Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin)

Journal of Invertebrate Pathology 109 (2012) 248–259

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Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin) D.M. Leemon a,⇑, N.N. Jonsson b a b

Agri-Science Queensland, DEEDI Ecosciences Precinct, GPO Box 267, Brisbane Qld 4001, Australia The University of Glasgow, College of Medical, Veterinary and Life Sciences, 464 Bearsden Rd., Bearsden G61 1QH, United Kingdom

a r t i c l e

i n f o

Article history: Received 5 September 2011 Accepted 1 December 2011 Available online 8 December 2011 Keywords: Lucilia cuprina Rhipicephalus microplus Metarhizium anisopliae Pathogenesis SEM Microscopy

a b s t r a c t Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods’ surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved.

1. Introduction Observations with in vitro assays analysed through probit analysis showed markedly different mortality patterns when isolates of Metarhizium anisopliae invaded ticks or blowflies (Leemon and Jonsson, in press). The invasion of ticks by fungi is strongly affected by the concentration of conidiospores in topically applied solutions. In contrast, blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of the concentration of conidia in the mix. These different patterns have implications for the development of fungal biopesticides for tick and blowfly control. The invasion of insects by M. anisopliae has been summarised by a number of authors (Butt, 1990; Boucias and Pendland, 1998; Butt et al., 2001) and has been thought to include the following components. Conidia attach to the host integument and, under suitable conditions, germinate to produce a germ tube and sometimes an appressorium. Penetration of the host integument occurs either directly via germ tubes or by penetration pegs produced from ⇑ Corresponding author. E-mail addresses: [email protected] (D.M. Leemon), nicholas. [email protected] (N.N. Jonsson).

appressoria. Penetration is effected through the action of a range of enzymes and mechanical pressure. Once inside the host, the fungus multiplies in the haemocoele as hyphal bodies, the yeast-like growth form. Death of the host follows after the production of toxins or exhaustion of nutrients, after which the fungus reverts to a mycelial phase to invade host organs. When the fungal mycelium has filled the host body cavity the hyphae will, under suitably moist conditions, penetrate from the interior to the exterior of the insect to produce new conidia. The interaction of host, pathogen, environment and time will influence how this general scheme of pathogenesis is expressed for any given host and pathogen resulting in the mortality patterns observed. Understanding the basis of the mortality pattern observed for ticks is critical for developing suitable formulation and application strategies for the in vivo use of M. anisopliae for tick control. Understanding why the time taken for blowflies to die when fed spores mixed with food does not seem to be directly influenced by dose is important for evaluating whether the control of adult blowflies with M. anisopliae is practical. The aim of this study was to compare the temporal and spatial progression of the invasion of the cattle ticks (Rhipicephalus microplus) and sheep blowflies (Lucilia cuprina) by M. anisopliae using light and scanning electron microscopy.

0022-2011/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.12.001

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2. Materials and methods 2.1. Outline Unengorged adult ticks were inoculated with two concentrations of conidia (5.0  106 and 5.0  107 conidia/ml) and ticks were processed for microscopy regularly over 102 h. One group of blowflies was exposed to conidia for 6 h while another group was continuously exposed to conidia in food. Flies were observed daily. Scanning electron microscopy was used to record the sequence of events on the surface of each host including the locations at which conidia adhered to the host surface, and where and when germination occurred. Stereo light microscopy showed any gross changes to the host over time while compound light microscopy revealed the progress of events inside each host. 2.2. Ticks and blowflies All ticks used (larvae, unengorged female adults and engorged female adults) were from the Yeerongpilly non-resistant field strain (NRFS) obtained from the Queensland Government Animal Research Institute (ARI), Yeerongpilly, Queensland. Adult female ticks are collected from the moated pens after dropping from the cattle. These ticks are incubated at 27 °C and 90% RH in batches of 50 for egg laying. Eggs are weighed into 0.25 g, 0.5 g or 1.0 g lots and incubated at 27 °C and 90% RH for approximately four weeks until they hatch into larvae. The larvae are then stored at the same temperature and humidity for a further 1–4 weeks until used to infest cattle. Small containers with 0.5 g (approximately 10,000) larval ticks are applied to the cattle in infestation collars. Approximately one week after infestation the larvae will have developed and moulted into nymphs. After a further week the nymphs will have moulted into young adults. The adult ticks spend a week on the cattle in which time the females will mate before engorging and dropping off the host animal. The developmental times can vary slightly with the season. Sheep blowflies (L. cuprina) were obtained from the Queensland Government laboratory susceptible reference colony maintained at ARI. Newly emerged adult flies are kept in cages (30  30  40 cm) and given a protein meal of fresh sheep or ox liver for 3 d. The liver is then removed and the flies are fed only sugar and water for the following 5 d. When fresh liver is again placed into the cages the female blowflies oviposit on it for 4 h, after which the eggs are removed manually and left on moist filter paper overnight to hatch. Next morning the first instar larvae are placed on fresh liver to develop through to third instar larvae over 3–4 d. After this time the third instar larvae will move off into clean sieved sand to pupate for 8–9 d before emerging as adults. 2.3. Fungal infection of ticks Two conidial suspensions were prepared by mixing conidia of M. anisopliae isolate ARIM16 into 0.1% Tween 80 to give approximately 5.0  106 and 5.0  107 conidia/ml. Unengorged female ticks (4–7 mm) were immersed in the different conidial suspensions for 1.5 min, blotted to remove excess liquid, placed individually in 24 well microtitre plates and incubated at 27 °C for up to 4 d. Microtitre trays were prepared with 1 ml water agar amended with 0.01% chloramphenicol per well. Control ticks immersed in 0.1% Tween 80 were incubated for 4 d. Treated ticks were removed at 6 h intervals for the first 24 h, then at 12 h intervals until 102 h, and fixed in a 3% glutaraldehyde 4% paraformaldehyde mix in 0.1 M sodium cacodylate buffer (pH 7.4). Ticks were stored in the fixative at 4 °C until further processing for

