The effect of temperature and relative humidity on theformation of Metarhizium anisopliae chlamydosporesin tick eggs

Dana MENTa,b, Galina GINDINa, Itamar GLAZERa, Shmuel PERLc, Daniel ELADc,Michael SAMISHc,*aARO, The Volcani Center, (ARO), P.O.B. 6, Bet-Dagan 50250, IsraelbThe Robert H. Smith Faculty of Agricultural, Food & Environmental Quality Sciences, The Hebrew University of Jerusalem,P.O.B. 12, Rehovot 76100, IsraelcKimron Veterinary Institute, P.O.B. 12, Bet-Dagan 50250, Israel

a r t i c l e i n f o

Article history:

Received 16 March 2009

Received in revised form

11 October 2009

Accepted 19 October 2009

Available online 24 October 2009

Corresponding Editor:

Richard A. Humber

Keywords:

Abiotic stress conditions

Entomopathogenic fungi

Infection pathways

Resting structures

Rhipicephalus (Boophilus) annulatus

eggs

a b s t r a c t

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 mm

long, 2.5" 0.2 mm wide); its wall comprises three distinct layers. The ability of M. anisopliae

to produce chlamydospores under harsh conditions is advantageous and should be consid-

ered in application.

ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction

The physiological adaptations of entomopathogenic fungi tochanging climatic conditions increase their potential as bio-logical control agents against insects. Various fungi have dif-fering survival strategies, many of which involve theformation of resting structures. Entomophthoralean fungimay survive stress conditions as resting spores, thick-walled

hyphal bodies or ‘‘loriconidia’’ (Elliot et al. 2002; Hajek & Shi-mazu 1996; Keller 1997;Weiser & Batko 1966), whereas hypho-mycete fungi form thick-walled hyphae or chlamydospores(Pendland 1982; Pendland & Boucias 1987). The widely usedentomopathogenic fungus, Metarhizium anisopliae, is physio-logically adaptable both to pathogenic development inside ar-thropod hosts and saprobic growth (Wang et al. 2005). Thisfungus can persist in soil or in insect cadavers for a long

* Corresponding author. Tel.: #972 3 9681690; fax: #972 3 9681753.E-mail address: samishm@int.gov.il

journa l homepage : www.e lsev ier . com/ loca te / funb io

f u n g a l b i o l o g y 1 1 4 ( 2 0 1 0 ) 4 9 – 5 6

0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.mycres.2009.10.005

time, even under unfavorable environmental conditions(Arthurs et al. 2001; Milner et al. 2003; Sallam et al. 2007), how-ever, resting morphological structures have very seldom beendetected and there are no data on factors that might inducetheir formation. Zacharuk (1971) described the formation ofM. anisopliae chlamydospores from hyphal bodies in the he-molymph of Elateridae larvae that become broader and sep-tate. M. anisopliae chlamydospores were also observed inhaemolymph of the greater wax moth, Galleria mellonella,4–5 d post infection (PI) (Sewify & Hashem 2001). Accordingto Hanel (1982), formation of M. anisopliae chlamydosporesfrom dying mycelium served as a defence response againstcolonization of termite cadavers by other saprobic organisms.Another survival strategy was described for M. anisopliae var.acridum (formerly identified as Metarhizium flavoviride) whichsurvives the dry season as desiccated hyphal fragments ininfected grasshopper cadaver (Thomas et al. 1996). Recently,Jackson & Jaronski (2009) discovered the formation ofM. aniso-pliae microsclerotia in liquid cultures, a trait well known forplant-pathogenic fungi and some entomopathogenic fungiand possessing high tolerant to desiccation.

