Jacquelyn C. Pendland

Attachment of Mycopathogens to Cuticle

Representative members of all major fungal groups have evolved mechanisms required for infection and subsequent development within invertebrates (for review see McCoy et al., 1988). To date, over 700 species within 90 different genera have been determined to be pathogenic to insect and mite hosts (Roberts and Humber, 1981; Samson et al., 1988). In the majority of cases, the spore stage represents the infectious propagule which attaches to and actively gains entry through the exoskeleton by producing penetrant hyphae (Charnley, 1984; see Chapter 6). In nature, the success of an invertebrate mycopathogen will be determined by its ability to produce and deliver infectious propagules to susceptible host systems. In turn, successful delivery will depend on the particular spore-bearing structure and the physical and chemical properties of the infectious propagule. In part, the array of spores and spore structures observed among the pathogenic fungi reflects their adaptation to the diversity of both the target hosts and their respective habitat.

Ultrastructural studies on the fungus, Nomuraea rileyi, infecting the velvetbean caterpillar, Anticarsia gemmatalis.

Abstract Combined scanning and transmission electron microscopy was used to study the fine structure of the developmental stages of Nomuraea rileyi infecting host larvae of Anticarsia gemmatalis. Larvae were dusted with large numbers of fungal conidia, which germinated and penetrated the cuticle within 6 hr post-treatment. Within 24 hr, penetration hyphae had reached the cuticular epidermis and, via a budding process, initiated the hyphal body stage in the hemocoel. The hyphal bodies, suspended in hemolymph, multiplied and spread throughout the host larvae. By 6–7 days post-treatment, the majority of larvae were mummified. Within 12 hr postmortem numerous conidiophores emerged producing a confluent mycelial mat over the entire cuticular surface. Numerous hydrophobic conidia were formed on phialides present on the aerial conidiophores.

Characterization and description of a virus causing salivary gland hyperplasia in the housefly, Musca domestica.

Abstract. A double‐stranded DNA virus was isolated from hyperplasic salivary glands of male and female houseflies, Musca domestica L. (Diptera: Muscidae), collected from a dairy in Alachua County, Florida, U.S.A.

Hemagglutinin activity in the hemolymph of Anticarsiagemmatalis larvae infected with the fungus Nomuraearileyi

Hemolymph samples from larvae of 3 lepidopteran species (Anticarsia gemmatalis, Trichoplusia ni and Spodoptera frugiperda) were tested for hemagglutination activity. Samples from A. gemmatalis larvae which had been injected 12-24 hrs previously with hyphal bodies of the entomopathogenic fungus Nomuraea rileyi showed agglutination activity against human 0, rabbit and sheep erythrocytes. Little or no activity was detected in samples from the other 2 larval species. Low titers (approximately 1:2) were observed when rabbit and sheep erythrocytes were tested with hemolymph from non-injected or water-injected A. gemmatalis control larvae. Higher titers (1:256-1:1024) were obtained when human erythrocytes were incubated with control hemolymph, but values were greater in the hyphal body-injected samples (1:2048-1: greater than 32,000). These results indicate a direct correlation between agglutinin production and the presence of fungal cells in the larval hemolymph. Injection with heat-killed or homogenated hyphal bodies did not cause increased activity. Decreases in titer values after these injections and for 1-12 hrs after injection with viable hyphal bodies suggest that the agglutinin(s) may function in immune surveillance. Agglutination of rabbit erythrocytes was inhibited by lactose, galactose and L-fucose. N-acetylneuraminic acid inhibited agglutination of human erythrocytes.

Characteristics of a galactose-binding hemagglutinin (lectin) from hemolymph of spodopteraexigua larvae

Hemolymph from Spodoptera exigua larvae agglutinates rabbit and human O erythrocytes. The agglutinin appears to be naturally occurring i.e., injury (by injection) or injection of larvae with fungal cells does not induce an increase in titer of hemolymph samples. Hemagglutinin activity is destroyed by heat or EDTA, and galactosidic carbohydrates inhibit agglutination of both red blood cell types. The agglutinin was purified by affinity chromatography using an Affi-Gel ovalbumin column. Binding to the column is calcium (cation) dependent. SDS gel electrophoresis shows that the agglutinin is a minor component of whole hemolymph represented by two bands with molecular weights of 30,500 and 31,000 daltons. Fluorescence microscopy using rhodamine-labeled agglutinin indicates that the agglutinin binds to fungal cell wall surfaces known to have galactose residues (e.g., Paecilomyces ochraceus), and that binding is specifically inhibited by galactose. There is no specific binding to fungal walls known to lack galactose residues (e.g., Paecilomyces ochraceus, Nomuraea rileyi). The agglutinin may be involved in the immune response of the insect e.g., by opsonization of microbial (fungal) surfaces which render the invading cells more susceptible to phagocytosis or agglutination.

Phagocytosis of lectin-opsonized fungal cells and endocytosis of the ligand by insect Spodoptera exigua granular hemocytes: an ultrastructural and immunocytochemical study

Abstract.Phagocytosis of blastospores of the fungal entomopathogen Paecilomyces farinosus by granular hemocytes from larvae of Spodoptera exigua (beet armyworm) was studied. Blastospores were opsonized with a galactose-specific lectin purified from S. exigua hemolymph or with peanut agglutinin prior to incubation with hemocytes. Observations of thin sections revealed that pseudopodia extending from granulocytes attached to ligands (lectins, lectin conjugates) on the blastospores, and that the ligands became detached from the fungal surfaces and were endocytosed by granulocytes via coated pits on the plasma membrane. Coated vesicles bearing the endocytosed molecules appeared to be transported to the hemocytic granules. In other cases, ligand still coated the blastospores after phagocytosis and may have later concentrated within the phagosome along with digested fungal cell wall components. Phagocytosis of blastospores and clustering of a biotinylated lectin conjugate on or within the granulocytes were inhibited by drugs targeting cytoskeletal elements. Actin was concentrated in the pseudopodia of phagocytic granulocytes and may be directly associated with lectin receptor(s). Microtubules were abundant in the granulocytes, sometimes in specific regions.

