Introduction

Locust outbreaks have been documentedin Africa, North America, Asia, and Australia, and can be responsible for billions of dollars worth of agricultural losses (Prior et al. 1992). The oriental migratory locust Locusta migratoria, has been listed as one of the major pests of crops in China since 707 B.C. There is considerable interest in developing the entomopathogenic fungus Metarhizium anisopliae as an environmentally friendly alternative to chemical pesticides for the control of locusts (Zhang & Hunter 2005). Effective deployment of the fungus as a control agent will depend on a thorough understanding of the interactions between the fungal pathogen and its insect host, including the mechanisms of invasion and the development of the fungus within the host.

Structural studies of host-pathogen interactions have helped to improve our understanding of the invasion process of fungal pathogens, and helped in screening for more virulent strains. Entomopathogenic fungi penetrate the insect host's cuticle and grow rapidly in the host lymph, and the cuticle is therefore regarded as an important barrier to fungal infection (Leger 1991, Hajek & Leger 1994, Butt et al. 1995). Numerous studies have investigated the infection process demonstrated by the entomopathogenic fungus M. anisopliae, e.g., in vitro production of appressoria, and germination and prepenetration events on the host cuticle (Zacharuk 1974, Hajek & Leger 1994, Leger 1991, Butt et al. 1995, Leger et al. 1989). Several microscopic studies of M. anisopliae infection of Schistocerca gregaria have been carried out (Gunnarsson 1988), with a focus on the haemocytic reaction. Hajek and Leger (1994) reviewed the interactions between insect hosts and fungal pathogens.

However, no detailed ultrastructural study has yet examined the penetration process of the entomopathogenic fungus M. anisopliae. This study therefore focused on the penetration of the L. migratoria cuticle by M. anisopliae. The results may help to reveal the mechanisms behind M. anisopliae infection and its development within the layers of the insect cuticle, as well as providing a potential means of screening fungal strains.

Materials and Methods

Maintenance of insect and fungal cultures.— L. migratoria were reared in the Department of Entomology, China Agricultural University (Beijing) at a temperature of 28 to 30°C, a relative humidity of 60% and under a photoperiod of 18:6 h (light:dark). Fresh wheat shoots were provided daily. Fourth instar nymphs were used in the experiments.

The isolate M. anisopliae var. acridum was used in this study and was kindly supplied by Becker Underwood, Australia. The product was stored as spore powder at 4°C until use.

Inoculation.—Approximately 20,000 M. anisopliae spores were applied to each locust nymph by placing a 2-µ1 dose of 1 × 10⁸ conidia ml^-1 in 0.03% Tween 80, onto the dorsal part of the abdomen using a pipetteman. The insects were then transferred to an illumination box at 28°C under a 18:6-h photoperiod in humid conditions (>80%), and supplied daily with fresh wheat seedlings.

Scanning electron microscopy (SEM).—Fourth-instar larvae inoculated with M. anisopliae were prepared for the SEM at 1, 2, 3, 4, and 5 d post-inoculation. The insects were chilled on ice. The infected area of the abdomen was then excised using a scalpel blade and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer solution (PBS), pH 7.4, overnight, rinsed for 30 min in PBS (four changes), postfixed with 2% OsO₄ in 0.1 M PBS and dehydrated in an ethanol series. The specimens were critical-point dried, mounted on aluminum stubs and sputter-coated with gold/palladium (40:60) in a Polaron E5400 high-resolution sputter coater. The samples were examined using a Hitachi S570 SEM (Hitachi Ltd., Tokyo, Japan).

Transmission electron microscopy (TEM).—The cuticle from the dorsal part of the abdomen was excised and fixed at daily intervals after treatment (1, 2, 3, 4 and 5 d postinoculation), using the same process as for the SEM samples, with the addition of a final rinse with 100% acetone. The fixed cuticle was embedded in Epon 618 and 0.500 to 0.6 µm ultrathin sections were cut using a glass knife on an LKB V Ultramicrotome (LKB Company, Bromma, Sweden). The sections were stained with uranyl acetate and lead citrate in a LKB ultrastainer, mounted on Formvar-coated grids, and examined using a Hitachi H-7500 TEM (Hitachi Ltd).

Results

Attachment and germination of fungal conidia.— Conidia of M. anisopliae were capable of binding to any site on the locust cuticle, but were frequently trapped by the setae (Fig. 1a). Many conidia also attached to the intersegmental folds, particularly to the intersegmental membranes, or to the tergum and sternum. Secretion of adhesive mucus from the conidia and the hyphae on the cuticle surface could be observed as the conidia swelled during pregermination.