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either scanning electron microscopy or compound light microscopy. Additional ticks were prepared for stereo light microscopy. Female engorged ticks immersed in a suspension of ARIM16 conidia (8  107 conidia/ml), were incubated as above in microtitre trays. 2.4. Fungal infection of blowflies For scanning electron microscopy adult blowflies were fed conidia of isolate ARIM16 mixed with sugar (0.5 g conidia to 3.0 g sugar). Flies were incubated in round plastic takeaway containers (500 ml) with gauze inserts in the lid for aeration and water was provided in a serum tube with a wick. One group of flies was continuously exposed to the conidial food mix for 5 d. Another group was exposed to the conidia for 6 h then transferred to clean/fresh containers with only sugar and water. Both groups of flies were kept in an insectary at 27 °C and 72% RH with 16:8 h light: dark. Samples of flies exposed to the conidia for 6 h were removed daily from day two until day-5 and fixed. On day-5 the dead and live flies were fixed in separate groups. Blowfly fixation was as for ticks. Additional blowflies were prepared for light microscopy by feeding with ARIM16 conidia mixed into 80% sucrose solution (1  109 conidia/ml) dyed with either blue or red food dye. Flies were incubated in standard blowfly assay containers at 27 °C for up to four days. Two groups of 20 flies were removed each day, one group was fixed in 10% formalin, and the other group was frozen at 20 °C. 2.5. Scanning electron microscopy Fixed specimens were washed twice in 0.1 M sodium cacodylate buffer and post-fixed in 1% osmium tetroxide in cacodylate buffer for 20 min. Specimens were then washed three times in the cacodylate buffer and dehydrated through a graded ethanol series (70–100% ethanol) with 20 min at each step. Air-drying of specimens was preceded by two 10 min washes in hexamethyl disilazene (HMDS). Specimens were mounted on stubs using doublesided carbon tape before sputter coating in platinum for 200 s at 15 mA. Specimens were examined with either a JEOL 6300 or JEOL 6400 scanning electron microscope. 2.6. Light microscopy 2.6.1. Ticks Unengorged ticks were dehydrated in 80%, 90% and 100% ethanol, with three changes at 100% ethanol, cleared with three changes in xylene (1 h each step), then impregnated and embedded in paraffin wax. Sections were cut on a microtome then stained with Grocott’s Methenamime Silver (GMS) stain to highlight fungal tissues (Luna, 1968). Specimens were examined under a Motic BA200 compound microscope and photographed with a Moticam 2000 digital camera. The treated female engorged ticks were photographed with a NIKON Coolpix 995 camera mounted on a NIKON SMZ800 Stereomicroscope every 6 h for 5 d. 2.6.2. Blowflies Blowflies were dissected with the aid of a NIKON SMZ800 stereomicroscope and photographed with a NIKON Coolpix 995. Structures and tissues of interest were mounted in and stained with lactophenol cotton blue on glass slides. Slides were examined under an Olympus BH2 Compound microscope and photographed with a DP12 Olympus digital camera.

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3. Results 3.1. Ticks 3.1.1. Stereo light microscopy Treated ticks sampled regularly for 102 h showed the progression of the external symptoms of the fungal infection. Uninfected ticks have a translucent cuticle through which the movement of the contrasting pale malpighian tubules can be seen against the dark tick interior (Fig. 1a). As the fungal infection progressed the cuticle became dull, then developed opaque areas where it was also weak (Fig. 1b and c). Opaque areas were visible 42 h post-inoculation and by 48 h had covered both ventral and dorsal surfaces. By 48 h most ticks were dead. Tick death was considered to occur with the cessation of malpighian tubule movement. At

this stage the weakened cuticle was prone to bursting if handled, and in some cases, liquid leaked from opaque regions (Fig. 1b and c). Hyphae growing across the cuticle surface became visible to the naked eye 54 h post-inoculation (Fig. 1d). By 72 h post-inoculation the entire tick surface was covered in a white mycelium that continued to thicken (Fig. 1e) before sporodochia became visible, signalling the initiation of conidiogenesis at 96 h post-inoculation. Six hours later sporulation was obvious from the patches of green appearing across the sporodochia (Fig. 1f). 3.1.2. Compound light microscopy Compound light microscopy (LM) revealed the internal progress of the Metarhizium infection in ticks. In the sectioned tick material the fungal tissues appear black as a result of silver molecules

M

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 1. (a) Control tick (M = malpighian tubules). (b) Dorsal surface 48 h post-inoculation showing fluid. (c) Higher magnification of dorsal surface 48 h post-inoculation (arrow). (d) Higher magnification of hyphal growth 54 h post-inoculation. (e) Mycelium on ventral surface 78 h post-inoculation. (f) Areas of green indicating sporulation 102 h post-inoculation.

