Ammoniacal Silver Research Articles

Innervation of cercarial tegumentary receptors investigated by the Sevier-Munger method

The investigation of the sensory nature of tegumentary receptors in platyhelminths remains a challenge due to technical difficulties related to nerve tissue exposure and its experimental handling. Neuromorphological studies have been carried out but few demonstrated the association of these receptors with the nervous system. This paper introduces the Sevier-Munger method as an alternative approach to study the innervation of tegumentary receptors in larval flatworms. Twenty heterophyid cercariae were fixed in hot 5% formalin, with all washes performed in tap and distilled water. They were developed in a solution of ammoniacal silver and 2% formalin under the microscope for 10 min, with preparations shaken gently throughout the procedure. In all specimens, nerve cells stained black against a pale gold background. Fine nerve fibers of the subsurface nerve plexus were observed. These fibers sent distal branches from the plexus to the cercarial tegument. The branches became fine nerve endings, projecting as receptors in the cephalic (5CIV 5, 2CV 2, and 2CV 4), anterior (4AIL, 3AIIL, 2AIIIL), midbody (1ML, 3MV), posterior (1PIL, 1PIIL, and 1PIIIL), and caudal (2UD) regions of the cercaria. These results indicate that the Sevier-Munger method is useful to demonstrate the association of cercarial tegumentary receptors with the subsurface nerve plexus. They also recommend the use of alternative methods to further investigate flatworm nervous systems. Moreover, there is a pressing urge for a standardized protocol, combining a plethora of methods and techniques. Interdisciplinary collaboration aiming at a better understanding of the function of flatworm nervous systems is particularly encouraged.

In Vitro Evaluation of Antimicrobial Efficacy of Iontophoresis against Enterococcus faecalis, Candida albicans, Pseudomonas aeruginosa and Bacillus subtilis

This in vitro study examined the antimicrobial efficacy of iontophoresis against Enterococcus faecalis, Candida albicans, Pseudomonas aeruginosa and Bacillus subtilis, all of which are often found in the root canals of teeth with refractory apical periodontitis. An experimental periapical lesion model was made from a transparent plastic root canal model. Iontophoresis was performed with Kantop Junior®; with (1) diammine silver fluoride solution (2) ammoniacal silver hydroxide solution and (3) iodine zinc iodide solution as the iontophoretic solutions. Iontophoresis using diammine silver fluoride solution eradicated all four microorganisms whereas iontophoresis using ammonium silver solution and iodine zinc iodide solution could not. The results of this study suggested that iontophoresis with diammine silver fluoride is an effective treatment for E. faecalis, C. albicans, P. aeruginosa and B. subtilis, all of which often found in the root canals of teeth with refractory apical periodontitis.

Quantitative comparison of permeability in the adhesive interface of four adhesive systems

The purpose of this study was to perform quantitative comparisons of water permeable zones in both the adhesive and the hybrid layer before and after thermo cycling in order to assess the integrity of the bonding interface. Twenty eight flat dentin surfaces were bonded with a light-cured composite resin using one of four commercial adhesives [OptiBond FL (OP), AdheSE (AD), Clearfil SE Bond (CL). and Xeno III (XE)]. These were sectioned into halves and subsequently cut to yield 2-mm thick specimens; one specimen for control and the other subjected to thermocycling for 10,000 cycles. After specimens were immersed in ammoniacal silver nitrate for 24 h and exposed to a photo developing solution for 8 h, the bonded interface was analyzed by scanning electron microscopy (SEM) and wavelength dispersive spectrometry (WDS) at five locations per specimen. Immediately after bonding. the adhesive layer of OP showed the lowest silver uptake, followed by CL, AD. and XE in ascending order (p

Nanoleakage, ultramorphological characteristics, and microtensile bond strengths of a new low-shrinkage composite to dentin after artificial aging