By one day after inoculation, the conidia had germinated and started to grow on the surface of the cuticle. Most conidia produced a single short germ tube from their tip or middle region (Fig. 1b, c), but a few exhibited bipolar germination. The mode of infection of M. anisopliae on the host cuticle appeared to consist of production of a germination tube by the conidia (Fig. 1c), followed by production of an appressorium (Fig. 1d). The appressorium varied in morphology, including cupped, clavate, curly and globose-shaped structures, and ranged in diameter from 2–7 µm. All the infection structures combined with the host cuticle without producing any melanotic lesions.

Penetration of the locust cuticle.—Cross-sections of uninfected locust cuticle showed that it consisted of several layers (Fig. 2a): the epicuticle, the procuticle comprised of the exocuticle and endocuticle, and the innermost epidermis. The epicuticle was very thin (0.1–3 µm) but stained very darkly. The exocuticle beneath the epicuticle was thicker (3–5 µm), while the innermost layer, the endocuticle, was even thicker (8–12 µm). The endocuticle was composed of lamellae, each comprising a mass of microfibers arranged in sheets.

Examination of the ultrastructure of the infected cuticle at 2 days postinoculation, showed that the fungus initially penetrated across the exocuticle and then developed between the layers of the endocuticle (Fig. 2 a, b).

Penetration of the epicuticle was achieved via a narrow peg produced by an appressorium (Fig. 1c, d). The penetrant structures subsequently reached the endocuticle, where they developed laterally between the sheets of endocuticle (Fig. 2a, b). The shape of the penetrant structures varied at this stage, but were mainly comprised of fusiform cells (Fig. 2a–d). The cells typically had a clear, large nucleus, with high densities of mitochondria and lysosomes. The cells had unseen or very thin cell walls, no vacuoles and no septa (Fig. 2d). Several layers of endocuticle surrounding the penetration structure were deformed, and parts of the layers directly contacting the penetrating structure were lysed, with serious infections also causing lysis of neighboring layers (Fig. 2a–c). The spaces around the penetrant structure were enlarged. Three days after inoculation, the penetrant structures had started to invade the epidermis, haemocoele and other tissues.

In summary, the ultrastructural events associated with M. anisopliae infection demonstrated several stages (Fig. 4). After landing on the host, the conidia started to penetrate the epicuticle by forming an appressorium (stage 1). Tube-like penetrant structures then directly invaded the epicuticle and exocuticle, but did not develop there (stage 2). On reaching the endocuticle, however, the fusiform penetrant structures developed laterally, between the procuticle layers. The layers close to the penetrant structures were lysed. The fungus was more active at this stage, having a larger nucleolus and more mitochondria and lysosomes, as well as more electron-dense cytoplasm (stage 3). The fungus then penetrated the endocuticle and developed within the haemocoele (stage 4). The form of the fungus varied at each stage of the penetration process.

Discussion

Insect pathogenic fungi such as M. anisopliae invade their hosts directly through the external skeleton or cuticle (Jarrold et al. 2007). The cuticle appears to influence the adhesion, germination and appressorium differentiation of the fungus (Butt 1990). This represents one of the four stages of fungal penetration of the cuticle noted in the current study; conidia were most often observed at the bases of hair sockets or on the intersegmental membranes. The exocuticle is more highly sclerotized than other layers of the cuticle, and is therefore harder and more rigid; it is absent in areas of the integument where flexibility is required, e.g., at joints and intersegmental membranes. These zones may be more suitable for attachment, recognition, germination and penetration of conidia.

However, the frequent penetration in these areas (Gunnarsson 1988, Vestergaard et al. 1999) may not be only a result of their structural weakness (Davies 1988); Wang and Leger (2005) found abundant appressorial formation by M. anisopliae conidia placed against plastic coated with locust-cuticle extract, indicating the importance of chemical factors in spore germination on the host cuticle. Pedrini et al. (2007) suggest that the surface structure, as well as the chemical composition of the cuticle, may be important. The cuticle contains numerous microenvironments within its folds and cleavages where humidity and temperature regimes may be more conducive to fungal germination and growth (Bidochka et al. 1997). The outermost epicuticular surface layer of the locust consists of complex polar and nonpolar lipids, and the fungus can use a wide range of aromatic organic compounds as nutrients during germination and other prepenetrant stages of growth (Jarrold et al. 2007). The absence of mechanical lesions on the cuticle suggests that the interaction between M. anisopliae and its host occurs via chemical dissolution of structures, ratherthan by purely mechanical breaching of tissues.