Characterization of monoclonal antibodies against cell wall epitopes of the insect pathogenic fungus, Nomuraea rileyi: differential binding to fungal surfaces and cross-reactivity with host hemocytes and basement membrane components

Monoclonal antibodies (MAbs) were generated against epitopes on yeast-like hyphal bodies and hyphae of the entomopathogenic hyphomycete, Nomuraea rileyi. Two MAbs (4C10, 2H4) bind to epitopes common to both hyphal bodies and hyphae, whereas MAb 4E9 binds only to hyphal surfaces. 4C10 and 2H4 appear to be directed towards carbohydrate portions of cell surface mannoproteins, as evidenced by similarities in staining patterns between these MAbs and Concanavalin A on Western blots of N. rileyi cell wall extracts. These MAbs cross-react with antigens on blastospore and hyphal surfaces of two other entomopathogenic fungi, Beauveria bassiana and Paecilomyces farinosus in fluorescence microscopy assays, but do not cross-react with a non-entomopathogenic strain of Candida albicans or with Saccharomyces cerevisiae yeasts. MAb 4C10 also cross-reacts with immunocompetent granular hemocytes from Spodoptera exigua (beet armyworm) and Trichoplusia ni (cabbage looper) larvae and with S. exigua plasmatocytes. Electron microscopy revealed that this MAb binds to a component in cytoplasmic granules in the hemocytes, and that surface labeling may be due to the release of this MAb-positive component upon degranulation. MAb 2H4 does not cross-react with granular hemocytes, but does bind to plasmatocytes and hemocytes that tightly adhere to the substrate in monolayer assays. Additionally, MAb 4C10 specifically labels a basement membrane epitope on S. exigua fat body, suggesting that this antibody binds to mannose residues on extracellular matrix glycoproteins. Cross-reactivity of these N. rileyi MAbs with insect hemocyte and tissue components indicates that fungal surface epitopes can mimic host surface molecules, which could explain why N. rileyi hyphal bodies are not recognized by granulocytes and are able to circulate freely in the hemolymph without binding to basement membranes lining the hemocoel.

Characterization of an iridovirus isolated from the southern mole cricket, Scapteriscus vicinus

Abstract A new iridovirus has been detected from diseased southern mole crickets, Scapteriscus acletus , collected in Brazil during the spring of 1986. This icosahedral virus measuring 146 nm (side-side) to 172 nm (apex-apex) has been purified via Ficoll gradient centrifugation and demonstrated to be infectious to 1st instar Scapteriscus vicinus nymphs. The cytopathology of this virus is typical of the pattern documented for other iridovirus isolates. Characterization of the structural polypeptides by SDS-polyacrylamide gel electrophoresis revealed an array of 3 major and 17 minor polypeptides ranging in molecular weight from 15.1 to 152.0 kDa. Electrophoresis in agarose gels of purified DNA revealed a single band of high molecular weight. Analysis of various restriction endonuclease (REN) digests of this DNA demonstrated it to have an approximate molecular weight of 144 kilobase pairs. Based on differences in the polypeptide profile and REN profiles we believe this virus is distinct from previously characterized invertebrate iridovirus isolates.

Comparative analysis of phagocytosis of fungal cells by insect hemocytes versus horse neutrophils.

In this study, the phagocytic ability of Spodoptera exigua hemocytes was compared to horse neutrophils. In vitro assays showed that the insect granulocytes and horse neutrophils actively phagocytose FITC-labeled Paecilomyces farinosus blastospores opsonized with S. exigua hemolymph lectin or horse serum, respectively. Killing of fungal cells by the neutrophils and hemocytes was analyzed under in vitro conditions. Neutrophils reduced the growth of P. farinosus up to 65% whereas no fungicidal activity was observed with hemocyte monolayers. The production of oxygen metabolites by both phagocytic cells incubated with various elicitors (fungal cells, bacteria, phorbol myristate acetate) was examined using luminol-enhanced chemiluminescence. Phagocytosis of opsonized microbes by horse neutrophils resulted in marked increase of chemiluminescence activity whereas no chemiluminescence was detected in similarly challenged phagocytic insect hemocytes. Electron microscopy was used to examine phagocytic events and confirmed that insect phagocytes were unable to kill tested microbes.

Use of labeled lectins to investigate cell wall surfaces of the entomogenous hyphomycete Nomuraea rileyi

Nomuraea rileyi is an entomogenous fungus infecting lepidopterous defoliators; host range of the pathogen varies according to strain. Identifying surface components of infectious stages of different strains of the fungus may be an important step in understanding host-parasite interactions. A variety of fluorescein and ferritin-conjugated lectins, including concanavalin A, peanut, soybean, winged pea and wheat germ were used to investigate surface components on germ tube and hyphal body walls from two strains (FL 74, FL 78) of N. rileyi. Binding was observed on outer wall layers when both Con A and wheat germ agglutinin conjugates were tested. There was no apparent difference in binding between the two strains or between germ tubes and hyphal bodies. These results indicate the presence of mannose (and/or glucose) residues (Con A specifity) and N-acetyl-D-glucosamine residues (wheat germ agglutinin specifity) on outer wall surfaces. Extracellular sheath material especially noticeable on germ tubes from the FL 74 strain was not labeled by any of the lectins tested, but was well stained with ruthenium red, indicating the presence of polysaccharides.