenetic analysis based on a partial nifH gene sequence and an assay for nitrogenase activity showed its nitrogen-fixing capacity. Phylogenetic analysis based on full 16S rRNA gene sequences suggested that strain BJ-18^T is a member of the genus Paenibacillus. High similarity of 16S rRNA gene sequence was found between BJ-18^T and Paenibacillus peoriae DSM 8320^T (99.05 %), Paenibacillus jamilae DSM 13815^T (98.86 %), Paenibacillus brasiliensis DSM 13188^T (98.55 %), Paenibacillus polymyxa DSM 36^T (98.74 %), Paenibacillus terrae DSM 15891^T (97.99 %) and Paenibacillus kribbensis JCM 11465^T (97.92 %), whereas the similarity was below 96.0 % between BJ-18^T and the other Paenibacillus species. DNA–DNA relatedness between strain BJ-18^T and P. peoriae DSM 8320^T, P. jamilae DSM 13815^T, P. brasiliensis DSM 13188^T, P. polymyxa DSM 36^T, P. kribbensis JCM 11465^T and P. terrae DSM 15891^T was determined to be 43.6 ± 2.7, 34.2 ± 5.3, 47.9 ± 6.6, 36.8 ± 3.5, 27.4 ± 4.3 and 23.6 ± 4.1 % respectively. The DNA G+C content of BJ-18^T was determined to be 45.8 mol %. The major fatty acid was identified as anteiso-C_15:0 (67.1 %). The polar lipids present in strain BJ-18^T were identified as diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The phenotypic and genotypic characteristics, and DNA–DNA relatedness data, suggest that BJ-18^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus beijingensis sp. nov. (Type strain BJ-18^T=DSM25425^T=CGMCC 1.12045^T) is proposed.     

Zinc oxide nanoparticle inhibits the biofilm formation of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Biofilms are structured consortia of microbial cells that grow on living and non living surfaces and surround themselves with secreted polymers. Infections with bacterial biofilms have emerged as a foremost public health concern because biofilm growing cells can be highly resistant to both antibiotics and host immune defenses. Zinc oxide nanoparticles have been reported as a potential antimicrobial agent, thus, in the current study, we have evaluated the antimicrobial as well as antibiofilm activity of zinc oxide nanoparticles against the bacterium Streptococcus pneumoniae which is a significant cause of disease. Zinc oxide nanoparticles showed strong antimicrobial activity against S. pneumoniae, with an MIC value of 40 μg/ml. Biofilm inhibition of S. pneumoniae was also evaluated by performing a series of experiments such as crystal violet assay, microscopic observation, protein count, EPS secretion etc. using sub-MIC concentrations (3, 6 and 12 µg/ml) of zinc oxide nanoparticles. The results showed that the sub-MIC doses of zinc oxide nanoparticles exhibited significant anti-biofilm activity against S. pneumoniae, with maximum biofilm attenuation found at 12 μg/ml. Taken together, the results indicate that zinc oxide nanoparticles can be considered as a potential agent for the inhibition of microbial biofilms.     

Marked enhancement in the artemisinin content and biomass productivity in Artemisia annua L. shoots co-cultivated with Piriformospora indica

A significant enhancement in artemisinin content, an important anti-malarial compound, has been achieved in Artemisia annua L. shoots by co-cultivating with Piriformospora indica, a mycorrhiza-like fungus. The in vitro shoots derived from nodal cultures of A. annua were implanted on four different culture media namely, (i) Murashige & Skoog (MS) basal, (ii) MS + 5 μM indole-3-butyric acid (IBA), (iii) MS + P. indica and, (iv) MS + 5 μM IBA + P. indica. After 2 months, it was observed that the cultures reared on MS + 5 μM IBA + P. indica showed optimum growth in terms of shoot and root proliferation over those cultured without P. indica. The average shoot number on MS + 5 μM IBA + P. indica was 17.83 ± 1.01 and on MS + P. indica alone was 12.75 ± 1.10. A drastic decline in shoot number was observed without P. indica which was 2.0 ± 0.12 on basal and 4.9 ± 1.52 on 5 μM IBA. Similarly, a maximum average of 16.83 ± 0.82 roots were achieved on MS + 5 μM IBA + P. indica which declined to 10.75 ± 1.02 on MS + P. indica. A further decrease in root number occurred in shoots without P. indica, their average being 2.5 ± 0.12 on basal and 8.91 ± 1.57 on 5 μM IBA. HPLC analysis of the aforesaid cultures revealed that the quantity of artemisinin was significantly higher (1.30 ± 0.03 %) in shoots cultured on 5 μM IBA + P. indica compared to those of control (0.80 ± 0.01 %).     