Theadvance of tick control based on entomopathogenic fun-gal applications requires deep knowledge of the pathogenicprocessand itscharacteristics correspondingduringalldevelop-mental stages. Thenatural environments inwhich ticksovipositare relatively favorable for fungal infection. Most female ixodidticks protect their sensitive eggs by laying them in large cohortsin well protected, sheltered areas, e.g., under stones, leaves,deep in sand, cracks, etc. This behavior provides an environ-ment of relatively high humidity and ‘‘buffered’’ temperaturearound the eggs. Eggs of many tick species need RH of over85 % for their normal development (Daniel & Dusbabek 1994).

Tick eggs are highly susceptible to infection byM. anisopliae(Bittencourt et al. 1994, 2000; Gindin et al. 2001, 2002; Monteiroet al. 1998; Souza et al. 1999). The fungus causes a very highpercentage of egg mortality and dramatically reduce theirhatchability. Moreover, many of the newly hatched larvaeare contaminated with conidia from infected eggs and oftendie within several days (Bittencourt et al. 1994; Gindin et al.2001; Souza et al. 1999). The penetration pattern ofM. anisopliaeinto Rhipicephalus sanguineus eggs under optimal ambient con-ditions, as well as the lesions caused inside the infected eggs,were described by Garcia et al. (2005). The successful infectionof tick eggs by pathogenic fungi involves adherence of conidiato the egg surface, germination and penetration of the eggs’chorion. Before penetration, appressoria may be formed ashyphal structures for better attachment of the fungus to itshost’s surface, and this is followed by enzymatic degradationof the host cuticle (St Leger et al. 1989; St Leger 1993). Theseearly steps of infection and the final external conidiogenesiswere observed on R. sanguineus eggs soon after they hadbeen inoculated with M. anisopliae and incubated at 27 !Cand RH above 80 % (Garcia et al. 2005). However, there isa lack of published information on fungal development andsurvival in tick eggs under less favorable conditions that oftenoccur in the tick environment. The aim of the present studywas to investigate the interaction between tick eggs andM. anisopliae under stressful environmental conditions andto demonstrate the putative physiological adaptation mecha-nism that guarantees fungal survival in the tick environment.

Materials and methods

Fungus

The strain used – Metarhizium anisopliae var. anisopliae-7 – wasisolated from unidentified beetles in Israel and was shown tobe the most virulent strain against eggs of three tick species:Rhipicephalus annulatus, Rhipicephalus sanguineus andHyalommaexcavatum (Gindin et al. 2001, 2009). This strain was passedthrough R. annulatus tick females and then re-isolated on Sab-ouraud dextrose agar (SDA) (Becton Dickinson, Sparks, MD)twice per year. For inoculation of eggs, recently re-isolatedfungi, i.e., after one or two passages on SDA, were used. Thefungus was grown on SDA for 2 weeks at 25 !C in the dark.Conidia were harvested by scraping and were suspended in10 ml of sterile distilled water containing 0.01 % (v/v) TritonX-100, in glass tubes. The suspension was vortexed in anMS2 minishaker (IKA Works, Inc., Wilmington, NC), sonicatedfor 5 min in a model D80H ultrasonic cleaner (Chemist Co.,Taipei Hsien, Taiwan) to break up clumps of conidia and fil-tered through sterile Miracloth (Calbiochem, La Jolla, CA).The conidial concentrations were then determined with a -haemacytometer. The suspension was adjusted to 1$ 107con-idia ml%1 by addition of sterile distilled water containing0.01 % (v/v) Triton X-100, and the percentage of viable conidiawas determined according to conidial germination on SDA24 h after incubation prior to eggs inoculation. Only suspen-sions with at least 95 % germinating conidia were used.

Ticks

Rhipicephalus annulatus ticks have been fed on calves and keptunder laboratory conditions (28 !C and 70–80 % RH) since 1984.The fully engorged females were surface sterilized by dippingin 0.5 % (w/v) methyl p-hydroxybenzoate and 0.1 % (w/v) so-diumbenzoate, each for 5 min, and rinsedwith sterile distilledwater in a vortex. The females were incubated for ovipositionat 25 !C in sterile Petri dishes lined with moist filter paper(Whatman no. 1). The females started to lay eggs 2–3 d afterdrop-off, and only eggs laid during the first 6–7 d after drop-off were used.

