Efficacy of the entomopathogenic fungus Metarhizium brunneum in controlling the tick Rhipicephalus annulatus under field conditions.

High infectivity of entomopathogenic fungi to ticks under laboratory conditions has been demonstrated in many studies. However, the few reports on their use under field conditions demonstrate large variations in their success, often with no clear explanation. The present study evaluated the factors affecting the efficacy of the fungus Metarhizium brunneum against the tick Rhipicephalus (Boophilus) annulatus. It demonstrates how environmental conditions and ground cover affect the efficiency of the fungus under field conditions. During the summer, 93% of tick females exposed to fungus-contaminated ground died within 1 week, whereas during the winter, only 62.2% died within 6 weeks. Nevertheless, the hatchability of their eggs was only 6.1% during the summer and 0.0% during winter. Covering the ground with grass, leaves or gravel improved fungal performance. Aside from killing female ticks, the fungus had a substantial effect on tick fecundity. Fungal infection reduced the proportion of female ticks laying full-size egg masses by up to 91%, and reduced egg hatchability by up to 100%. To reduce the negative effect of outdoor factors on fungal activity, its conidia were mixed with different oils (olive, canola, mineral or paraffin at 10% v/v) and evaluated in both laboratory and field tests for efficacy. All tested oils without conidia sprayed on the sand did not influence tick survival or weight of the laid eggs but significantly reduced egghatchability. Conidia in water with canola or mineral oil spread on agarose and incubated for 18 h showed 57% and 0% germination, respectively. Comparing, under laboratory conditions, the effects of adding each of the four oils to conidia in water on ticks demonstrated no effect on female mortality or weight of the laid egg mass, but the percentage of hatched eggs was reduced. In outdoor trials, female ticks placed on the ground sprayed with conidia in water yielded an average of 175 larvae per female and there was no hatching of eggs laid by females placed on ground sprayed with conidia in water with canola or mineral oils.

On-host control of the brown dog tick Rhipicephalus sanguineus Latreille (Acari: Ixodidae) by Metarhizium brunneum (Hypocreales: Clavicipitaceae).

Ticks are obligatory blood-sucking arthropods. Their life cycle includes a relatively short period of feeding on a vertebrate host and a long off-host period spent in the upper layer of the soil. Entomopathogenic fungi are known to be highly effective tick pathogens and the on-host application of these fungi may be a promising economic approach for tick control. In this study, we evaluated the tick control provided by spraying Metarhizium brunneum onto the tick's vertebrate host, specifically gerbils (Meriones tristrami) and rabbits (Oryctolagus cuniculus). The efficacy of the fungal treatment was not limited to a direct effect on the mortality of feeding ticks, but continued during molting (off host) and, in the case of female ticks, the treatment reduced the production of eggs and their hatching rate. The direct control of the on-host stages was relatively low (from 19 to 38%); whereas the effects of the applied fungus on subsequent tick development reduced the yield of the following engorged stages up to 30-63%. Engorged females that dropped from rabbits sprayed with M. brunneum laid 21.5% fewer eggs than the control females. Moreover, these ticks transmitted conidia by contact to the eggs which they laid, resulting a 3-fold reduction in the rate of hatching relative to the control. Based on theoretical cumulative calculations, these results suggest that if the progeny of each unfed stage feed on fungus-sprayed hosts, there will be a 92% reduction in the tick population within one generation. Two spray formulations, one based on mineral oil and another based on a starch-sucrose mixture, significantly enhanced on-host tick control, in comparison with an unformulated conidial suspension. The reduction in the number of nymphs that fed on the treated host and later developed into unfed adults was 54.9% for unformulated conidia, 70.4% for the oil formulation and 86.4% for the starch-sucrose formulation. Increasing the environmental humidity around the gerbils while the ticks fed on them to 90% RH significantly improved the control of the on-host developmental stages, reducing the number of engorged ticks that dropped from fungus-sprayed gerbils 3-fold in comparison with the same animals kept at 30-60% RH. There was no difference between the efficacy of the observed tick control at an ambient temperature of 21°C and that observed at 28°C.