Objectives To study the microtensile bond strengths and nanoleakage of low-shrinkage composite to dentin. The null hypotheses tested were (1) aging does not affect the bonding of low-shrinkage composite; (2) there is no difference in microtensile bond strengths and nanoleakage using different bonding strategies. Methods 32 extracted molars were assigned to one of four groups: LS System Adhesive (LS, 3M ESPE); dentin etched for 15 s with phosphoric acid + LS System Adhesive (LSpa); Adper Single Bond Plus (SB, 3M ESPE); SB + LS Bond (SBLS). Occlusal dentin was exposed and restored with Filtek LS (3M ESPE). The samples were tested after 24 h or after 20,000 thermocycles and 6 months of aging. Teeth were sectioned with a cross-section of 0.8 ± 0.2 mm 2 and fractured at a crosshead speed of 1 mm/min. The data were submitted to ANOVA/Duncan's post hoc test, at p < 0.05. Five slabs from each group were selected and immersed in 50 wt% ammoniacal silver nitrate. Then, specimens were processed for SEM, the silver penetration was measured and data analyzed with Kruskal–Wallis at p < 0.05. Results No statistically significant difference was found among the experimental groups for the factor dentin treatment ( p = 0.165) and aging ( p = 0.091). All experimental groups exhibit some degree of nanoleakage. There was no adhesion of Filtek LS applied directly over dentin surfaces treated with SB. Significance The new low-shrinkage resin composite showed compatibility only with its dedicated adhesive. Pre-etching did not improve the bond strengths to low-shrinkage resin composite. Some degree of nanoleakage was evident in all groups.

Analysis of the RNA Content of the Yeast Saccharomyces cerevisiae

The principles involved in the storage and expression of genetic information are now well established and have been incorporated into biology curricula for elementary, secondary, and college students (National Research Council, 1996). Almost every biology textbook now describes the structure of DNA and the roles of messenger RNAs, transfer RNAs, and ribosomal RNAs in protein synthesis. Many different laboratory experiments have been developed in which students isolate genomic DNA (Dollard, 1994; Helms et al., 1998), subject fragments of DNA generated by restriction endonucleases or polymerase chain reactions to gel electrophoresis (Jenkins & Bielec, 2006; Kass, 2007), transform bacteria with DNA plasmids carrying genes for antibiotic resistance (Guifoile & Plum, 2000), or combine several of these methods to clone particular genes (Becker et al., 1996; Micklos et al., 2003; Winfrey et al., 1997). However, very few classroom experiments focus specifically on RNA. Bregman (2002) has described two experiments in which students study cellular RNA microscopically either by staining whole cells in a blood smear with a combination of methyl green and pyronin or by staining tissue culture cells with the ammoniacal silver method for ribosomal RNA. However, neither of these experiments is quantitative and both require microscopy skills beyond those of many beginning students. Direct measurement of RNA formation by transcription of a DNA template or analysis of RNA function during translation normally involves the use of chemiluminescent or radioactively-labeled nucleotides or amino acids (Ausubel et al., 2002; Grandi, 2007; Martin, 1998; Sambrook & Russell, 2001). Although kits for doing these types of studies are commercially available, most schools do not have either the liquid scintillation counters or X-ray film developers need to detect the products, and lack the support staff necessary to meet federal and state requirements for safely using radioactive compounds. These limitations are unfortunate in light of the growing body of scientific information about the pre-biotic RNA world. It now seems clear that the basic steps in protein synthesis were established before DNA became important as a way of storing genetic information in a stable way (Gesteland et al., 2006; Gilbert, 1986; Muller, 2006; Spirin, 2002; Woese, 2001). In addition to having self-catalytic activity, RNA molecules are involved in all the steps of translation, from the initial activation of amino acids by attaching them to transfer RNAs to the association of messenger RNAs and aminoacyltransfer RNAs with ribosomes to the actual polymerization of amino acids into polypeptide chains. Table 1 summarizes the characteristics and sizes of the major types of RNA commonly found in prokaryotic and eukaryotic cells. The Howard Hughes Medical Institute (2006) has produced a DVD from its Holliday Lectures on Science series which discusses the many roles of RNA and can be used to introduce students to this topic. In this article, we describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. The general goals of these experiments are to emphasize the importance of RNA in cell biology and to provide practice in basic biochemical and molecular analysis. Overview of Experiments This set of experiments focuses on RNAs from the yeast Saccharomyces cerevisiae, a unicellular budding microorganism that has served as a model of cellular and molecular processes in eukaryotes (Davis, 2003). S. cerevisiae can be grown easily in the laboratory, has a relatively small genome that has been completely sequenced, and is susceptible to genetic analysis using both classical and molecular techniques. In these experiments, students: 1. study yeast cells by light microscopy 2. …