Eggs inoculation

Eggs deposited on sterile filter paper were collected witha camel-hair brush, and were transferred into Petri dishes(50 mm diameter) lined with filter paper that has previouslybeen impregnated with 0.5 ml of 0.01 % (v/v) Triton X-100,with or without (control) conidia suspension (1$ 106 conidiaml%1, i.e., about 2.5$ 104 conidia cm%2). Fifty to 100 eggswere distributed on the impregnated filter paper, and thedishes were placed in desiccators over the saturated solutionappropriate for maintaining the required humidity: sodiumchloride for 75 % RH; D (#)-glucose-monohydrate for 55 %RH; and water for 100 % RH. The incubation temperaturewas 25 or 30 !C. For each combination four or five Petri disheswere prepared, and about 50 eggs from each dish were exam-ined by light microscopy on day 14 PI.

50 D. Ment et al.

Histology

Infected and control eggswere sampled for fixation at 6, 12, 18,and 24 h PI and then daily for 2–10 d PI. The eggs were fixed in3.6 % formalin in 0.1 M sodium cacodylate buffer (pH 7.2) andprocessed overnight in a Histoprocessor Tissue Tek VIP-5(Sakura Finetek, CA, USA, Inc.). The samples were embeddedin paraffin wax and sectioned into 3- to 5-mm slices, whichwere then stained with haematoxylin-eosin and with peri-odic-acid-Schiff (PAS). The slideswere coveredwithmountingmedium (DPX) and coverslips and left to dry. The sectionswere examined under a light microscope, photographed andmonitored for measurements with a Leica DC200 digital cam-era and the IM1000 program.

TEM

Chlamydospores that were formed in infected eggs at 30 !Cand 100 % RH were observed with a transmission electron mi-croscope (TEM), 10 d PI. Samples of infected eggswere fixed for2 h in 3.5 % (v/v) glutaraldehyde in PBS buffer, pH 7.2; theywere later fixed for 1 h in buffered (pH 7.2) 1 % (v/v) OsO4

and dehydrated in a graded ethanol series and an acetone se-ries. Then, samples were embedded in Agar 100 resin (AgarScientific, Cambridge, UK). Sections were cut with a diamondknife on an LKB-ultramicrotome and stained with 5 % (v/v)aqueous uranyl acetate followed by lead citrate. The sampleswere examined with a Tecnai G2 electron microscope (Fei Co.,Philips), and micrographs were taken with a Megaview IIIcamera (SIS, Munster, Germany). Same procedure was carriedout for Metarhizium anisopliae conidia.

Results

Influence of ambient conditions on fungus developmentin tick eggs

At 25 !C and 100 % RH, mortality of inoculated eggs was closeto 100 %, and conidia always developed on the surfaces of eggsinfected with Metarhizium anisopliae. The fungus started toemerge as white hyphae on egg surfaces at 5–7 d PI, and con-idiogenesis was observed on the surfaces of all dead eggs by 8–10 d PI (Table 1).When inoculated eggswere incubated at 30 !C

and 100 % RH, their mortality was also very high – up to 73 % –but no fungus was observed to emerge on egg surfaces. Themicroscopic examination at 14 d PI indicated that many eggs– 36.7–100 % of the dead ones – that had been incubated at30 !C contained chlamydospore-like structures, and this indi-cation was confirmed by histological and TEM observations(see below: Morphological examination). Other dead eggswere full with hyphae undergoing septation. Incubation of in-oculated eggs at 25 !C at the lower RH of 75 % caused 100 % eggmortality. There was also no conidiogenesis on eggs surfaceand most eggs were filled with hyphae undergoing septationand/or chlamydospores (Table 1). At the same temperatureof 25 !C but 55 % RH, part of the dead eggs dried before fungusdevelopment; conidia were not present but chlamydosporeswere observed inside eggs that had undamaged outer shells.