Control of tick populations by spraying Metarhizium anisopliae conidia on cattle under field conditions.

Conidia of the entomopathogenic fungus, Metarhizium anisopliae, in oil/water formulation (1 × 10(8) 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.

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.

Cross-stress tolerance and expression of stress-related proteins in osmotically desiccated entomopathogenic Steinernema feltiae IS-6.

Infective juveniles (IJs) of the entomopathogenic nematode (EPN) Steinernema feltiae IS-6 can survive exposure to 24% glycerol solution by entering an osmotically desiccated state. Exposure of osmotically desiccated nematodes to extreme temperature assays (40 degrees C for 10 h and -20 degrees C for 360 h) resulted in gradual reduction in survival, whereas non-desiccated IJs died within a short exposure to the assay conditions. Through SDS-PAGE, a stress-related protein UNC-87 was found in osmotically desiccated IJs exposed to 40 degrees C for 3, 6, and 8 h, whose survival rates were 98.9+/-1.43, 78.5+/-5.87 and 20.9+/-4.93%, respectively. The protein was not found in IJs following exposure of osmotically desiccated individuals to 40 degrees C for 10 h, in which none of the IJs survived. After exposure to -20 degrees C for 360 h, the survival of osmotically desiccated EPNs with a weak band of UNC-87 was 13.0+/-3.32%. To identify other responsive proteins that are required for osmotic stress, we used 2-dimensional electrophoresis to analyse the proteins in osmotically desiccated EPNs. The results revealed that 10 novel protein spots and 10 up-regulated protein spots in osmotically desiccated IJs were detected by digital image analysis. Mass spectrometry analysis of 7 significant spots indicated that osmotic stress in desiccated IJs was associated with the induction of actin, Proteasome regulatory particle (ATPase-like), GroEL chaperonin, GroES co-chaperonin and transposase family member. It seems to show actin, UNC-87 and Proteasome regulatory particle may play distinct roles in specific aspects of organization of macromolecular structures under desiccation stress. GroEL and GroES are members of the Hsp60 family of chaperons.

Protein interaction of nucleosome assembly protein 1 and casein kinase 2 during desiccation response in the insect-killing nematode Steinernema feltiae IS-6.

The change in gene expression induced by desiccation in the semiarid, entomopathogenic nematode Steinernema feltiae IS-6, includes induction of transcription of a nucleosome assembly protein, NAP1 homolog, and of casein kinase 2 (CK2) genes. Therefore, one of the events during the dehydration response of S. feltiae IS-6 may be transcriptional activation by S. feltiae IS-6 NAP1 homolog (Sf-Nap1), which is regulated by S. feltiae IS-6 CK2 (Sf-CK2). This regulation necessitates physical interaction between the Sf-Nap1 and Sf-CK2 proteins. In the present study we used yeast 2-hybrid analysis to demonstrate physical interaction between the 2 proteins, thus confirming the involvement of a protein interaction-based step in the desiccation response mechanism of S. feltiae IS-6.

Biological control of ticks.

Ticks have numerous natural enemies, but only a few species have been evaluated as tick biocontrol agents (BCAs). Some laboratory results suggest that several bacteria are pathogenic to ticks, but their mode of action and their potential value as biocontrol agents remain to be determined. The most promising entomopathogenic fungi appear to be Metarhizium anisopliae and Beauveria bassiana, strains of which are already commercially available for the control of some pests. Development of effective formulations is critical for tick management. Entomopathogenic nematodes that are pathogenic to ticks can potentially control ticks, but improved formulations and selection of novel nematode strains are needed. Parasitoid wasps of the genus Ixodiphagus do not typically control ticks under natural conditions, but inundative releases show potential value. Most predators of ticks are generalists, with a limited potential for tick management (one possible exception is oxpeckers in Africa). Biological control is likely to play a substantial role in future IPM programmes for ticks because of the diversity of taxa that show high potential as tick BCAs. Considerable research is required to select appropriate strains, develop them as BCAs, establish their effectiveness, and devise production strategies to bring them to practical use.