Ultrastructural and cytochemical aspects of Schistosoma mansoni cercaria

An alternative to identify the critical processes necessary to the parasite establishment of the host is to focus on the evolutionary stage responsible for the primary invasion, i.e. the infection structure. The objective of this study was to ultrastructurally characterize Schistosoma mansoni cercariae, using cytochemical techniques. In order to identify basic proteins, techniques such as ethanolic phosphotungstic acid (EPTA) and ammoniacal silver staining were used. Calcium sites location was achieved using the Hepler technique and to evidence anionic groups, we used cationic ferritin particles and enzyme treatment with trypsin Vibrio cholerae, chondroitinase and neuraminidase. The EPTA technique highlighted the presence of basic tegument proteins, nucleus and nucleolus from subtegumental cells, inclusion bodies and preacetabular glands. After using ammoniacal silver, we observed a strong staining in all infective larvae, particularly in the nuclei of muscle cells, circular muscle tissue and preacetabular glands. Calcium site locations were shown to be uniform, thereby limiting the inner spaces of the larvae, especially muscle cells. Samples treated with cationized ferritin particles presented strong staining at the cuticular level. Neuraminidase treatment did not alter the stained shape of such particles on the trematode surface. However, trypsin or chondroitinase treatment resulted in absence of staining on the larval surface. This information on the biochemical composition of the infecting S. mansoni larvae provides data for a better understanding of the biology of this parasite and background on the intriguing parasite–host relationship.

Polysaccharide Templated Silver Nanowire for Ultrasensitive Electrical Detection of Nucleic Acids

An ultrasensitive electrical detection method of nucleic acids has been developed on a nanogapped biosensor. In this study, peptide nucleic acid (PNA) probes were immobilized in the gaps of a pair of finger microelectrodes first and were then hybridized with their complementary target DNA. After that, pectin molecules were introduced into the DNA strand via zirconium-phosphate and zirconium-carbonate chemistries and were oxidated by periodate in acetate buffer (pH 3.98). The newly produced aldedyde groups act as a reactant to reduce ammoniacal silver ion to produce silver nanoparticles, which bridged the gap of the interdigitated microelectrode. The conductance of the metallic nanoparticles correlated directly with the amount of the hybridized DNA. A much higher sensitivity was achieved at 3 femtomolar (S/N > 3) under optimal conditions. This biosensor is also applicable to the direct detection of RNA.

Ultrasensitive electrical detection of nucleic acids by hematin catalysed silver nanoparticle formation in sub-microgapped biosensors

An ultrasensitive electrical detection method of nucleic acids has been demonstrated on sub-microgapped biosensor. In this method, peptide nucleic acid (PNA) probes were firstly immobilized in the gap areas of a pair of interdigited microelectrodes and then were hybridized with their complementary target DNA. After hybridization, hematin molecules were introduced into the DNA strand via zirconium-phosphate and zirconium-carbonate chemistries. The newly attached hematin molecules act as a catalyst to accelerate reducing ammoniacal silver ion to form silver nanoparticles, which span the gap of the interdigitated microelectrode. The conductance of the silver nanoparticles directly correlated with the number of the hybridized DNA molecules. Nearly 1 fM sensitivity was achieved under optimal conditions. This approach is also applicable to the detection of RNA.

Micropermeability of current self‐etching and etch‐and‐rinse adhesives bonded to deep dentine: a comparison study using a double‐staining/confocal microscopy technique

Water sorption decreases the mechanical properties and the bond strengths of resin-bonded dentine. The aim of this study was to evaluate the micropermeability of several self-etching and etch-and-rinse adhesives. Optibond FL, Silorane, Scotchbond 1XT, G-Bond, and DC-Bond were bonded under simulated pulpal pressure. A 10 wt% solution of ammoniacal silver nitrate and a 1 wt% solution of rhodamine B were injected into the pulp chamber at 20 cm of water pressure. The dentine-adhesive interfaces were examined using a confocal scanning microscope. Micropermeability was detected in all the adhesives. DC-Bond, G-Bond, and Scotchbond 1XT showed voids along the resin-bonded interface. Silorane and Optibond FL showed an adhesive layer that was free from water trees and micropermeability. The double staining technique is a method that gives accurate results in the study of the resin-dentine micropermeability. Each class of adhesive has a different distribution of micropermeability. The higher the micropermeability, the higher the risk of defects at the resin-dentine interface, which may represent the pathway for hydrolytic and enzymatic degradation of resin-dentine bonds over time.