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min^–1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105 ± 16 μm up to 246 ± 67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.     

Culture and Motion Analysis of Diatom Bacillaria paradoxa on a Microfluidic Platform

We proved the feasibility of using a microfluidic chip to culture diatom Bacillaria paradoxa, and analyzed the gliding characteristics of its self-organized colony in detail. The optimal cultivation parameters of B. paradoxa for the designed chip made with polydimethylsiloxane are as follows: the preferable cells injecting rate for keeping the cells alive is 0.2 mL/h, the initial cell density for fast reproduction is 5.5 × 10⁴ cells/mL, and the optimal replacement period of culture medium is 4 days. B. paradoxa tends to form a colony during their growth, and the colony can glide with a steady period of 29 ± 3 s along its axial direction in a constant stream, the amplitude of the colony will not decay (e.g., 24 μm of two-cell colony at 1.1 mm/s flow rate), and the colony rapidly adjusts its direction of gliding to the direction of water flow. The successful culture of diatoms on a microfluidic platform may be used for biosensing chips and the creation of gasoline-producing diatom solar panels.     

Motility in liquid and semisolid media of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> associative rhizobacteria differing in exopolysaccharide yield and properties

Paenibacillus polymyxa rhizobacteria associate with a wide range of plants and promote plant growth and development. These bacteria produce exopolysaccharides, which are very important for P. polymyxa adaptation to changing environmental conditions. In this study, five P. polymyxa strains differing in exopolysaccharide yield and properties (CCM 1465, CCM 1460, CCM 1459^T, 88A, and 92) were investigated for motility in a liquid and a semisolid nutrient medium with either glucose or sucrose as the carbon source. In the liquid medium, all strains except CCM 1460 were motile and swam by peritrichous flagella. After being stab inoculated into the semisolid (0.4% agar) medium, all strains except CCM 1460 switched to collective swarming and formed concentric macrocolonies with different diameters, depending on the strain and the carbon source used. On the semisolid medium containing the vital dye Congo red, P. polymyxa adsorbed the dye, forming stained colonies. No changes in colony morphology were observed. At 37.5 μg ml^?1 of Congo red, swarming was strongly suppressed: the swarming ring diameters of strains 92, CCM 1459^T, 88A, and CCM 1465 ranged from 12.4 to 17% (glucose) and from 9.5 to 16.0% (sucrose) of the colony diameters obtained from bacterial growth without the dye. The results suggest that the speed of collective migration of P. polymyxa on agarized media may be affected, among other factors, by the yield and physicochemical properties of the bacterial exopolysaccharides. Further, the results suggest that Congo red influences the collective migration speed of P. polymyxa by forming a complex with the bacteria’s carbohydrate polymers – an event that alters bacterial surface structure and affects intercellular interactions.     

Paenibacillus thermophilus sp. nov., a novel bacterium isolated from a sediment of hot spring in Fujian province, China

A Gram-positive, thermophilic, strictly aerobic bacterium, designated WP-1^T, was isolated from a sediment sample from a hot spring in Fujian province of China and subjected to a polyphasic taxonomic study. Cells of strain WP-1^T were rods (~0.6–0.8 × 2.5–3.5 μm) and motile by means of peritrichous flagella. Endospores were ellipsoidal in terminal or subterminal positions. Strain WP-1^T grew at 37–60 °C (optimum 42–45 °C), 0–3 % NaCl (optimum 1 %, w/v) and pH 3.0–9.0 (optimum pH 6.5–7.0). The predominant menaquinone was MK-7. The major fatty acids were anteiso-C_15:0, iso-C_16:0, C_16:0 and anteiso-C_17:0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, two glycolipids, two unidentified phospholipids and two unknown polar lipids. The cell-wall peptidoglycan contained meso-diaminopimelic acid (meso-DAP). The G + C content of the genomic DNA was 52.5 %. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain WP-1^T is a member of the genus Paenibacillus and exhibited sequence similarity of 99.3 % to Paenibacillus macerans DSM 24^T and both strains represented a separate lineage from all other Paenibacillus species. However, the level of DNA–DNA relatedness between strain WP-1^T and P. macerans DSM 24^T was 34.0 ± 4.7 %. On the basis of phylogenetic, physiological and chemotaxonomic analysis data, strain WP-1^T is considered to represent as a novel species of the genus Paenibacillus, for which the name Paenibacillus thermophilus sp. nov., is proposed, with the type strain WP-1^T (=DSM 24746^T = JCM 17693^T = CCTCC AB 2011115^T).     