Entomopathogenic nematodes for the biocontrol of ticks.

Entomopathogenic steinemematid and heterorhabditid nematodes are increasingly used to control insect pests of economically important crops. Laboratory and field simulation trials show that ticks are also susceptible to these nematodes. The authors review the potential of entomogenous nematodes for the control of ticks.

Alterations in the levels of glycogen and glycogen synthase transcripts during desiccation in the insect-killing nematode Steinernema feltiae IS-6.

The ability to withstand desiccation by entering anhydrobiosis is important for the survival of many nematode species. We are interested in the metabolic changes that occur during dehydration in the semiarid strain IS-6 of the insect parasitic nematode Steinernema feltiae. These changes may enable IS6 to be more tolerant to desiccation than temperate strains. We identified genes of IS-6 that exhibit changes in transcript levels during dehydration. These included glycogen synthase (Sf-gsy-1), which is the rate-limiting enzyme in the synthesis of glycogen, which is likely to play a role in desiccation survival. We established the changes in the steady state level of Sf-gsy-1 transcripts upon dehydration and determined the biochemical changes in the level of its product, glycogen, during the dehydration and rehydration of nematodes. Our results suggest a shift from glycogen to trehalose synthesis during dehydration, which is regulated at least in part by suppression of glycogen synthase transcription.

Factors affecting the virulence of entomopathogenic nematodes to engorged female Boophilus annulatus ticks.

The laboratory trials rank the virulence of entomopathogenic nematode strains (3 heterorhabditids and 6 steinernematids) to engorged female Boophilus annulatus ticks according to 3 parameters of the infection process: the effect of exposure time on tick mortality, the quantity of nematodes that penetrate ticks, and the rate of tick mortality after the injection of 1, 2, or 3 nematodes. Exposure of the ticks to heterorhabditid strains for 6 hr resulted in >80% mortality, but only 20 or 65% mortality after exposure to most steinernematids. The quantity of nematodes recovered per tick exposed to nematodes for 6 days averaged from 16 to 141. For steinernematids, a negative correlation was obtained between tick mortality and the average quantity of nematodes recovered. Injecting 1 infective juvenile from 1 of 2 heterorhabditid strains into each tick resulted in close to 100% mortality. Increasing the quantity of nematodes injected into each tick had little or no additive effect on tick mortality.

Pathogenicity of entomopathogenic fungi to different developmental stages of Rhipicephalus sanguineus (Acari: Ixodidae).

The pathogenicity of 4 species of entomopathogenic fungi (Hyphomycetes species: Beauveria bassiana, Metarhizium anisopliae, Metarhizium flavoviride, and Paecilomyces fumosoroseus) to various developmental stages of Rhipicephalus sanguineus (Acari: Ixodidae) ticks was compared under laboratory conditions. The most virulent isolate, M. anisopliae-108, caused 92-96% mortality to unfed larvae and nymphs on day 7 postinfection (PI) and 100% mortality to unfed adults and engorged females on day 21 PI. The pathogenicity of M. anisopliae-108 to engorged larvae and nymphs was lower--82.6 and 60%, respectively. All tested B. bassiana, M. flavoviride, and P. fumosoroseus isolates were significantly less virulent (P < 0.05) or avirulent toward most life stages of R. sanguineus. The M. anisopliae and M. flavoviride isolates also prevented or reduced the ability of the ticks to lay eggs several days before their deaths. Female ticks infected by the fungi achieved only 11.3-60.8% of their egg-laying capacity compared with the controls.

Desiccation stress of entomopathogenic nematodes induces the accumulation of a novel heat-stable protein.