Egg mortality and, correspondingly, larval hatching in thecontrol depended on ambient conditions; under favorableconditions (100 % RH and 25 !C) egg mortality in the controlwas about 10–15 % during three weeks PI and larval hatchingreached 85.1" 6.5 %.

In unfavorable conditions, egg mortality in the control was7.8" 2.0 % during the first 3–4 d PI. However, the percentage ofdead eggs increased on 5-d PI and varied from 11.8" 2.3 % at30 !C and 100 % RH to 36.5" 4.6 % at 25 !C and 55 % RH.Then, during 2–3 weeks, all control eggs that were incubatedunder unfavorable conditions dried and larval hatching wasnot observed.

When inoculated eggswere initially incubated for 3–5 d un-der favorable conditions for conidiogenesis, i.e., 25 !C and100 % RH, and then transferred to desiccators at the sametemperature but RH 75 % until 14 d PI, the number of eggs onwhich conidia developed was very low, but some of the deadeggs were filled with hyphae undergoing septation and chla-mydospores (Table 2). Chlamydospores were found only ineggs that had been transferred to low humidity after been in-cubated for not more than 3 d at 100 % RH. The percentage ofeggs with chlamydospores in this case was low (14" 12 %)compared with that in eggs that were exposed to 75 % RH im-mediately after inoculation (74" 15.4 %) (Tables 1 and 2).

When eggs with chlamydospores (14 d PI) due to exposureto high temperature or low RHwere placed in Petri dishes andincubated at 25 !C and 100 % RH, fungus emerged on egg sur-faces within 3–4 d, and formed conidiophores with conidiaabout 6 d after transfer. We observed the conidia on the

Table 1 – Dead Rhipicephalus annulatus eggs (% ±SD)a with Metarhizium anisopliae conidiogenesis on egg surfaces or withchlamydospores inside eggs under different ambient conditions.

Eggs kept for 14 d PI at

Temperature 25 !C 30 !C

RH

Fungal stage and location 55 % 75 % 100 % 100 %

Conidiogenesis on egg surface 0 0 Nearly 100 0Chlamydospores inside eggs 10.9" 4.9b 74" 15.4 0 073.1" 32.7

a Average % was based on four or five egg masses, 50–100 eggs in each.b At 55 % RH, part of the dead eggs had dried before fungus development.

Metarhizium anisopliae chlamydospores in tick eggs 51

surface of intact eggs only, but not on dry, damaged eggs thatwere dead at 55 % RH and that showed no signs of internalfungal growth.

Morphological observation of infection and the formation ofchlamydospores in tick eggs

Fresh, uninfected Rhipicephalus annulatus eggs are brown, oval,and covered with a protective wax layer, in contrast to thelighter coloration and opacity of eggs with internal coloniza-tion by Metarhizium anisopliae hyphae. These visual changesstarted to appear 48–72 h after inoculation under all tempera-ture and humidity regimes tested.

Conidia attached firmly to the waxy coating of the tick eggsshortly (1–2 h) after inoculation and were observed both inhistology and TEM samples. The conidia started to germinateon the egg surfaces 6–8 h PI, and by 24–48 h PI most of the con-idia observed had germinated. The average germ-tube length,24–48 h PI, was 5.1" 0.9 mm. Appressoria were formed at theend of the germ tubes close to the germinated spore at24–48 h PI. The appressoria produced narrow pegs (about0.5 mm thick), which penetrated the egg membranes (Fig 1A).The invasion of the fungi into the eggs vitellus occurredmainly during 48–72 h PI, via several (usually one to three)penetration points in every histological section (Fig 1A). Beforepenetration no hyphal development on the egg surfaces could

Table 2 – Dead Rhipicephalus annulatus eggs (% ±SD)a with Metarhizium anisopliae conidiogenesis on egg surfaces or withhyphae undergoing septation or chlamydospores inside. Total incubation time of infected eggs – 14 d.