Enhanced Control of Cucumber Wilt Disease by Bacillus amyloliquefaciens SQR9 by Altering the Regulation of Its DegU Phosphorylation

Bacillus amyloliquefaciens strain SQR9, isolated from the cucumber rhizosphere, suppresses the growth of Fusarium oxysporum in the cucumber rhizosphere and protects the host plant from pathogen invasion through efficient root colonization. In the Gram-positive bacterium Bacillus, the response regulator DegU regulates genetic competence, swarming motility, biofilm formation, complex colony architecture, and protease production. In this study, we report that stepwise phosphorylation of DegU in B. amyloliquefaciens SQR9 can influence biocontrol activity by coordinating multicellular behavior and regulating the synthesis of antibiotics. Results from in vitro and in situ experiments and quantitative PCR (qPCR) studies demonstrate the following: (i) that the lowest level of phosphorylated DegU (DegU∼P) (the degQ mutation) impairs complex colony architecture, biofilm formation, colonization activities, and biocontrol efficiency of Fusarium wilt disease but increases the production of macrolactin and bacillaene, and (ii) that increasing the level of DegU∼P by degQ and degSU overexpression significantly improves complex colony architecture, biofilm formation, colonization activities, production of the antibiotics bacillomycin D and difficidin, and efficiency of biocontrol of Fusarium wilt disease. The results offer a new strategy to enhance the biocontrol efficacy of Bacillus amyloliquefaciens SQR9.     

Adhesion of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> biofilms to glass,stainless steel and cellulose

Objectives

The adhesion of colloidal probes of stainless steel, glass and cellulose to Pseudomonas fluorescens biofilms was examined using atomic force microscopy (AFM) to allow comparisons between surfaces to which biofilms might adhere.

Results

Biofilm was grown on a stainless steel substrate and covered most of the surface after 96 h. AFM approach and retraction curves were obtained when the biofilm was immersed in a tryptone/soy medium. On approach, all the colloidal probes experienced a long non-contact phase more than 100 nm in length, possibly due to the steric repulsion by extracellular polymers from the biofilm and hydrophobic effects. Retraction data showed that the adhesion varied from position to position on the biofilm. The mean value of adhesion of glass to the biofilm (48 ± 7 nN) was the greatest, followed by stainless steel (30 ± 7 nN) and cellulose (7.8 ± 0.4 nN).

Conclusion

The method allows understanding of adhesion between the three materials and biofilm, and development of a better strategy to remove the biofilm from these surfaces relevant to different industrial applications.

     

A combination of ellagic acid and tetracycline inhibits biofilm formation and the associated virulence of Propionibacterium acnes in vitro and in vivo

Propionibacterium acnes is an opportunistic pathogen which has become notorious owing to its ability to form a recalcitrant biofilm and to develop drug resistance. The current study aimed to develop anti-biofilm treatments against clinical isolates of P. acnes under in vitro and in vivo conditions. A combination of ellagic acid and tetracycline (ETC; 250 μg ml^?1 + 0.312 μg ml^?1) was determined to effectively inhibit biofilm formation by P. acnes (80–91%) without affecting its growth, therefore potentially limiting the possibility of the bacterium attaining resistance. In addition, ETC reduced the production of extracellular polymeric substances (EPS) (20–26%), thereby making P. acnes more susceptible to the human immune system and antibiotics. The anti-biofilm potential of ETC was further substantiated under in vivo conditions using Caenorhabditis elegans. This study reports a novel anti-biofilm combination that could be developed as an ideal therapeutic agent with broad cosmeceutical and pharmaceutical applicability in the era of antibiotic resistance.     