The present study describes a novel heat-stable, water-stress-related protein with a molecular mass of 47 kDa (designated Desc47) in the entomopathogenic nematode Steinernemafeltiae (IS-6). The protein was accumulated about 10-fold (from 7.84 +/- 1.85 to 74.09 +/- 4.35 % relative content level [RCL]) in dehydrated clumps of infective juveniles (IJs), which had lost 344.% of their initial water content (from 65.1 +/- 1.7% to 427 +/- 0.72%) in a desiccation-tolerance-inducing treatment (97% relative humidity [RH] for 3 days). The appearance of Desc47 was accompanied by trehalose accumulation (from 300 to 600 mg trehalose/g protein) during the process of inducing the IJs into a quiescent anhydrobiotic state. A second cycle of IJ dehydration did not alter the RCL of Desc47 (79.3% for the first cycle and 73.3% for the second cycle). Desc47 retained its high RCL (69.7%) in rehydrated active IJs for 3 days, reaching 51.2% of its initial RCL only after a week. No homology to other known proteins was found by mass-spectrometry electrospray-ion-trap analysis. However, of the 5 sequences obtained from the 11protein(ranging from to 21 amino acids), the 21-amino-acid peptide N V A S D A V E T V G N A A G Q A G (D/T) A V showed excellent homology (74% identity in 19 amino acids) to the cold-responsive protein COR14b (g6564861) from Triticum aestivum. In the Caenorhabditis elegans predicted proteome database search, the N21 yielded the first-best identity score (59 % identity in 17 amino acids) to the CE-LEA homologue protein (g2353333). In plants, COR and LEA are related proteins, heat-stable, which are expressed in response to both dehydration and cold acclimation. The implication of the involvement of Desc47 and the osmoprotectant trehalose in the desiccation-tolerance mechanisms of S. feltiae is discussed.

Mortality rate of adult ticks due to infection by entomopathogenic nematodes.

The mortality of Boophilus annulatus, Hyalomma excavatum, Rhipicephalus bursa, and Rhipicephalus sanguineus adult ticks was recorded after their exposure in petri dishes to 5 entomopathogenic nematode strains. The strains used were DT and Mexican of Steinernema carpocapsae, Hb HP88 of Heterorhabditis bacteriophora, and IS-3 and IS-5 of Heterorhabditis sp. The most rapid killing rates (LT50 and LT90) were observed for B. annulatus ticks (0.8-5.0 days). Most unfed males died 0.3-2.8 days after the unfed females, whereas even more time (0.8-8.0 days) passed before engorged females died. In most bioassays, the IS-3 and IS-5 strains of Heterorhabditis sp. nematodes appear to be the most pathogenic for ticks and, in most cases, killed ticks several days before the other 3 nematode strains. Unfed adult ticks exposed to nematodes died within less time than it takes for adults to complete their prefeeding period after molting.

Environmental factors affecting sexual differentiation in the entomopathogenic nematode Heterorhabditis bacteriophora.

The present study was aimed at determining the influence of various environmental factors on sex differentiation (SD) in the entomopathogenic nematode Heterorhabditis bacteriophora HP88 strain, under in vivo and in vitro culture conditions. Injection of individual nematodes into last instars of Galleria mellonella resulted in development of a similar number of females and hermaphrodites (35-40%) and 20-25% males. Increasing the number of nematodes injected into the insect did not change these proportions. In smaller insects (0.7-1.5 cm long), an increase in the proportion of hermaphrodites was recorded as compared with larger size cadavers (2.4-2.7 cm long). When individual hermaphrodites were placed on NGM, the proportion of hermaphrodites, females and male progeny was 63%, 31%, and 6%, respectively. Rearing on richer medium ("Dog-food" agar) resulted in reduction in the proportion of hermaphrodites. Nematodes introduced to the symbiotic bacterium obtained from other nematode strains (IS-5 and IS-33) developed similarly to the culture reared on the HP88 bacteria. Rearing the nematodes at a temperature range between 21 degrees C to 30 degrees C also did not have a significant effect on the sexual differentiation among nematodes cultured on NGM. The proportion of hermaphrodites increased as the starvation period of hatching nematode juveniles lengthened (>6 hr). The data obtained in the present study strongly suggest that the main factor affecting sex differentiation in H. bacteriophora is the nutrition source. The practical and biological implications of the results are discussed.

