Comparative response of Metarhizium brunneum to the cuticles of susceptible and resistant hosts.

Earlier studies demonstrated that Metarhizium brunneum, usually a broad-host pathogen of arthropods, is unable to complete its pathogenic life cycle when inoculated on the fungus-resistant tick, Hyalomma excavatum engorged females. While the fungus penetrates the cuticle of fungus-susceptible tick, Rhipicephalus annulatus females, it is unable to penetrate the cuticle of fungus-resistant tick, and even perishes on its surface. This is probably due to high concentration of antifungal fatty acids and probably also due to a hypersensitive-like response of the tick. To understand the metabolic pathways occurring in the fungal hyphae upon encountering the cuticles, we compared the response of the fungus to cuticle from susceptible and resistant tick cuticles by 2D-gels. The intracellular proteomes of M. brunneum Mb7 exposed to cuticle of the fungus-susceptible tick, R. annulatus, and to the fungus-resistant tick, H. excavatum engorged females were compared after exposure to either cuticles. By means of liquid chromatography-mass spectrometry/mass spectrometry we identified in both proteomes common proteins involved in biological processes as well as unique proteins identified only in the proteome of fungus exposed to fungus-resistant tick cuticle. These proteins were identified in high probability as heat shock proteins, four key enzymes of the glyoxylate cycle, and proteins associated with hypoxia, and exposure to antifungal drugs. These findings are discussed within the M. brunneum-tick pathosystem in relation to tick resistance and host resistance in general.

Resistant ticks inhibit Metarhizium infection prior to haemocoel invasion by reducing fungal viability on the cuticle surface.

We studied disease progression of, and host responses to, four species in the Metarhizium anisopliae complex expressing green fluorescent protein (GFP). We compared development and determined their relative levels of virulence against two susceptible arthropods, the cattle tick Rhipicephalus annulatus and the lepidopteran Galleria mellonella, and two resistant ticks, Hyalomma excavatum and Rhipicephalus sanguineus. Metarhizium brunneum Ma7 caused the greatest mortality of R. annulatus, Metarhizium robertsii ARSEF 2575 and Metarhizium pingshaense PPRC51 exhibited intermediate levels of virulence, and Metarhizium majus PPRC27 caused low mortality of cattle ticks. Conidia of all four species germinated on all hosts examined, but on resistant hosts, sustained hyphal growth was inhibited and GFP emission steadily and significantly decreased over time, suggesting a loss of fungal viability. Cuticle penetration was observed only for the three most virulent species infecting susceptible hosts. Cuticles of resistant and susceptible engorged female ticks showed significant increases in red autofluorescence at sites immediately under fungal hyphae. This is the first report (i) of tick mortality occurring after cuticle penetration but prior to haemocoel colonization and (ii) that resistant ticks do not support development of Metarhizium germlings on the outer surface of the cuticle. Whether reduced Metarhizium viability on resistant tick cuticles is due to antibiosis or limited nutrient availability is unknown.

Characterization of Onthophagus sellatus as the major intermediate host of the dog esophageal worm Spirocerca lupi in Israel.

Spirocerca lupi is a parasitic nematode of dogs, that causes significant morbidity and mortality. Its intermediate hosts in Israel have never been described. The aim of this study was therefore to identify the intermediate hosts of S. lupi in Israel and to describe their abundance and annual infection rate with the nematode, in different microenvironments (i.e., the effects of irrigation and shade) in an endemic area. Dog dung pads were collected every 2 months from two different public parks for 1 year. Dung beetles were identified to the species level in infested dog feces and were examined for the presence of S. lupi larvae through dissection. The Scarabid beetle Onthophagus sellatus was the most abundant dung beetle species in dog dung pads and the only one infected with S. lupi larvae. The minimal period for development of the S. lupi L3 infective stage was 7 days. Significant differences were observed between the two different microenvironments and along the year. The highest risk for infection of dogs with the nematode was during the summer, in an irrigated, shady microenvironment.

The effect of temperature and relative humidity on the formation of Metarhizium anisopliae chlamydospores in tick eggs.

The influence of ambient conditions on the development of Metarhizium anisopliae chlamydospores in tick eggs is reported for the first time. The infection of tick eggs by M. anisopliae involves common events, such as adhesion, conidial germination, appressoria formation, invasion, and development within the eggs. However, the final stage of fungal development differs according to the environmental conditions. At high humidity (close to 100%) and moderate temperature (25°C) the fungus emerged from the eggs and formed conidiophores and conidia externally on the dead eggs. Elevating the temperature to 30°C or reducing humidity to 55-75% induced the production of chlamydospores inside the eggs, without conidiogenesis. When eggs with mature chlamydospores were returned to the appropriate conditions (25°C and 100% RH), conidiogenesis was recovered. Formation of chlamydospores, observed by means of histology and TEM, began with the thickening and septation of hyphae. As the chlamydospore wall thickened a new external undulated wall layer appeared. The mature chlamydospore in eggs has an oval shape (5.3 ± 0.9 microm long, 2.5 ± 0.2 microm wide); its wall comprises three distinct layers. The ability of M. anisopliae to produce chlamydospores under harsh conditions is advantageous and should be considered in application.