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(GMS) binding to unique fungal cell wall components (Luna, 1968). Although ticks were treated with two different concentrations of conidia (5.0  106 and 5.0  107 conidia/ml), there was no discernible difference seen in the histology, thus sections from the two groups are not presented separately. In sections from ticks samples 6 h post-inoculation, conidia adhering to the tick surface are visible (Fig. 2a), and the lamellate inner endocuticle and non-lamellate outer endocuticle regions of the tick alloscutum (Hackman and Filshie, 1983) are discernible. Conidia germinating on the tick surface were evident in sections taken 12 h post-inoculation and at 24 h post-inoculation fungal hyphae had penetrated the cuticle (Fig. 2b), the region around penetrating hyphae staining lighter than surrounding tissues. Hyphae were also seen growing extending into a canal in the tick cuticle 24 h post-inoculation.

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At 36 h post-inoculation fungal hyphae are well established in the outer endocuticle (Fig. 2c). By 48 h post-inoculation the fungal growth has destroyed the integrity of the outer endocuticle and penetrated into tissues beyond the cuticle (Fig. 2d). At 60 h postinoculation the simultaneous destruction of the outer endocuticle and penetration of the inner tissues by fungal hyphae is well developed (Fig. 2e). Areas of tick cuticle with widespread hyphal growth in the outer endocuticle but limited growth in the inner endocuticle are visible 72 h post-inoculation (Fig. 2f); other areas of cuticle have extensive hyphal growth on the exterior. Different patterns of fungal growth are visible in tick sections 80 h post-inoculation; in some areas hyphae have grown right through the internal tissues while in other areas the hyphal growth is mostly confined to the cuticle and immediate underlying tissues. These patterns are also visible in sections from ticks sampled 96 h post-inoculation. For

Ep

OE

IE

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 2. (a) Conidia visible on cuticle 6 h post-inoculation (arrowed), the lamellate inner endocuticle (IE) and the non-lamellate outer endocuticle (OE) are visible below the epicuticle (Ep). (b) Penetration into the endocuticle 24 h post-inoculation, zone of lighter staining tissues around penetrating hypha (arrowed). (c) Fungal growth in the cuticle 36 h post-inoculation (400). (d) Cuticle destruction and penetration beyond the cuticle 48 h post-inoculation (400). (e) 60 h – Destruction of cuticle with extensive fungal growth in the endocuticle and proliferation through tissues. (f) 72 h – Extensive hyphal growth in and on cuticle.

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both patterns the exterior tick cuticle supported a thick layer of fungal hyphae. 3.1.3. Scanning electron microscopy (SEM) The proliferation of fungal hyphae across the surface of infected ticks was clearly visible with SEM. As in the compound light microscopy investigation, two groups of ticks were inoculated with different concentrations of conidia. However results are treated together because there was no evident difference between the two conidial concentrations seen under the scanning electron microscope. When ticks were immersed in a suspension of conidia the conidia readily adhered to the surface (Fig. 3a) and were distributed across ventral and dorsal tick surfaces. Although conidia are unevenly distributed there does not appear to be any clearly pre-

ferred part of the tick surface for conidial adhesion. By 6 h conidia had commenced germination and 12 h post-immersion many conidia had germinated (Fig. 3b). By this time some germ tubes appeared to be directly penetrating the tick surface while others showed directional growth towards pores associated with the setae on the tick surface. Some conidia had produced shorter germ tubes ending in appressoria (Fig. 3b). Twenty-four hours post-inoculation the hyphal growth on the tick surface had developed a variety of hyphal formations (Fig. 3c). Hyphae had begun to anastomose to form groupings surrounded by extracellular mucilage (ECM). The tick cuticle under the extracellular mucilage surrounding the fungal hyphae appeared to be eroded in some parts. By 36 h post-inoculation clusters of anastomosing hyphae had developed on the surface with hyphae growing out to either directly penetrate the tick surface

A C C A

(a)

(b)

(c)

(d)

(e)

(f)

ECM

Fig. 3. (a) Conidia on tick surface immediately after immersion in conidial suspension. (b) 12 h post-immersion germination of conidia and germ tube penetration of ticks surface and directional growth. A = appressorium; C = conidium. (c) 24 h post-immersion ECM = extracellular mucilage. (d) 48 h post-immersion (e) Hyphae growing through fungal sheets around and over a tick seta 60 h post-immersion. (f) Hyphae growing through a large crystal (9–10 lm) of calcium oxalate.; S = seta.