Fungal structure Location Days PI when eggs were incubated at 100 % RHbefore transferred to 75 % RH

- 3 5 7

Conidiogenesis On eggs surface 0 0 0 100Hyphae undergoing septation Inside eggs 0 85.3" 12.4 100 0Chlamydospores Inside eggs 100 14.7" 12.4 0 0

a Average % was based on four or five egg masses, 50–100 eggs in each.

Fig 1 – Light micrographs of an egg of Rhipicephalus annulatus infected with Metarhizium anisopliae-7. PAS stain. (A) Thefungus is penetrating the egg membrane. The germ tube develops from conidia and terminates in appressorium from whichpenetration pegs project. (B) The egg vitellus is fully colonized by fungal mycelium. (C) Fungus (F) extrusion from the infectedegg in candelabrum-like structure. (D) Large fungal cells on the egg surface (arrows) and conidiogenesis of fungus. (E) For-mation of chlamydospores inside the egg vitellus. (F) Chlamydospores (arrows) inside the egg. Ap, appressorium; C, conidia;Chl, chlamydospores; E, egg; GT, germ tube; M, membrane of egg; PP, penetration peg.

52 D. Ment et al.

be detected. Nomelanotic lesions on the egg chorion were ob-served at sites where fungi penetrated.

Hyphae ofM. anisopliae, 1.5–2 mmindiameter, were observedinside eggs from 2–3 d PI. At 3–4 d PI, the numbers of fungalhyphae in the eggs increased sharply, and on day 4–5 PI thevitellus of over 90 % of the eggs was fully colonized by welldeveloped mycelium (Fig 1B). After colonization of the eggvitellus, the fungus developed in two differentways, dependingonambient conditions: a) conidiogenesis on the egg surfaces, orb) formation of chlamydospores inside the eggs.

Conidiogenesis on egg surfaces

Hyphae emerged from inside the egg on days 5–7 PI and colo-nized the outer surface of the egg, forming a dense layer oflarge cells, 3–7 mm in length, 2–6 mm in width, which producedcandelabrum-like conidiophores 6–7 d PI (Fig 1C and D). Coni-diogenesis was observed on the egg surface between 7 and 9 dPI. The fungus formed cylindrical conidia, 5.6" 0.7 mm inlength, 2 mm in width, in compact long chains. No chlamydo-sporeswere observed in eggs onwhich conidiawere produced.

Chlamydospore formation in eggs

The first prepenetration events of infection under unfavorableconditions were similar to ones under favorable conditions

(see above). However, on 3–4 d PI, hyphae inside the tick eggthickened and underwent transverse septation for chlamydo-spores formation (Figs 1E,F and 2A,B). At the beginning of thehyphal divisions, the nuclei became granular because of theextensive activity in the fungal cell, and exhibited condensedcytoplasm. The dense cells were observed as individual, sepa-rate cells as well as short chains (2–8 cells). As the chlamydo-spores matured, the nucleus filled most of the cells’ cavity.The new chlamydospores separated from the emptied hy-phae, the cell wall thickened, and storage vacuoles wereseen (Fig 2C). The mature chlamydospore was oval in shape,5.3" 0.9 mm long and 2.5" 0.2 mm wide. An outer osmophiliccell wall, about 60 nm thick with an undulate structure, andadditional middle and inner cell walls, 30 and 15 nm thick, re-spectively, were visible. The nucleus comprised most of thecell volume, and mitochondria and nucleoli were visible (Fig2D). The conidial cell wall as opposed to a chlamydospore iscomprised of two cell-layers; an outer osmophilic wall andan inner wall, both are about 150 nm thick (Fig 2E and F).The vitellus of infected eggswas often densely filledwith chla-mydospores, as many as 100 chlamydospores per histologicalsection. No rupturing of egg chorion by emerging hyphae wasobserved when the fungus formed chlamydospores inside theeggs. Neither emergent hyphae nor conidiogenesis were everobserved on the surfaces of eggs containing chlamydospores(Fig 1F).