The nanoleakage patterns of experimental hydrophobic adhesives after load cycling

The purpose of this study was: (1) to compare nanoleakage patterns of a conventional 3-step etch and rinse adhesive system and two experimental hydrophobic adhesive systems and (2) to investigate the change of the nanoleakage patterns after load cycling. Two kinds of hydrophobic experimental adhesives, ethanol containing adhesive (EA) and methanol containing adhesive (MA), were prepared. Thirty extracted human molars were embedded in resin blocks and occlusal thirds of the crowns were removed. The polished dentin surfaces were etched with a 35 % phosphoric acid etching gel and rinsed with water. Scotchbond Multi-Purpose (MP), EA and MA were used for bonding procedure. Z-250 composite resin was built-up on the adhesive-treated surfaces. Five teeth of each dentin adhesive group were subjected to mechanical load cycling. The teeth were sectioned into 2 mm thick slabs and then stained with 50 % ammoniacal silver nitrate. Ten specimens for each group were examined under scanning electron microscope in backscattering electron mode. All photographs were analyzed using image analysis software. Three regions of each specimen were used for evaluation of the silver uptake within the hybrid layer. The area of silver deposition was calculated and expressed in gray value. Data were statistically analyzed by two-way ANOVA and post-hoc testing of multiple comparisons was done with the Scheffe's test. Silver particles were observed in all the groups. However, silver particles were more sparsely distributed in the EA group and the MA group than in the MP group (p

Hybridization quality in cervical cementum and superficial dentin using current adhesives

Objectives The aim of this research was to determine the hybridization quality of adhesives to gingival cementum and close superficial dentin using both total-etch and self-etch, one-step and two-step adhesives in vitro. Methods Five adhesive systems were used and evaluated in this study; three kinds of two-step adhesives (total-etch—Single Bond and self-etch—Clearfil SE Bond and Clearfil Protect Bond) and two one-step adhesives (Clearfil S 3 Bond, G Bond). Fifteen extracted intact human third molars were used in this study. A diagonal cut which was approximately 45° to the long axis of the roots, with the initiating point located 2 mm below the buccal enamel–cementum junction and ascending towards the pulp chamber was prepared on each tooth. Flat cervical cementum and dentin surfaces were ground with wet 600-grit silicon carbide paper, and bonded with one of the adhesives and finished by applying a flowable resin composite. After 24 h storage at 37 °C in water, the bonded assemblies were sectioned into approximately 1 mm thick slabs. Two central slabs from each tooth were chosen. One slab was totally demineralized in 0.5 M EDTA and the other was not demineralized and immersed into 50% (w/v) solution of ammoniacal silver nitrate for 24 h, and successively exposed to photodeveloping solution for 8 h. The specimens were then processed for TEM observation. Both the stained demineralized silver unchallenged and unstained non-demineralized silver challenged resin–cervical cementum/proximal superficial dentin interface were observed and evaluated under a transmission electron microscope. Results The nanoleakage pathway and extent vary among the different adhesives used and also between the resin–cementum interface and resin–dentin interface. Two-step self-etch adhesives showed better hybridization quality both in cementum and proximal superficial dentin as compared to those of two-step total-etch adhesive and one-step self-etch adhesives. Significance Two-step self-etch adhesives may provide a better sealing in cervical cementum and the proximal superficial dentin region.