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or follow the topography into the tick via a pore canal. By 48 h post-inoculation there were hyphae embedded in large fungal sheets that covered areas of the tick surface and setae (Fig. 3d). By 60 h post-inoculation these sheets had become more extensive and the hyphal network denser, obscuring large sections of the tick surface (Fig. 3d and e). The first signs of conidiogenesis were seen 60 h post-inoculation. From 60 h post-inoculation crystals of calcium oxalate di-hydrate (weddelite) with their characteristic bipyramidal shape began to appear and with time both their prevalence and size increased (Fig. 3f). By 72 h post-inoculation some areas of the tick surface were covered in a solid mass of hyphae embedded in fungal sheets and by 84 h post-inoculation other areas of the tick surface supported dense hyphal networks littered with crystals. After a further 12 h a second type of crystal, looking like stacks of disc-shaped plates, became visible amongst the large (>30 lm) calcium oxalate crystals and some hyphae were encrusted with crystals. Ninety-six hours after inoculation well developed conidiophores and conidia were abundant. The identity of the crystals was established using the energy dispersive X-ray spectroscopy capability of the JEOL 6400 scanning electron microscope. Characteristic radiation in the X-ray backscatter from the crystals was used for compositional analysis, which indicated that the crystals were mostly composed of calcium and oxygen. The CRC Handbook of Chemistry and Physics (2004) was consulted to confirm that the crystals were calcium oxalate (Lide, 2004). 3.2. Blowflies 3.2.1. Scanning electron microscopy SEM was used to determine where conidia were adhering to the surface of blowflies and where germination and invasion were occurring in blowflies infected by M. anisopliae. After 6 h of exposure conidia were seen adhering to various parts of the fly surface, especially in regions such as the legs, abdomen and wings (Fig. 4a). However few conidia could be seen around the mouthparts (Fig. 4b). Blowflies exposed to conidia for 5 d did not appear to have more conidia adhering to their surfaces compared to flies exposed only for 6 h (Fig. 4f and a respectively). Large masses of conidia embedded in a matrix material were visible around the anal region of flies sampled after five days continuous exposure to conidia. There did not appear to be much variation in the number and distribution of conidia adhering to the surface of flies, sampled on successive days. Despite comprehensive examination of the entire surface of all flies exposed to conidia, only one germinating conidium was found on a fly sampled 4 d post-exposure. The conidium, with a short germ tube ending in an appressorium, was attached within the mouthparts at the entry to the buccal cavity (Fig. 4b and c). No conidia were visible on the surrounding outer mouthparts. This blowfly also had a large number of un-germinated conidia on various other parts of its surface. By 5 d post-exposure to conidia most blowflies had died and the dead and remaining live flies were sampled for SEM examination. The pattern of conidial attachment on the surfaces of both groups of flies was similar to that observed on all flies sampled after exposure to conidia in food. Conidia adhered to many areas of the blowflies but particularly to areas with lots of hairs such as around the legs and feet (Fig. 4d and e). 3.2.2. Light microscopy Blowflies were exposed to conidia mixed into liquid sugar stained with food dye to highlight the crop and digestive system. Flies were sampled daily until most of the flies had died (day four). The external appearance of dead infected flies was no different to uninfected control flies (Fig. 5a). However, hyphae growing

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through the internal tissues of dead infected flies were readily visible upon dissection and a thick hyphal growth emerged from dead infected flies within 30 h when surface sterilised, plated on water agar and incubated at 25 °C (Fig. 5b). Flies preserved in formalin were dissected and selective tissue samples taken for LM examination rather than sectioning flies embedded in resin. The SEM studies suggested that fungal infection was likely to be initiated in restricted areas. When a crop full of dyed sugar solution was dissected, dark areas with large numbers of Metarhizium conidia were seen. The inner crop surface was examined for germinating conidia, but none were found. However in some flies that had died after four or five days exposure to conidia in sugar the crop appeared to have broken open or disintegrated. The mouthparts of blowflies that had been exposed to conidia in food for two, three and four days were removed and the interior tissues examined. A large number of conidia were visible on the internal surface of mouthparts from flies that had been exposed to conidia in food for two days (Fig. 5c). Extensive hyphal growth was seen around the mouthparts of flies dead after four days of exposure to conidia in food. Hyphae were visible growing out from the tissues around the labellum and across the internal surface of the labellum (Fig. 5d and e). In addition large numbers of hyphal bodies were visible in the haemolymph bathing tissues around the mouthparts, in the head and in the upper areas of the thorax (Fig. 5f, top). The hyphal bodies appeared to be more common around the mouthparts, decreasing in concentration in tissues further away from this area. No hyphal bodies were seen in haemolymph removed from the lower thorax or abdomen. Oxalate crystals were present in the haemolymph of flies dead after four days exposure to conidia. The crystals varied in shape with single crystal and druse-like crystal aggregates (Fig. 5f, bottom). Crystals of calcium oxalate dihydrogen with their characteristic bi-pyramidal shape were the most common. 4. Discussion The pattern of invasion by M. anisopliae seen in ticks and blowflies is markedly different. Conidia adhering to the surface of ticks immersed in a conidial suspension germinated within 12 h, and soon after, hyphae began penetrating the tick cuticle while other hyphae grew across the surface. Forty-eight hours post-inoculation, ticks were dead and there was clear evidence of severe degradation of the cuticle followed by the proliferation of hyphae throughout the internal tissues of ticks. The invasion of ticks by M. anisopliae was characterised by simultaneous surface and internal hyphal growth causing rapid death. Blowflies exposed to conidia mixed with food became contaminated with large numbers of conidia adhering across the body surface. However, evidence of conidia germinating on any exterior surface could not be found with scanning electron microscopy. Only one germinating conidium could be found within the entry to the buccal cavity. The light microscopy findings suggest the invasion of adult blowflies by M. anisopliae begins in the mouthparts, penetrates into the adjacent tissues, and spreads throughout the rest of the body via hyphal bodies in the haemolymph. There is no visible hyphal growth on the exterior of the blowflies until they are exposed to moist conditions post-death. 4.1. Ticks The process occurring during the rapid invasion and destruction of R. microplus adults by M. anisopliae is revealed with scanning electron microscopy together with stereo and compound light microscopy. In this study conidia appeared to adhere easily to

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(b)

(a) A

GT

C

(d)

(c)

(f)

(e)