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.

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface.

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae (M.an.an.-7) and M. anisopliae var. acridum (M.an.ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M.an.an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M.an.an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M.an.an.-7 conidia. Conidia of the non-virulent M.an.ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M.an.an.-7, but not M.an.ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.

Pathogenicity of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) to tick eggs and the effect of egg cuticular lipids on conidia development.

The ovicidal efficacy of two entomopathogenic hyphomycetes fungi--Metarhizium anisopliae variety acridum (M. an. ac.) Driver and Milner (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae variety anisopliae (M. an. an.) (Metschn.) Sorokin (Hypocreales: Clavicipitaceae)--was evaluated against eggs of three tick species (Acari: Ixodidae)--Hyalomma excavatum (Koch), Rhipicephalus (Boophilus) annulatus (Say), and Rhipicephalus sanguineus (Latereille)--by placing eggs, laid by surface-sterilized females, on conidia-impregnated filter paper. Although M. an. an. strains differed in their virulence to the tested ticks, they reduced the hatching percentages of eggs of all three tick species to 0-32% compared with 80-90% in the control eggs. The M. an. ac. strains were found highly virulent to H. excavatum and R. sanguineus eggs, reducing the hatching percentages to 2-6% but had no influence on hatching of R. annulatus eggs. Older tick eggs were more susceptible to fungal infection than newly laid ones. The effects of polar and nonpolar lipid fractions, extracted from the surface of tick eggs, on the development of conidia were tested. Both germination of M. an. an. conidia and formation of appressoria were stimulated by extracts from egg cuticles of all three tested tick species. However, the stimulating effect was lower when the conidia were exposed to lipids from relatively less susceptible R. annulatus eggs than when exposed to lipids from H. excavatum or R. sanguineus eggs. Unlike those of M. an. an., conidia of M. an. ac. exposed to such lipid extracts did not germinate and did not form appressoria.

Life cycle of Hepatozoon canis (Apicomplexa: Adeleorina: Hepatozoidae) in the tick Rhipicephalus sanguineus and domestic dog (Canis familiaris).

The life cycle of the apicomplexan protozoon Hepatozoon canis in its natural hosts Rhipicephalus sanguineus (tick) and Canis familiaris (domestic dog) was studied in an experimental infection. Tick nymphs were fed on a naturally infected dog, or they were infected by percutaneous injection of blood. Dogs were inoculated by ingestion of adult ticks containing mature oocysts. Gamonts were in syzygy 24 hr after percutaneous injection of ticks. Early oocysts were detected 96 hr after nymph repletion, and mature oocysts in adult ticks were infective to dogs 40 days postmolt. Merogony was detected in dog bone marrow from 13 days postinoculation (PI) and included meronts containing 20-30 micromerozoites, and a second type with 2-4 macromerozoites. Monozoic cysts were observed in the spleen in conjunction with merogony. Gamontogony with infection of leukocytes by micromerozoites occurred from 26 days PI, and gamont parasitemia, which completed the life cycle, was detected 28 days PI. The length of the life cycle from nymphal attachment to parasitemia in dogs was 81 days. Increased body temperatures were evident from 16 to 27 days PI and paralleled the time of intensive bone marrow merogony. Skeletal pain and recumbency were manifested in 2 dogs. This study further elucidates the life cycle of H. canis and provides a sequential morphologic description of H. canis merogony, gamontogony, and sporogony.

Immunity against Boophilus annulatus induced by the Bm86 (Tick-GARD) vaccine.

Friesian cattle were immunized with two inoculations of anti-tick Bm86 (Tick-GARD) vaccine and were challenged 30 or 90 d later with Boophilus annulatus larvae derived from 1.2 g of eggs. No nymphs or adult ticks were found on the immunized cattle during four weeks after challenge. Repeated infestations (2 to 4) with larvae on three other calves during a period of 160 and 390 d after the immunization did not result in development of nymphal and adult stages. In control, non-immunized cattle infested with corresponding batches of larvae 1380 to 4653 replete adult female ticks were collected. Larvae issued from Babesia bovis-infected female ticks transmitted the infection to Bm86-immunized cattle, but the progeny of B. bigemina-infected females did not. Since B. bigemina is transmitted exclusively by nymphal stages of Bo. annulatus these results support the observation that immunity induced by Bm86 affects the larval stage of this tick.

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 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. excalatimn ticks, which were relatively resistant.