The Susceptibility of Different Species and Stages of Ticks to Entomopathogenic Fungi

Boophilus annulatus, Hyalomma excavatum and Rhipicephalus sanguineus ticks were shown to be susceptible to different entomopathogenic fungi under laboratory conditions. Comparative results of bioassays using five different fungal species showed that some strains of Metarhizium anisopliae are highly pathogenic against various tick stages tested. In contrast to their activity against insects, fungi also affected tick eggs. All tested tick stages including those feeding on a host were found to be susceptible to these fungi, except for adult H. excavatum ticks, which were relatively resistant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Novel Technique for Quantifying Adhesion of Metarhizium anisopliae Conidia to the Tick Cuticle

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of Metarhizium anisopliae var. anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged Rhipicephalus annulatus (Say) (Arachnida: Ixodidae) females and was also verified for M. anisopliae var. acridum Driver et Milner (Hypocreales: Clavicipitaceae) and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female R. annulatus and tick mortality. However, no correlation was observed between a tick''s susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.The entomopathogenic fungus Metarhizium anisopliae var. anisopliae (Metschn.) Sorokîn (1883) infects a broad range of arthropod hosts and can be used as a biopesticide against different insect and tick species (8, 22, 35, 36). The adhesion of the conidia of entomopathogenic fungi to the host cuticle is the initial stage of the pathogenic process and includes both passive and active events (5, 10). The hydrophobic epicuticular lipid layer plays an important role during both the attachment process and the germination of the conidia on the surface of the host (15, 19). According to Boucias et al. (7), the attachment of conidia to the host cuticle is based on nonspecific hydrophobic and electrostatic forces. The conidia of most entomopathogenic fungi, including M. anisopliae, have an outer cell layer made up of rodlets (6). The hydrophobins, specific proteins present in the rodlet layer, mediate the passive adhesion of conidia to hydrophobic surfaces, such as the cuticles of arthropods (16, 45, 46). However, as germination commences, the hydrophobins are replaced by an adhesion-like protein, Mad1, which promotes tighter and more-specific adhesion between the conidia and the host (44). Many factors may affect the adhesion and persistence of conidia on the host cuticle (i.e., characteristics of the pathogen, including its virulence [2, 18, 48], conditions under which the pathogen is cultured [17], type of spores [7, 16], topographical and chemical properties of the host cuticle [9, 38, 42], host surface hydrophobicity [15, 23], host behavior [31, 33], and environmental conditions [33]). Conidia of M. anisopliae have shown an affinity to cuticular regions containing setae or spines (7, 38) and to highly hydrophobic cuticle regions, such as mosquitoes'' siphon tubes (23) and intersegmental folds (43). Sites with higher numbers of adhered conidia varied among host species. However, in general, the membranous intersegmental regions were often particularly attractive sites for conidial attachment (26). Variation in the distribution of conidia across different anatomical regions has also been noted in studies of several tick species inoculated with entomopathogenic fungi (3, 21, 22). An evaluation of the attachment of Beauveria bassiana conidia to three tick species, Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis, demonstrated that the distribution patterns of the different conidia on the ticks'' bodies were not uniform (22). The density of the conidia and their germination varied dramatically across different anatomical regions of Amblyomma maculatum and A. americanum that had been inoculated with B. bassiana (21). Arruda et al. (3) demonstrated that mass adhesion of M. anisopliae conidia to engorged Boophilus microplus females occurs predominantly on ticks'' legs, suggesting its association with the presence of setae.There are a few approaches for assessing the adhesion of conidia to the host cuticle that are based on direct observation of the conidia on the arthropod cuticle. They involve examining a few areas on the surface of an arthropod by means of scanning electron microscopy (SEM) (11, 15, 30), transmission electron microscopy (TEM) (4), or fluorescence microscopy following vital staining of the conidia (2, 28, 29, 37). These methods are expensive, time-consuming, and relatively inaccurate due to the uneven distribution of conidia on the host surface.In this work, we describe a quantitative method for determining the total amount of conidia that have adhered to a whole host cuticle. This method is based on removing adhered conidia from the tick cuticle using an organic solvent, separating the conidia from the extract by centrifugation, resuspending the conidia in a detergent solution, and then counting the conidia in a hemocytometer. The efficacy of the method was evaluated by comparing the results of this procedure with those of a supplementary examination of conidial removal using SEM.The term “adhered” is often used to define conidia in different states: washed or unwashed after inoculation, present on the host cuticle immediately after inoculation, or kept for several hours (1, 2, 38). In this paper, the term “adhered conidia” refers to conidia that remained on the cuticle after washing by vortexing the inoculated and dried host in an aqueous solution of Triton X-100 and rinsing of the material under tap water.     

TMKink: a method to predict transmembrane helix kinks

A hallmark of membrane protein structure is the large number of distorted transmembrane helices. Because of the prevalence of bends, it is important to not only understand how they are generated but also to learn how to predict their occurrence. Here, we find that there are local sequence preferences in kinked helices, most notably a higher abundance of proline, which can be exploited to identify bends from local sequence information. A neural network predictor identifies over two-thirds of all bends (sensitivity 0.70) with high reliability (specificity 0.89). It is likely that more structural data will allow for better helix distortion predictors with increased coverage in the future. The kink predictor, TMKink, is available at http://tmkinkpredictor.mbi.ucla.edu/.     