Fig. 4. (a) Clusters of conidia adhering to a blowfly wing surface of blowflies after 6 h exposure to conidia (b and c) Surface of blowfly 4 d post-exposure to conidia. (b) Mouthparts with the only germinating conidium found (arrow). (c) Higher magnification of (b) showing location of germinating conidium. C = conidium, GT = germ tube, A = Appressorium. (d) Blowfly tarsus of dead blowfly 5 d post-exposure to conidia with conidia caught in crevices and between hairs (arrows). (e) Higher magnification of (d) showing conidia adhering to hairs. (f) Conidia on wing of dead blowfly after 5 d of continuous exposure to conidia.

the tick surface with no preferred sites for adhesion. Rapid germination was seen, with a germ tube initiating from at least one conidium 6 h post-inoculation and many conidia with well developed germ tubes 12 h post-inoculation (Fig. 3b). Some germ tubes seem to directly penetrate the cuticle, some grow into pore canals or into the base of setae, while others end in appressoria. Occasionally separate germ tubes grow together to form the complex appressoria reported in previous studies (Leemon and Jonsson, 2008; Vestergaard et al., 1999). Light microscopy studies confirmed that by 24 h post-inoculation hyphae had penetrated the surface and were established in the outer endocuticle (Fig. 2b). Hyphae were also seen entering the tick through a pore canal. By 48 h post-inoculation ticks were dead, with fluids leaking from discoloured and weakened cuticles (Fig. 1b and c). Extensive degradation

of the cuticle, particularly the outer endocuticle, is apparent at this stage (Fig. 2d) and well developed hyphal growth is evident on the tick surface (Fig. 3d). The development of a thick mycelial growth on the surface of ticks is synchronous with the internal invasion and development of hyphae. In some LM sections the internal hyphal growth appeared to be restricted to the cuticle (Fig. 2f), while in other sections hyphae had proliferated throughout the tissues. However, this may have been an artifact of section location. While the invasion described in this work is much faster than that described previously for M. anisopliae invading R. microplus (Arruda et al., 2005; Bittencourt et al., 1999), there are a number of features in common. Similarly, in studies with both M. anisopliae and Beauveria bassiana infecting other species of ticks and acarines (Garcia et al., 2008, 2004, 2005; Kirkland et al., 2004a, 2004b;

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(a)

(b)

(c)

(d)

~17 µm

~5 µm

(e)

(f)

Fig. 5. (a) Infected fly dead after 4 d exposure to conidia in food (5). (b) Fungal hyphae growing from a dead blowfly 30 h after surface sterilisation and plating on agar (3). (c) Conidia on internal surface of mouthparts after 2 d exposure to conidia in food (400). (d) Inner surface of labellum with hyphae on tissues and hyphal bodies washed out of tissues (200). (e) Hyphae in tissues at the top of the labellum (400). (f)Top: Hyphal bodies from fluids in head and upper thorax (right 400, left 600) Bottom: Oxalate crystals from tissues of a blowfly dead after 4 d exposure to conidia in food. Slides stained with lactophenol cotton blue.

Maketon et al., 2008) there are common findings. All of these SEM studies show conidia adhering to the tick surface and germinating, and hyphae proliferating across the tick surface. Bittencourt et al. (1999) concluded that penetration of the tick was primarily through the cuticle and showed germinating conidia producing both germ tubes and appressoria. Arruda et al. (2005) also showed germinating conidia with germ tubes of varying lengths ending in appressoria. In addition they showed the undersurface of the cuticle with well developed penetrating hyphae 24 h post-infection. Gunnarsson (1988) reported a similar time frame for the invasion of Schistocerca gregaria by M. anisopliae to that described in the present study. Other SEM studies have also recorded very similar events on the surface of insects being invaded by M. anisopliae as those described above. Schabel (1978) described the extensive growth of germ tubes and hyphae across the surface of larval Pales

weevils (Hylobius pales) being invaded by M. anisopliae. He also observed that, when germ tubes and hyphae had reached other hyphae and appressoria, they fused with them producing larger hyphal aggregates. Schabel (1978) suggested the fungus may thus enhance its invasive properties by mass action. Extracellular material or mucilage (ECM) was commonly seen around appressoria and hyphae, and as large sheets in which hyphae are embedded in a number of scanning electron micrographs (Fig. 3c–e). ECM is a common feature of many fungi with important roles in adhesion, hydration and enzymic digestion (Abu et al., 1999; Boucias and Pendland, 1991; Jones, 1994; Nicholson and Epstein, 1991). Abu et al. (1999) used SEM to study the extracellular mucilaginous materials produced by wood decay fungi. They noted that the morphological appearance of the mucilage depends on the preparatory procedures used for microscopic examination, which