Fig 2 – Transmission electron micrographs of Rhipicephalus annulatus egg infected with Metarhizium anisopliae-7, 10 d PI andmicrographs of M. anisopliae-7 conidia. (A) Egg vitellus filled with chlamydospores. (B) The beginning of chlamydosporeformation inside the egg. The septum (arrows) is formed as chlamydospore formation begins. (C) Young chlamydospores areformed from mycelium inside the egg. (D) A mature chlamydospore inside the egg. The three wall layers include an externalundulated layer. (E) M. anisopliae conidia. (F) The cell wall of conidia. LB, lipoid body; M, mitochondrion; My, mycelium;N, nucleus; YChl, young chlamydospores.

Metarhizium anisopliae chlamydospores in tick eggs 53

Discussion

Survival of pathogens under unfavorable conditions can becritical for their success as biological control agents, and un-derstanding of the survival mechanism of fungal pathogensin their various host environments is an important step to-wards the development of mycopesticides. The present paperis the first report on factors that induce the production ofMeta-rhizium anisopliae chlamydospores. Though M. anisopliae chla-mydospores have rarely been described in the literature, it isprobable that they are a common developmental stage of thispathogen under warm or dry ambient conditions. Chlamydo-spores are defined as ‘‘swollen, thick-walled resting cells’’ or,in greater detail, as ‘‘an asexual single-celled spore originatingendogenously and singlywithinpart of a preexisting cell by thecontraction of the protoplast, and possessing an inner second-ary andoften thickenedhyalineor brownwall, usually impreg-nated with hydrophobic material’’ (Hawksworth et al. 1995; deHoog et al. 2000). They are formed under certain conditionswithin hyphae or at hyphal tips. A similar survival strategy isknown for many fungal pathogens of plants and animals, in-cluding some entomopathogens (Darmono & Parke 1990; Jou-venaz & Kimbrough 1991; Paraud et al. 2005). The ability ofBeauveria bassiana to produce chlamydospores both in vivoand in vitro was described by El-Sinary & Rizk (2007) and byMalo & Pardey (1997), whereas M. anisopliae chlamydosporeswere found only in vivo (Zacharuk 1971; Hanel 1982; Sewify &Hashem 2001). The factors that induce the formation of chla-mydospores differ among fungal species; they include: nonop-timal or stress-inducing changes innutrient compounds (Konoet al. 1995), temperature (Gronvold et al. 1996), pH (Saxena et al.2001), ultraviolet light (Englander & Turbitt 1979), and toxins(Abou-Gabal & Fagerland 1981). Thus, the chlamydosporeservesmainly as a survival structure under unfavorable condi-tions (Couteaudier & Alabouvette 1990; Armengol et al. 1999;Croan et al. 1999). To the best of our knowledge, the present pa-per forms thefirst report of factors that induce the formationofM. anisopliae chlamydospores in the host.

According to Yoder et al. (2006), Amblyomma americanumunfed females infected with M. anisopliae lose twice as muchwater as uninfected ones, i.e., fast dehydration of an infectedhost is probably one of themajor causes of tick death. It is pos-sible that a similar fast dehydration process occurs in M. ani-sopliae-infected eggs also. The infection process developsrelatively fast and at moderate temperature and 100 % RH,the fungus colonized the egg vitellus, then emerged onto theegg surface and formed conidiophores with conidia. Both thestressful ambient factors, higher temperature and lower hu-midity, can cause additional lack of moisture in eggs cyto-plasm, increasing the dehydration effect of fungus ininfected eggs. We suppose that the rapid dehydration ofeggs due to these abiotic factors may induce or hasten the for-mation of chlamydospores. The fact that chlamydosporesappeared in infected eggs that were incubated under condi-tions unfavorable for conidiogenesis indicates that they areprobably alternative survival structures. No eggs were foundwith both structures: chlamydospores inside eggs and conidiaon their surface. However, changing the ambient conditionsby transferring eggs already containing chlamydospores to

100 % RH and moderate temperature for several days resultedin the appearance of conidia on the egg surface. We hypothe-size that, by analogy with chlamydospores of other fungi(Urbasch 1991; Armengol et al. 1999), the thick-walledM. aniso-pliae chlamydospores are a type of resting structure that cansurvive under suboptimal conditions and then renew the lifecycle under favorable conditions.