Knowledge-based potential for positioning membrane-associated structures and assessing residue-specific energetic contributions

The complex hydrophobic and hydrophilic milieus of membrane-associated proteins pose experimental and theoretical challenges to their understanding. Here, we produce a nonredundant database to compute knowledge-based asymmetric cross-membrane potentials from the per-residue distributions of C(β), C(γ) and functional group atoms. We predict transmembrane and peripherally associated regions from genomic sequence and position peptides and protein structures relative to the bilayer (available at http://www.degradolab.org/ez). The pseudo-energy topological landscapes underscore positional stability and functional mechanisms demonstrated here for antimicrobial peptides, transmembrane proteins, and viral fusion proteins. Moreover, experimental effects of point mutations on the relative ratio changes of dual-topology proteins are quantitatively reproduced. The functional group potential and the membrane-exposed residues display the largest energetic changes enabling to detect native-like structures from decoys. Hence, focusing on the uniqueness of membrane-associated proteins and peptides, we quantitatively parameterize their cross-membrane propensity, thus facilitating structural refinement, characterization, prediction, and design.     

Protection of Metarhizium anisopliae conidia from ultra-violet radiation and their pathogenicity to Rhipicephalus evertsi evertsi ticks

Metarhizium anisopliae conidia were formulated in water or in olive oil containing 3% commercial sunscreens (Everysun or E45 Sun Block 50) and exposed to an artificial UV source for up to 5 hours. Survival of conidia after 5 h of exposure to UV in oil formulation was 29% when protected with Everysun, 40% when protected with E45, and 4% in control. In comparison, survival of conidia formulated in water was 13% when protected with Everysun, 24% when protected with E45, and 0% in control. Furthermore, the influence of sunscreens on conidia viability and virulence to Rhipicephalus evertsi evertsi larvae and unfed adult ticks was evaluated. Adding these compounds to the conidial formulations did not reduce the viability of the conidia. Larval mortality was 95 and 100%, while unfed adult mortality was 90 and 97% after being exposed to unprotected conidia formulated in water or in oil, respectively. Conidia protected by Everysun or E45 formulated in water, induced 88 and 83% mortality in larvae, and 92 and 90% mortality in unfed adults, respectively. Conidia suspended in oil and protected by Everysun or E45 induced 94 and 91% mortality in larvae, and 83 and 81% in unfed adults, respectively. These observations indicate that olive oil and the two sunscreens confer protection to conidia against damages by UV radiation without interfering with their pathogenicity to ticks.     

The Role of Light in Leaf and Flower Bud Break of the Peach (Prunus persica)

The effect of light on peach leaf and flower bud break was examined. It was found that leafless dormant shoots were light-perceptive organs. Darkness, after light preconditioning during dormancy, reduced leaf bud opening; however, light was obligatory when the shoots were preconditioned in the dark. Relatively short exposures to light were sufficient to stimulate leaf bud break. Terminal buds were less inhibited by darkness than were laterals. Flower bud break was inhibited in light after dark preconditioning. The red region of the spectrum was found to be active; the phytochorome system seems to be involved in the light reactions, as the red light effect was reversible with subsequent far-red illumination. Supplementary light, producing long-day conditions, could partly compensate for insufficient chilling. A possible sequence of reactions in the plant is suggested.     

Control of tick populations by spraying <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> conidia on cattle under field conditions

Conidia of the entomopathogenic fungus, Metarhizium anisopliae, in oil/water formulation (1 × 10⁸ conidia/ml) were sprayed at 3 weekly intervals on Rhipicephalus evertsi evertsi and Rhipicephalus (Boophilus) decoloratus ticks while feeding on Afrikana bulls grazing in paddocks for a period of 1 year. The fungus reduced the on-host tick populations by 83% 3 month after commencement of the experiment. The formulation by itself had only minimal effect on the tick population. Tick populations and fungal efficacy were highest at the peaks of rainfall and relative humidity or soon thereafter. Fed and unfed adult R. e. evertsi and R. (B.) decoloratus collected at the end of the experiment from the fungus-sprayed and from the control cattle and incubated in the laboratory exhibited a mortality of 93% in oil formulated conidia and 14% in oil control. The corresponding mortality in R. (B.) decoloratus was 100% in fungus and 11% in oil control. Ticks on the fungus-sprayed groups had significantly higher mortality (P < 0.05) than on the control groups. Furthermore, no significant difference (P < 0.05) was observed in fungus-induced mortality between the two tick species. Mortalities induced by Triton X-100 (0.05%), sunflower oil (20%) and water alone were low, suggesting that they were non-toxic to ticks at the concentrations used and no significant difference was observed among them. No physical or behavioral abnormalities were observed in the fungus-sprayed cattle at any time during the course of the experiment. All groups of cattle gained weights during the experimental period.     

Entomopathogenic fungi as a potential control agent against the lesser mealworm, <Emphasis Type="Italic">Alphitobius diaperinus</Emphasis> in broiler houses

We tested the pathogenicity of 18 Metarhizium anisopliae (Metschn.) Sorokin isolates and 22 Beauveria bassiana (Balsamo) Vuillemin isolates against Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae and adults. The efficacy of the most virulent isolate—M. anisopliae K—was evaluated in containers with a concrete bottom covered with wood shavings, under simulated poultry house conditions. Application of conidia of this isolate to the shavings or directly to the concrete bottom reduced the yield of larvae in 8–15 time compared with the control. In another test, the mortality of mature larvae placed on previously inoculated shavings or bottom reached 80–90% within 14 days, compared with 14% in the control. The residual activity of conidia kept at 28°C retained its initial level during 14 days post-inoculation, but declined after three weeks. Based on our data M. anisopliae has considerable potential for the control of A. diaperinus.

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.