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may account for these materials being variously named as extracellular matrix, mucin, sheath or extracellular membranous structures. Fungi from various taxonomic and ecological groups have been reported to produce extracellular mucilage. Many fungal hyphae are extensively coated in this mucilage enabling adhesion to surfaces as well as other hyphae (Jones, 1994). The mucilage consists of organic polymers such as carbohydrates, proteins and lipids exuded by the fungi, but the composition varies among fungi (Cooper et al., 2000; Jones, 1994). Daniel (1994) suggested that the ECM associated with wood degrading basidiomycetes plays an important role in the decay process and may explain why lignin and polysaccharide degradation can occur at a distance from the invading hyphae. Abu et al. (1999) extended this idea by postulating that the ECM plays a key role in acting both as a local and a ‘diffuse’ transfer medium for decay agents and decomposition products between hyphae and wood cell walls. They also suggested ECM might be involved in important regulatory functions (e.g. pH, dissolved O2) for the extra-hyphal environment, which is necessary for the successful functioning of complex biochemical breakdown systems. St. Leger et al. (1996) observed an extracellular mucilaginous sheath around the hyphae of M. anisopliae adhering to the insect cuticle. They suggested that the mucilage could have a role in the support and transport of cuticle-degrading enzymes to their target. Boucias and Pendland (1991) noted that many arthropod pathogenic fungi actively secrete mucilage during the formation of germ tubes and appressoria to bind these structures to the cuticle. Schabel (1978), Madelin et al. (1967) and Zacharuk (1970a, 1970b) all describe mucilaginous sheaths around hyphae and appressoria of M. anisopliae invading insects. The integument of ticks, although similar to that described for insects in the general sense (Chapman, 1969), has some important differences, particularly in the structure and development of the cuticle of the alloscutum of the female ixodid ticks (Hackman and Filshie, 1983). R. microplus is referred to as a hard tick (Ixodidae) because it has a sclerotised capitulum, appendages, scutum and other small areas. An unsclerotised alloscutum covers most of the surface of female nymphs and adult ticks and affords an enormous capacity for growth, expansion and stretching during engorgement of blood by the female adult. Therefore the alloscutum of adult R. microplus lacks the pigmented and sclerotized outer exocuticle layer of the insect procuticle. Hackman and Filshie (1983) noted that two distinct layers of endocuticle are visible beneath the epicuticle in both histology and transmission electron microscopy sections; a non-lamellate outer endocuticle and a lamellate inner endocuticle. The outer layer is composed almost entirely of amorphous material that is continuous with the inner layer. They also noted the pore canal system traversing the endocuticle layers seems to be a far more elaborate network than that of insects. Two distinct layers of endocuticle, one apparently lamellate, are visible in histology sections in this study (Fig. 2a). Another major difference between ticks and insects is the composition of the cuticle. Chitin and protein account for more than 95% of the dry weight of non-sclerotized tick cuticle. The remaining few percent is largely made up of lipid which functions to control the movement of water through the cuticle (Hackman and Filshie, 1983). The proportion of chitin is much lower than that in insects. Basal and Hefnawy (1972) reported the cuticle of unfed Hyalomma dromedarii ticks contained 11.6% chitin which dropped to 3.8% after engorgement. Hackman (1974) also found the dry lipid-free alloscutum cuticle of the fully fed female R. microplus contains 3.8% chitin. In contrast, Charnley (1989) noted that chitin can constitute 17–50% of the dry weight of insect cuticle; more pliant cuticles have higher chitin content than stiff cuticles. The larval cuticle of Calliphora vicina contains 44.2% chitin (Hackman and Goldberg, 1977). Chitin fibrils in insects are laid down parallel to the cuticular surface and present a barrier to penetration by entomopatho-

genic fungi (Charnley, 1989). The proteins in the tick alloscutum also differ markedly from those in non-sclerotized cuticles of other arthropods. Most of this protein (94%) is not covalently bound to other cuticular components such as chitin (Hackman, 1974, 1975; Hackman and Goldberg, 1977). Moreover, R. microplus protein has a different amino acid composition, higher alanine and histidine with lower aspartic acid and glutamic acid, compared to the proteins in non-sclerotized cuticle of other arthropods. Hackman and Filshie (1983) suggest that the difference in cuticle proteins is probably related to the ability of the tick cuticle to expand rapidly during feeding when the epicuticle stretches. Furthermore they speculated that these proteins probably do not have a periodic secondary structure to impede the two-dimensional expansion of the cuticle. Hackman and Filshie (1983) suggested that the non-lamellate layer of the outer endocuticle layer is mostly composed of protein while the lamellate inner endocuticle has chitin fibrils embedded in the protein matrix. It could be expected that a cuticle with such a structure and composition will be readily invaded by a fungal pathogen secreting large amounts of proteases. M. anisopliae has been reported to produce of a range of proteases which are critical to the invasion of insect cuticle (Goettel et al., 1989; St. Leger et al., 1986). Goettel et al. (1989) showed that M. anisopliae secretes protease into the cuticle from its infection structures. Initially the proteases are confined to the immediate vicinity of germ tubes, appressoria and invading hyphae, but diffuse throughout the cuticle during the later stages of pathogenesis. St. Leger et al. (1986) suggested that the digestion of protein by these proteases might loosen lamellae in the cuticle, allowing an easier mechanical passage through the insect cuticle. The rapid weakening and destruction of the tick cuticle (Fig. 1b and c) is most likely a result of the proteases secreted by M. anisopliae digesting the proteinaceous layers of a cuticle low in chitin or other components. This conclusion is supported by the histological sections showing extensive degradation of the protein rich outer layer of the endocuticle (Fig. 2d–f). The structure and composition of the R. microplus cuticle that allows for rapid expansion during engorgement may also make it extremely susceptible to attack from fungal pathogens such as M. ansiopliae that secrete large amounts of protease. All layers from the epidermis to the epicuticle of the tick play a role in defence against water loss to the environment (Hackman and Filshie, 1983). A consequence of extensive fungal degradation of the tick cuticle as seen in this study could be rapid water loss hastening death. 4.2. Blowflies Previous studies (unpublished) with blowflies fed M. anisopliae conidia mixed with food showed a rapid onset of mortality after an initial delay of 3–4 d regardless of conidial concentration. Earlier experiments carried out in the same laboratory showed that a mortality pattern similar to this occurred irrespective of whether blowflies were exposed to conidia for 4 h, 24 h or 5 d. Blowflies exposed to conidia of the most virulent M. anisopliae isolates started dying 3 d after the time of first exposure and the majority of flies were dead by 5 d. Examination of the dead blowflies with stereo light microscopy showed that external hyphae only appeared if the flies were exposed to moisture post-mortem (Fig. 5a and b). Scanning electron microscopy revealed many conidia adhering over the surface of blowflies up to 4 or 5 d after the source of the conidia had been removed (Fig. 4a, d–f). The numerous conidia seen adhering to body surfaces including wings, setae, legs, tarsi and eyes suggest a strong adherence of M. anisopliae conidia to blowfly integument. However, few conidia were seen adhering to the outer mouthparts. No germinating conidia were recorded on the external surfaces of