Anti-herpesvirus bovine type 5 activities of extracts obtained from Plocamium brasiliense

Bovine herpesvirus type 5 (BoHV-5) is an important etiologic agent of meningoencephalitis in cattle and has been frequently identified in outbreaks of neurological disease in bovine in the southern hemisphere including Brazil. This study aimed to evaluate the cytotoxic effect and the antiviral properties of extracts obtained from Plocamium brasiliense (Greville) Howe and Taylor in BoHV-5 RJ42/01 replication. The cytotoxic effects were measured in Madin-Darbin bovine kidney cells (MDBK) and cytotoxic concentration (CC₅₀) values have been determined for acyclovir (ACV) (223 μg?±?2.0), ethyl acetate extract from P. brasiliense (2,109 μg?±?10), hexane extract from P. brasiliense (7.181 μg?±?5), dichloromethane extract from P. brasiliense (2.356 μg?±?6.5), and hydroalcoholic extract from P. brasiliense (1.408 μg?±?5.8). As a first approach to characterize the action of these extracts on BoHV-5 RJ42/01, a virucidal assay activity was performed. A virus suspension containing 1?×?10⁵ plaque-forming units (PFU) of the BoHV-5 RJ42/01 was mixed with 600 μg of extract and acyclovir and kept at room temperature (24 °C) for 3 h. Meanwhile, a control of untreated infected virus was performed in the same conditions. Then, treated virus suspension and untreated control were diluted, and percentage of inhibition of infectivity was determined by plaque assay: ethyl acetate extract (99 %), hexane extract (90 %), dichloromethane extract (99 %), and hydroalcoholic extract (27 %). Acyclovir had a slight virucidal activity on viral particle. The inhibition of attachment was performed in MDBK cells inoculated with 100 PFU of BoHV-5 RJ42/01 in the presence or absence of various concentrations of extracts (0.3, 0.9, and 1.5 μg mL^?1). Acyclovir was also assayed at 2.8 and 11.25 μg mL^?1. The inhibition of adsorption was also tested in MDBK cells treated with the same concentrations of the extracts before virus inoculation. Results: hexane extracts inhibited virus attachment in pre-treated cell 0.9 μg (55 %) and 1.5 μg (71 %) and untreated MDBK cell only with 1.5 μg (63 %). Ethyl acetate extract on cell pre-treated with 0.3 μg (67 %), 0.9 μg (81 %), and 1.5 μg (91 %). Ethyl acetate extract on pre-treated cell 0.3 μg (67 %), 0.9 μg (81 %), and 1.5 (91 %) but discrete inhibition on cell untreated. Dichloromethane extract and acyclovir slightly inhibited virus binding on MDBK cell.     

Discontinuous ammonia excretion and glutamine storage in littoral Oniscidea (Crustacea: Isopoda): testing tidal and circadian models

A key evolutionary development facilitating land colonization in terrestrial isopods (Isopoda: Oniscidea) is the intermittent liberation of waste nitrogen as volatile ammonia. Intermittent ammonia release exploits glutamine (Gln) as an intermediary nitrogen store. Here, we explore the relationship between temporal patterns of ammonia release and Gln accumulation in three littoral oniscideans from Southern California. Results are interpreted in terms of water availability, habitat, activity patterns, and ancestry. A two-way experimental design was used to test whether ammonia excretion and Gln accumulation follow a tidal or diel periodicity. Ammonia excretion was studied in the laboratory using chambers with or without available seawater and using an acid trap to collect volatile ammonia. Ligia occidentalis releases ammonia directly into seawater and accumulates Gln during low tide (48.9 ± 6.5 μmol g^?1 at low tide, 24.1 ± 3.0 μmol g^?1 at high tide), indicating that excretion is tidally constrained. Alloniscus perconvexus and Tylos punctatus can excrete ammonia directly into seawater or utilize volatilization. Both species burrow in sand by day and show a diel excretory pattern, accumulating Gln nocturnally (31.8 ± 2.7 μmol g^?1 at dawn and 21.8 ± 2.3 μmol g^?1 at dusk for A. perconvexus; 85.7 ± 15.1 μmol g^?1 at dawn and 25.4 ± 2.9 μmol g^?1 at dusk for T. punctatus) and liberating ammonia diurnally. Glutaminase shows higher activity in terrestrial (0.54–0.86 U g^?1) compared to intertidal (0.25–0.31 U g^?1) species, consistent with the need to generate high PNH₃ for volatilization. The predominant isoform in Armadillidium vulgare is phosphate dependent and maleate independent; phosphate is a plausible regulator in vivo.