A new compact medical kit on El Al passenger aircraft.

El Al was one of the first international airlines to introduce a medical kit (MK) on board its passenger aircraft. Based on our own experience and considering the recent regulations of the European Joint Aviation Authority (JAA), the items are updated. The design of the kit is arranged so that all the medications with a short "kit life" are placed in a compartmented tray and can be removed in toto and replaced by a new tray. The equipment and medications in the MK are listed on the form which the physician on board is required to complete, checking the items used and filing a report. The contents of the MK will evidently be upgraded once ECG monitoring, transmission facilities and automatic external defibrillators (AED) are put on board.

Laboratory evaluation of pathogenicity of entomogenous nematodes to African tick species.

Five strains of entomogenous nematodes, Steinernema carpocapsae strains DD, Mexican, SR, and Heterorhabditis bacteriophora strains 1S5 and HP88, were tested for their pathogenicity to various developmental stages of five African tick species namely; Rhipicephalus appendiculatus, R. evertsi, Amblyomma variegatum, A. gemma, and Boophilus decoloratus. In engorged female R. appendiculatus, all nematodes at a concentration of 1,000 infective juveniles (IJ)/dish, except S. carpocapsae Mexican strain, induced high mortalities (56-100%), whereas in engorged female R. evertsi, only S. carpocapsae DD and H. bacteriophora HP88 induced high mortalities (78% and 56%, respectively). In engorged B. decoloratus, S. carpocapsae DD, Mexican, SR and H. bacteriophora HP88 (100 IJ/dish) induced mortalities of 85%, 65%, 80%, and 100%, respectively. In all cases, except for S. carpocapsae Mexican strain, a higher concentration (5,000 IJ/dish) did not result in higher mortality than occurred with 1,000 IJ/dish. Unfed females and immature stages of ticks were found to be generally resistant to the nematodes. The feasibility of using entomogenous nematodes for biological control of African tick species are briefly discussed.

Biocontrol of ticks by entomopathogenic nematodes. Research update.

Entomopathogenic nematodes (EPNs) are lethal to ticks even though they do not use their normal propagation cycle within tick cadavers. The tick Boophilus annulatus was found to be far more susceptible to EPNs than Hyalomma excavatum, Rhipicephalus bursa, or Rhipicephalus sanguineus. Ticks seem to be less susceptible to nematodes when feeding on a host. Preimaginal tick stages were less susceptible to nematodes than adult ticks. The mortality rate of unfed females was highest, followed by unfed males, and engorged females. The virulence of nematodes to ticks varied greatly among different nematode strains. In most cases, the Heterorhabditis sp. strains were the most virulent strains tested in petri dishes. In buckets containing sandy soil sprayed with 50 nematodes/cm2 and engorged B. annulatus females, the LT50 of the ticks was less than five days. The addition of manure to soil or a manure extract to petri dishes reduced nematode virulence. Since ticks spend most of their life cycle in the upper humid layer of the ground, and many nematode strains share this same ecological niche, the use of EPNs for biocontrol of ticks appears promising.

Efficacy of entomopathogenic nematode strains against engorged Boophilus annulatus females (Acari: Ixodidae) under simulated field conditions.

The biocidal efficacy of entompathogenic nematodes against engorged females of Boophilus annulatus (Say) was evaluated in soil-filled buckets in a greenhouse, where conditions resemble nature. The 9 tested nematode strains differed markedly in their effect upon tick mortality. The Mexican strain of Steinernema carpocapsae was most efficient, inducing 100% tick mortality at a concentration of 50 nematodes per square centimeter. An increase in the concentration of the S. carpocapsae DT strain to > 200 IJ/cm2 failed to kill more ticks. It appears that entomopathogenic nematodes show promise as tick control agents.