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the blowflies. It is possible that the conidia were unable to germinate due to inadequate moisture or fungitoxic agents on the exterior surface of blowflies. Schabel (1978) investigated the invasion of the Pales weevil (Hylobius pales) by M. anisopliae and noted low conidial germination (<10%) on the unsterile meta wings. However, if the wings were sterilised or put onto water agar, approximately 85% of conidia germinated. Schabel (1978) inferred that the unsterile wings had fungitoxic agents that could have dissolved away into the agar. Sosa-Gomez et al. (1997) also reported on the fungistatic effect of cuticular lipids and aldehydes on the large numbers of M. anisopliae conidia adhering to the surface of the Southern green stink bug (Nezara viridula). Only one germinating conidium was seen inside the buccal cavity with scanning electron microscopy (Fig. 4b and c). Evidence from light microscopy also suggested that conidia were germinating and invading blowflies via the buccal cavity (Fig. 4c–e). Veen (1966) noted that invasion through the buccal cavity commonly occurred in locusts (S. gregaria) inoculated per os. Other investigators also reported M. anisopliae invading insects through the buccal cavity (Dillon and Charnley, 1986; Schabel, 1976). Both reports noted that they did not find any evidence of germination and penetration through the intestinal tract. However, Schabel (1976) found that conidia remained viable after passing through the gut of H. pales, and conidia mixed with gut fluid were able to germinate in vitro despite the presence of yeasts and bacteria. Allee et al. (1990) found that conidia of B. bassiana were able to germinate in the gut of Leptinotarsa decemlineata regardless of the presence of gut microflora, although they only observed infection via the alimentary tract in one starved individual. The investigations in the present study were not exhaustive enough to discount the possibility of invasion of blowflies via the alimentary tract, but the evidence does support some invasion through the buccal cavity. The presence of hyphal bodies in haemolymph removed from the head and upper thorax, but absent from haemolymph removed from the lower thorax and abdomen also suggests that M. anisopliae invaded from the anterior end of blowflies. The scenario of M. anisopliae invasion of blowflies through the buccal cavity could be expected to involve the adhesion of conidia to buccal cavity surfaces followed by germination and penetration of hyphae through the tissues then by the growth and dissemination of hyphal bodies throughout the haemolymph until death from either toxicosis, organ invasion or nutrient depletion. This scenario is consistent with a virulence mechanism involving an incubation period or lag period before death of the host regardless of dose. 4.3. Oxalate Calcium oxalate crystals were found on and around hyphae on the surface of ticks and in the haemolymph of blowflies infected with M. anisopliae. This is the first report of calcium oxalate crystals associated with M. anisopliae invading and killing arthropod hosts. Arnott (1995) noted that calcium oxalate crystals are commonly found associated with fungi and went onto quote the great mycologist Anton de Bary who in 1887 stated ‘‘that calcium oxalate is a substance so generally found in the fungi that it is quite unnecessary to enumerate instances of its occurrence’’. De Bary was primarily referring to observations with basidiomycetous fungi, however, in more recent times there have been many reports of calcium oxalate crystals associated with the production of oxalic acid by fungi from different ecophysical groups including phytopathogens (Bateman and Beer, 1965; Noyes and Hancock, 1981; Pulim et al., 1994; Rao and Tewari, 1987; Stone and Armentrout, 1985), wood decay fungi (Abu et al., 1999; Shimada et al., 1997), lichens (Jackson, 1981) and mycorrhizae (Mahmood et al., 2001; Wallander et al., 2002). Oxalate crystals have also been reported