Staining Proteins in Gels with Silver Nitrate

INTRODUCTIONSilver staining is one of the commonly used procedures for visualizing proteins in acrylamide gels. All silver staining methods rely on the reduction of ionic to metallic silver to provide metallic silver images; the selective reduction at gel sites occupied by proteins compared to nonprotein sites is dependent on differences in the oxidation-reduction potentials at these sites. There are two broad methodologies for silver staining. One approach (nondiamine silver nitrate stains) uses silver nitrate as the silvering agent and formaldehyde in alkaline carbonate solution as the developing agent, whereas the other approach (diamine or ammoniacal stains) uses ammoniacal silver as the silvering agent and formaldehyde in dilute citric acid as the developing agent. Although protocols using ammoniacal silver are arguably more sensitive and give darker hues than those based on silver nitrate, they are more prone to negative staining, resulting in hollow or "doughnut" spots, give unacceptable backgrounds with tricine-based gel systems, and are not very robust because of their reliance on the ammonia-silver ratio. Additionally, ammoniacal silver staining is more sensitive for basic proteins but less so for very acidic proteins. This protocol describes a silver nitrate staining approach. Its sensitivity is in the low-nanogram range, which is 50-100 times more sensitive than classical Coomassie Blue staining, ~10 times better than colloidal Coomassie Blue staining, and at least twice as sensitive as the zinc/imidazole negative staining method.

Mass Spectrometry-Compatible Silver Staining

INTRODUCTIONThe ammoniacal silver staining method is one of the most sensitive methods used to detect proteins on an SDS-PAGE gel. However, this and other standard silver staining methods are not compatible with mass spectrometry (MS), which is fast becoming the best way to identify proteins isolated on 2D gels. Because the proteins in gels to be analyzed by mass spectroscopy cannot be modified, many of the common sensitizing agents (e.g., glutaraldehyde and strong oxidizing agents) cannot be used. This method is compatible with MALDI and ESI-MS, and it shows an increased ability to deal with semipreparative protein loads without negative staining as compared with other silver staining methods. However, this process is less sensitive than standard silver staining methods.

Studies on Nanocrystalline Ag2Se

Silver selenide nanocrystals were synthesized using a chemical route by the interaction of ammoniacal silver nitrate and sodium selenosulfate in aqueous medium. The prepared samples were characterized by XRD, EDXA, and TEM. UV-VIS absorption spectrum of the nanocrystals shows that the optical band gap of the material is 1.72 eV, which is quite larger than the bulk phase of the materials. Thermal analysis exhibits that the phase changes from β to α at the transition temperature 406 K. Electrical conduction and thermoelectric power measurements also show the presence of two distinct phases of the materials and characteristic changes in transport properties due to the nanosize of the materials.

Two-Dimensional Gel Electrophoresis Based Technologies for Potential Biomarkers Identification in Amniotic Fluid: A Simple Model

To assess the efficiency of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and of two-dimensional electrophoresis and ammoniacal silver staining (2D-E), different amounts of soybean trypsin inhibitor and horse myoglobin were added to amniotic fluid. In this model, a minimum of 5 to 10 ng of "artificial" biomarkers was detected.

Relationship between Canine Visceral Leishmaniosis and the Leishmania (Leishmania) chagasi Burden in Dermal Inflammatory Foci

The skin is the first point of contact with organisms of the genus Leishmania from sand fly vectors, and apparently normal skin of sick dogs harbours amastigote forms of Leishmania chagasi. In relation to canine visceral leishmaniosis (CVL), the ear skin was examined in 10 uninfected dogs (UDs) and in 31 dogs dogs naturally infected with L. chagasi. The infected animals consisted of 10 symptomless dogs (SLDs), 12 mildly affected dogs (MADs) and nine affected dogs (ADs). A higher parasite burden was demonstrated in ADs than in SLDs by anti-Leishmania immunohistochemistry (P<0.01), and by Leishman Donivan Unit (LDU) indices (P=0.0024) obtained from Giemsa-stained impression smears. Sections stained with haematoxylin and eosin demonstrated a higher intensity of inflammatory changes in ADs than in SLDs (P<0.05), and in the latter group flow cytometry demonstrated a correlation (P=0.05/r=0.7454) between the percentage of CD14(+) monocytes in peripheral blood and chronic dermal inflammation. Extracellular matrix assessment for reticular fibres by staining of sections with Masson trichrome and Gomori ammoniacal silver demonstrated a decrease in collagen type I and an increase in collagen type III as the clinical signs increased. The data on correlation between cellular phenotypes and histological changes seemed to reflect cellular activation and migration from peripheral blood to the skin, mediated by antigenic stimulation. The results suggested that chronic dermal inflammation and cutaneous parasitism were directly related to the severity of clinical disease.