In our study, the first steps of infecting Rhipicephalus annu-latus eggs withM. anisopliaewere similar under both favorableand unfavorable conditions. The fungus developed only on vi-tal intact eggs. The eggs penetration and colonization startedon 2–3 d, when mortality in control eggs was insignificant atall regimes tested. The infection process of R. annulatus eggstreated by M. anisopliae are practically the same as those de-scribed for Rhipicephalus sanguineus eggs: spore attachmentto the egg surface, germination, formation of appressoriaand penetration into the egg (Garcia et al. 2005). At 27 !C, con-idia germination on R. annulatus egg surfaces started before18 h PI, and appressorium formation, penetration and coloni-zation of the egg vitellus started 24–72 h PI. The first penetra-tion pegs and fungal propagules were observed inside the eggs48 h PI. The germination ofM. anisopliae on the cuticle of adultBoophilus microplus ticks was detected within 24–48 h PI at28 !C (Arruda et al. 2005), and on R. sanguineus eggs withinabout 18 h PI at 27 !C (Garcia et al. 2005). Complete coloniza-tion of R. sanguineus eggs incubated at 80 % RH and 27 !Cwas observed within 4–5 d PI (Garcia et al. 2005).

Extrusion of M. anisopliae conidia on host cadavers undermoist conditions has been demonstrated for many insectand ticks stages, including eggs (Gindin et al. 2001; Ekesiet al. 2002; Sun et al. 2002; Garcia et al. 2005). The productionof M. anisopliae chlamydospores has only seldom been men-tioned with regard to coleopteran or lepidopteran insects’ lar-vae as hosts (Sewify & Hashem 2001; Zacharuk 1971).

M. anisopliae is one of the more promising candidates foruse in tick control (Kaaya et al. 1996; Samish et al. 2000; Polaret al. 2005). In general, ticks are controlled on or off their ver-tebrate hosts by scattering the antitick agent on the host oron the ground. The large egg mass of hard ticks is laid understones, in soil, under leaves. Such habitats, with their rela-tively high humidity, moderate temperatures, and minimalUV irradiation are often well suited for fungal development.The eggs of many tick species are highly susceptible to fungalinfection (Souza et al. 1999; Bittencourt et al. 2000; Gindin et al.2001; Onofre et al. 2001; Fernandes et al. 2004), and the damageto eggs caused by penetrating fungi is irreversible, thereforetreating the ground with the fungal conidia might be a meanto reduce the amount of viable tick eggs, and might stronglysuppress tick populations. An additional advantage of M. ani-sopliae for controlling tick eggs lies in its ability to producechlamydospores under stressful conditions and thereby toovercome ‘‘difficult’’ periods and so to widen the time‘‘window’’ for producing infective conidia.

Acknowledgements

We are grateful to Dr. Natalia Sheichat (Department of Pathol-ogy, Veterinary Institute) for helping in the preparation of his-tological sections and slides, to Dr. Vered Holdengraber

54 D. Ment et al.

(Department of Plant Pathology and Weed Research, ARO) forthe TEM sections and slides preparation and to Benni Ginz-burg for adapting the figures for print.

This publication was partially made possible through sup-port provided by the Office of the Environment, Science, andTechnology Counselor, Bureau for Global Programs, Field Sup-port & Research Center for Economic Growth, Washington,D.C., U.S. Agency for International Development, under theterm of Award No. TA-MOU-03-C22-008.

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