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on the cadavers of insects killed by fungi (Dresner, 1950; Moino et al., 2002). Bidochka and Khachatourians (1991) explored the role of metabolic acids such as oxalic acid in the pathogenesis of the migratory locust (Melanoplus sanguinipes) by B. bassiana, while Bidochka and Khachatourians (1993) and Kirkland et al., 2005 investigated the effect of oxalic acid secretion on B. bassiana virulence. Bidochka and Khachatourians (1993) tested B. bassiana mutants selected for oxalic acid hyperproduction against the migratory grasshopper. Although one mutant strain caused a significantly lower LT50 than either the other mutants or the wild strain, they concluded that oxalic acid hyperproduction only occurred in media with certain carbohydrates and was not related to virulence in grasshoppers. However, Kirkland et al. (2005) presented evidence to support their hypothesis that oxalic acid secretion by B. bassiana, coupled to a reduction in the pH of the medium, acted as a potent acaricidal factor during pathogenesis of three tick species: Ambyloma americanum, A. maculatum and Ixodes scapularis. Bidochka and Khachatourians (1991) found that citric and oxalic acids acted synergistically with B. bassiana to cause mortality in the migratory locust M. sanguinipes. They speculated that the oxalic acid produced by B. bassiana bound calcium in the cuticle making the cuticle more amenable to hydrolysis by B. bassiana proteases. Bateman and Beer (1965) showed that with plant pathogens, oxalic acid binds calcium in the pectates of the bean cell wall making the substrate more easily hydrolysed by polygalacturonase. A general role postulated for oxalic acid production by fungi is the binding of divalent cations such as metal ions to protect fungal extracellular enzymes (Sayer and Gadd, 1997). While many metal ions are essential for fungal growth and metabolism, in excess they exert toxicity in a number of ways including blocking the functional groups of extracellular enzymes (Ramsay et al., 1997). Connolly et al. (1999) observed that the formation of biominerals such as calcium oxalate is commonly observed in fungal mucilage and has been suggested as a method by which fungi regulate the external calcium concentration. Arnott (1995) noted that, although not all fungi produce calcium oxalate crystals or even oxalic acid, calcium oxalate crystals can be expected in the Basidiomycetes and Mucorales. He further suggested that the very frequency of calcium oxalate’s association with fungi makes calcium oxalate significant and it is likely that the fabrication of this compound provides a selective advantage to organisms that produce it. 4.4. Comparative mechanisms Roberts (1981) outlined nine steps critical steps in the development of fungal disease in insect hosts. Initially, the attachment of the infective unit (conidium) to the insect epicuticle, followed by the germination of the infective unit on the cuticle and penetration of the cuticle, either directly by germ tubes or by infection pegs from appressoria. Once inside the host the fungus multiplies in the haemocoel in the yeast phase producing hyphal bodies. The production of toxic metabolites leads to the death of the host which is followed by mycelial growth with invasion of virtually all host organs. Finally hyphae penetrate from the interior through the cuticle to the exterior of the insect and produce infective units (sporulation) on the exterior of the insect. The development of disease in R. microplus caused by M. anisopliae is clearly different from that noted above. After the germination and penetration of the cuticle there is extensive degradation of the outer cuticle and simultaneous mycelial growth across the tick surface and into the tissues beneath the cuticle. There was no evidence of hyphal bodies and sporulation is initiated on the surface while mycelial growth continues throughout the internal tissues. It is possible that the different structure and composition of the tick cuticle from that of an insect results in such a markedly

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different pattern of invasion. Previous histological and SEM studies also show the simultaneous surface and interior growth of M. anisopliae mycelium on R. microplus and other ticks (Arruda et al., 2005; Garcia et al., 2004). However, their isolates of M. anisopliae appeared to take longer to kill ticks than the isolates used in the present study. The development of disease in blowflies is more consistent with the outline proposed by Roberts (1981) above, however penetration does not appear to be initiated through external cuticle, rather through cuticle lining the buccal cavity. There was also evidence of large numbers of hyphal bodies in the haemocoel at the time of death. The mycelium appeared to proliferate through the internal tissues, especially muscle, post-death. Hyphae did not penetrate from the interior to the exterior until mycosed blowfly cadavers were exposed to moist conditions (Fig. 5b). Sporulation only commenced after the blowfly surface was enveloped in thick mycelial covering. The rapid death of ticks invaded by M. anisopliae appears most likely related to the degradation of the outer tick cuticle. This unique invasion mechanism suggests that the rapid death of ticks on animals might be achieved if enough conidia of M. anisopliae can be applied to ticks on animals under optimal environmental conditions. However, the investigations into the invasion of blowflies suggest that the most likely route of entry of M. anisopliae conidia is through the buccal cavity. In addition, (the results from previous studies Leemon and Jonsson, in press) indicate that there will be a minimum time to death irrespective of dose. An attractant trap baited with a mixture of conidia and food would provide the best method of applying conidia to adult blowflies. Using conidia in an attractant trap would have disadvantages over the conventional chemicals previously used in such traps. Fungal spores in an outdoor trap under ambient conditions of temperature and humidity have the potential to rapidly lose viability, especially in hot weather. The time lag from conidial uptake to death means flies would probably die in the traps from the effects of heat and dehydration before the fungus kills them. Even if a spray application were practical, the time lag to death opens the possibility of flies having time to initiate strike before they die. Therefore further testing of M. anisopliae for the control of adult L. cuprina does not seem warranted. 5. Conclusion The morphological investigations in the study support the differences in virulence mechanisms for ticks and blowflies invaded by M. anisopliae proposed previously (Leemon and Jonsson, in press). Stark differences were observed in both the temporal and spatial aspects of invasion of adult ticks and adult blowflies by M. anisopliae. The invasion of blowflies through the buccal cavity appeared to cause a time lag until death, regardless of conidial dose. The direct invasion of M. anisopliae through the integument of ticks with the associated destruction of the endocuticle resulted in a more rapid death influenced by conidial concentration. These differences will have an influence on the decision to further investigate M. anisopliae as a potential biocontrol agent for ticks or adult blowflies. Acknowledgments Mr. Geoff Brown, for providing blowflies from the ARI blowfly colony. Mr. Peter Green and Mr. Ralph Stutchbury, for providing ticks from the ARI tick culture. UQ Centre for Microscopy and Microanalysis staff, for advice when needed.

Mr. Mo Amigh, Yeerongpilly Veterinary Laboratory for assistance with tick histology.

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