Activated fungal molecules can generally be classified as penetration effectors (toxins, enzymes) that reduce host defense, transporters that protect the fungus from host defenses, or components of signal transduction pathways that are required for fungal sensing of the host environment (Pedrini et al. 2007). However, host specificity is determined by the chemical signals utilized by the conidia to recognize their host and to promote germination on the host cuticle (Hall & Papierok 1982). Host specificity means that a fungus should have specific receptor systems to recognize their host's signals. The results of this study imply that the bases of hair sockets and the intersegmental membranes contained higher concentrations of the chemical signals recognized by the fungus.

Entomopathogenic fungi infect insects by breaching the host cuticle, which represents an important barrier to fungal infection (Leger 1991, Butt et al. 1995). The observations in this study showed that although the fungal tube-like penetrant structures directly penetrated the epicuticle and exocuticle, they never developed there, suggesting that locust epicuticle and exocuticle present more significant barriers to the fungus than endocuticle, even though this layer contains many chitin fiber sheets (Hajek & Leger 1994).

Once the fusiform penetrant structures reached the endocuticle, they then developed laterally between the layers, causing lysis of the layers close to the penetrating structures. Increased activity of the fungus at this stage was suggested by the presence of a larger nucleolus, more mitochondria and lysosomes, and more electrondense cytoplasm. The penetrant structures were able to separate the layers of the locust, and their lateral expansion, together with the holes made in the cuticle, may weaken cuticle integrity and so facilitate further penetration (Brey et al. 1986) and dispersal of the pathogen's cuticle-degrading enzymes (Goettel et al. 1989).

Fig. 1.

Conidia of M. anisopliae and their germ tube and appressorium on locust cuticle, observed by scanning electron microscopy; ultrastructure of conidium with germination tube observed by transmission electron microscopy, a. Conidia (C) of M. anisopliae with mucus surrounding the sensillum (S). Scale bar, 4 µm. b. Metarhizium is inserting into the cuticle (arrow). Scale bar, 4.5 µm. c. Germ tube (arrow) produced from the middle of a conidium. Scale bar, 2 µm. d. An appressorium (A) at the end of a short germ tube (G), C, conidium. Scale bar, 5 µm.

Observations in this study suggest that, once the fungus has penetrated the endocuticle, it is able to develop rapidly within the haemocoele, soon leading to the death of the host. Damage to host structures, as well as nutrient exhaustion and toxicosis, are all likely to be contributory factors to host mortality (Gillespie & Claydon 1989).

Entomopathogenic fungi invoke adaptive biochemical processes and cellular differentiation within their host, resulting in the formation of a series of specific morphological structures that favor fungal development (Hajek & Leger 1994). The current observations identified several ultrastructures during the four stages of M. anisopliae penetration of the locust cuticle: the germination tube and appressorium on the surface of the cuticle, the tube-like penetrant structure in the epicuticle and exocuticle, the fusiform penetrant structure in the endocuticle, and the hyphae in the haemocoele. It is possible that these forms of the penetrant structure of M. anisopliae may evolve from relationships between the pathogen and its host. Our study partially revealed ultratructural events of fungal pathogen-insect host interactions during cuticle penetration, that should help in screening for more virulent strains to control locusts. In our experimental results, we did find that the forms of the fungus in different layers of locust cuticle present differently.

Fig. 2.

The ultrastructure of M. anisopliae penetrant structures and locust cuticle during penetration process. a. Appearance in longitudinal section of penetrant strucuture of M. anisopliae in layers of locust cuticle, epi, epicuticle; exo, exocuticle; end, endocuticle; pns: longitudinal sections of fusiform penetration structures extend laterally between sheets of endocuticle. Scale bar, 5µm. b. Transverse section of the fusiform penetrant structures in locust cuticle. The penetrant structure had developed near to epidermis still in uninfected condition, pns, fusiform penetrant structure; exo, exocuticle; end, endocuticle. Scale bar, 5µm. c. The locust cuticle layers around penetrant structures were degraded. Scale bar 2µm. d. Ultrastructure of fusiform penetrant structure in cuticle, me, membrane of the cell; nu, nucleus; ly, lysosome; mt, mitochondrion. Scale bar, 1µm.

Fig. 3.

Schematic representation of infection structures of M. anisopliae and cuticular change in transverse section of locust cuticle. C, condium; A, appressorium; P, penetrant structure; H, hypha; epi, epicuticle; exo, exocuticle; end, endocuticle; m, epidermis.

We did not find the form taken by the fungus when penetrating epicuticle: it should be studied further.