Vapor phase formation of a well-ordered aldehyde-terminated self-assembled monolayer on a SiO 2 surface and formation of silver film on the surface based on the silver mirror reaction

A well-ordered aldehyde (CHO)-terminated self-assembled monolayer (SAM) was formed from a vapor of triethoxysilylundecanal (TESUD) onto both quartz glass plates and silicon substrates covered with native oxide. This vapor phase treatment produced a TESUD-SAM on both substrates without any marked change in surface morphology, as evidenced by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The thickness of this SAM was estimated by ellipsometry to be 1.2 ± 0.1 nm. Silver (Ag) metallization was also demonstrated on TESUD-SAM-covered quartz glass substrates based on the silver mirror reaction using an ammoniacal silver nitrate solution. XPS and AFM images confirmed that the deposited film was composed of densely packed Ag nanoparticles with an average diameter of 300 nm. The average thickness of the film was about 1–2 μm. Due to this particulate Ag film formation, the transparent insulating quartz glass plate became conductive with a mirror-like surface. The CHO terminal groups of the TESUD-SAM were found to serve effectively as a reducing agent to deposit Ag metal at the solid/liquid interface.

2-D native-PAGE/SDS-PAGE visualization of an oligomer's subunits: Application to the analysis of IgG

A 2-D native-PAGE/SDS-PAGE method for detecting the subunit components of protein oligomers at low picomole sensitivity is presented. IgG was electrophoresed in a native acidic polyacrylamide gel in amounts ranging from 51 pmol to 60 fmol. Silver-staining (native fast silver stain, ammoniacal silver stain, permanganate silver stain), Coomassie-staining (R-250, G-250), metal ion-reverse-staining (zinc, copper), and fluorescent chromophore-staining (SYPRO Ruby) methods were used to visualize the IgG oligomers. The protein zones were then excised, separated by SDS-PAGE, and subunits visualized with a permanganate silver stain. The Coomassie R-250/permanganate silver-staining combination detected IgG subunits using 2 pmol of sample. Coomassie G-250 and native fast silver staining in the first-dimensional gel produced detectable subunits in the second-dimensional separation at 3 and 13 pmol, respectively. Staining with silver (ammoniacal, permanganate), copper, zinc, or SYPRO Ruby in the first-dimensional gel did not produce discernible subunits in the second-dimensional gels due to protein streaking or protein immobilization in the native gel. When using a 2-D native-PAGE/SDS-PAGE system, Coomassie staining of the first-dimensional native gel combined with permanganate silver staining of the second-dimensional denaturing gel provides the most sensitive method (2-3 pmol) for visualizing constituent subunits from their oligomeric assemblies.

Improved mass spectrometry compatibility is afforded by ammoniacal silver staining

Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer.

Structure and function of the urnulae in Balaustium sp. (Parasitengona: erythraeidae) featuring secretion of a defensive allomone and alarm pheromone

In studying predator-avoidance tactics of adult Balaustium sp. mites, a characteristic red fluid was observed leaking from a single pair of tubercles (urnulae) that project from the dorsal idiosoma, just behind the eyes. Until now, the urnulae had no known function, but served a useful taxonomic purpose. When threatened, mites were observed actively secreting urnulae-derived fluid that was spread over their body surface by means of numerous setae. Subsequent application of the fluid to mealworm beetle larvae (Tenebrio molitor) resulted in sudden diminished ant attacks, suggesting that this secretion serves a defensive role as an allomone. True to semiochemical parsimony, this secretion also prompted an excited dispersal response among conspecific mites, indicating that it also functions as an alarm pheromone, perhaps as an injury cue. More pronounced predator defense and alarm activities are displayed by mite body fluids, indicating that the activity of the emission from the urnulae parallels that of hemolymph. Based on positive staining with ammoniacal silver nitrate, their capacity to secrete, and additional morphological and histological investigations carried out, the role of the urnulae is clearly glandular. Of interest is that the defensive mechanism appears to operate by a novel form of reflex bleeding reminiscent of aposematic beetles.