John M. Webster

Lipopolysaccharides of Xenorhabdus nematophilus (Enterobacteriaceae) and Their Haemocyte Toxicity in Non-immune Galleria mellonella (Insecta: Lepidoptera) Larvae

SUMMARY: Three varieties of Xenorhabdus nematophilus subsp. nematophilus released lipopolysaccharide (LPS) during bacteraemia in larval Galleria mellonella. Larval serum triggered release of LPS from the bacterial envelope. LPS activated the plasmatocytes and eventually damaged the haemocytes. LPS and its lipid A portion bound to the insect haemocytes through D-glucosamine-binding lectins on the haemocyte surfaces. The toxicity of LPS resides in the fatty acids of the lipid A moiety.

Ultrastructure of the hypersensitive reaction in roots of tomato, Lycopersicon esculentum L., to infection by the root-knot nematode, Meloidogyne incognita☆

Abstract The hypersensitive reaction in roots of Lycopersion esculentum L., cv. Nematex, infected by Meloidogyne incognita was studied by light and electron microscopy. An increase in cytoplasmic density characterized by increased numbers of ribosomes, proliferation of endoplasmic reticulum and increased stainability of the cytoplasmic ground substance was the first symptom of the resistance response to the nematode larvae. Concomitant with this change was a disappearance of dense osmiophilic inclusions from the vacuoles of hypersensitive cells. There followed a general loss in distinctness of the cell membranes resulting in the disappearance of mitochondria and Golgi bodies. The fibrillar structure of the nucleoplasm disappeared and numerous electron-dense inclusions appeared in the nucleoplasm. Organized arrays of ribosomes appeared on the outer membrane of the nuclear envelope. The plastid stroma lost its granular texture although large starch grains persisted. Changes in cell structure were restricted to cells close to the nematode, but the products of cell breakdown moved away from this region through intercellular spaces. Giant cell production was generally suppressed by the hypersensitive reaction. Heat-treatment of Nematex roots inhibited the hypersensitive reaction and thus enabled giant cell formation. Treatment of roots with the growth substance kinetin did not inhibit the hypersensitive reaction although it appeared to enhance giant cell formation.

Antihemocytic surface components of Xenorhabdus nematophilus var. dutki and their modification by serum of nonimmune larvae of Galleria mellonella

Abstract Modifying the outer membrane of Xenorhabdus nematophilus var. dutki , by dissociating outer membrane proteins and extracting lipopolysaccharide, accelerated bacterial removal from the hemolymph of nonimmune Galleria mellonella larvae. Larval serum modified the bacterial surface which enhanced adhesion to hemocytes and accelerated the removal of X. nematophilus from the hemolymph. This was independant of phenoloxidase activity. X. nematophilus inhibited the conversion of prophenoloxidase to phenoloxidase but did not inhibit phenoloxidase activity. The sugars α- d -mannose, d -galactose, and N -acetyl- d -glucosamine decreased the attachment of surface modified X. nematophilus to hemocytes and slowed their removal from the hemolymph. Evidence is presented suggesting that humoral factors, with preotolytic, lysozyme, and carbohydrase activity, may alter the outer membrane of X. nematophilus , and represent antibacterial factors in insect hemolymph.

Nematicidal metabolites produced by Photorhabdus luminescens (Enterobacteriaceae), bacterial symbiont of entomopathogenic nematodes

The secondary metabolites, 3,5-dihydroxy-4-isopropylstilbene (ST) and indole, from the culture filtrate of Photorhabdus luminescens MD, were shown to have nematicidal properties. ST caused nearly 100% mortality of J4 and adults of Aphelenchoides rhytium , Bursaphelenchus spp. and Caenorhabditis elegans at 100 mu g/ml, but had no effect on J2 of Meloidogyne incognita or infective juveniles (IJ) of Heterorhabditis megidis at 200 mu g/ml. Indole was lethal to several nematode species at 300 mu g/ml, and caused a high percentage of Bursaphelenchus spp. (J4 and adults), M. incognita (J2) and Heterorhabditis spp. (IJ) to be paralysed at 300, 100 and 400 mu g/ml, respectively. Both ST and indole inhibited egg hatch of M. incognita . ST repelled IJ of some Steinernema spp. but not IJ of Heterorhabditis spp., and indole repelled IJ of some species of both Steinernema and Heterorhabditis . ST, but not indole, was produced in nematode-infected larval Galleria mellonella after 24 h infection. Von Photorhabdus luminescens (Enterobacteriaceae), einem Symbionten entomopathogener Nematoden gebildete nematizide Metaboliten - Es wurde gezeigt, dass die Sekundarmetaboliten 3,5-Dihydroxy-4-isopropylstilben (ST) und Indol aus dem Kulturfiltrat von Photorhabdus luminescens MD nematizide Eigenschaften besassen. In einer Konzentration von 100 mu g/ml verursachte ST eine fast 100%ige Sterblichkeit bei J4 und Adulten von Aphelenchoides rhytium , Bursaphelenchus spp. und Caenorhabditis elegans , hatte aber bei 200 mu g/ml keine Wirkung auf J2 von Meloidogyne incognita oder auf Infektionsjuvenile (IJ) von Heterorhabditis megidis . Bei 300 mu g/ml war Indol fur etliche Nematodenarten todlich und fuhrte dazu, dass Bursaphelenchus spp. (J4 and Adulte) bei 300, M. incognita (J2) bei 100, und Heterorhabditis spp. (IJ) bei 400 mu g/ml zu einem grossen Teil gelahmt wurden. ST und Indol behinderten beide das Schlupfen von M. incognita . ST wirkte abstossend auf IJ einiger Steinernema -Arten aber nicht auf IJ von Heterorhabditis spp., und Indol wirkte abstossend auf IJ einiger Arten der beiden Gattungen Steinernema und Heterorhabditis . ST wurde in nematoden-befallenen Larven von Galleria mellonella 24 h nach der Infektion gebildet, Indol dagegen nicht.

Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

Abstract The lethal dose (LD)50 values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum. The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues. Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes. The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.

Mermis nigrescens: Physiological relationship with its host, the adult desert locust Schistocerca gregaria

Abstract Adult desert locusts were experimentally infected per os with 30, 50, or 60 Mermis nigrescens eggs, and changes in the host physiology were recorded. Larval nematodes were recovered from the hemocoel and counted at appropriate times after infection. The food consumption and blood volume of the host were unaffected by the parasitism, but the nematode significantly impaired the ability of male locusts to excrete the injected dye, amaranth, from their hemolymph. The total carbohydrate in the hemolymph of infected male and female locusts was severely depleted during the active growth period of the nematode and the possible utilization of these carbohydrates by the nematode are discussed. The total amino acid and protein levels in the blood of the host were unaffected by the nematode development, although concomittant changes in the levels of both these blood metabolites occurred in all locusts throughout the experimental period. However, although changes of this nature reflected the normal pattern of protein synthesis during oocyte development and oviposition in control locusts, the nematode suppressed oocyte development and caused oocyte resorption in the female host. The nematode did not significantly affect the level of total protein and amino acids in the flight muscles of male and female locusts, but a significant decrease in the level of fat body proteins and amino acids was recorded in infected hosts 16 and 21 days after infection. The possible effect of the nematode on protein metabolism by the host fat body is discussed in relation to the nutritional requirements of the nematode and involvement of the host endocrine system.

Partially Characterized Components of the Epicuticle of Dauer Juvenile Steinernema feltiae and Their Influence on Hemocyte Activity in Galleria mellonella

The epicuticle of dauer juveniles of the DD 136 strain of Steinernemafeltiae prevents encapsulation of the nematodes in vivo by hemocytes of Galleria mellonella larvae. The chemical composition of the epicuticle, which provides this protection, was determined using fluorescein isothiocyanate-conjugated lectins (concanavalin A, soybean agglutinin, wheat germ agglutinin) in conjunction with carbohydrases, lipase, and proteases. a-Mannose, 3-N-acetyl-D-galactosamine, and f8-N-acetyl-D-glucosamine were detected on the nematodes epicuticle. The absence of these sugars from enzyme-modified epicuticle did not affect hemocyte attachment. There was no evidence of epicuticular glycoproteins. In general, proteolytic enzymes did not alter the dauer juvenile epicuticle such as to elicit hemocyte-mediated encapsulation. Digestion of the epicuticle of dauer juveniles with lipase rendered the nematodes susceptible to hemocyte-mediated encapsulation. Many entomophilic nematodes live part of their lives in the hemolymph of their insect hosts, where the nematode cuticle is the interface between the nematode and the humoral and cellular components of the hemolymph. The function of the cuticle of these nematodes is incompletely known, but studies have shown that aspects of the transcuticular uptake of nutrients by entomophilic parasitic nematodes (Rutherford and Webster, 1974; Chen and Howells, 1979; Gordon et al., 1982) are related to cuticular structure (Nicholas, 1972; Batson, 1979; Platzer and Platzer, 1985). During the parasitic phase, the cuticle thickness of many entomophilic nematodes decreases (Bailey and Gordon, 1973), and in others, an outer microvillar layer develops (Riding, 1970) and functions as a nutrient absorptive layer. These nematodes encounter humoral (phenol oxidases) and/or cellular (hemocyte-mediated encapsulation) nonself resistance systems in the insect hemolymph (Ratcliffe and Rowley, 1979), but the contribution of the nematode cuticle to either resisting or evading these defenses is unknown, despite the reported occurrence of humoral (Welch and Bronskill, 1962; Andreadis and Hall, 1976) and cellular encapsulation (Nappi, 1975; Ratcliffe and Rowley, 1979). Vinson (1977) proposed that in the absence of active suppression of insect defenses, successful insect Received 5 September 1986; revised 3 December 1986; accepted 5 December 1986. * Present address: Department of Entomology, Macdonald College, McGill University, Ste. Anne de Bellevue, Quebec, Canada H9X 1CO. parasitoids would not be encapsulated due to (1) t e acquisition of a coat of host hemolymph components presenting the nonself agent as self, (2) the possession of heterophilic antigens, (3) the possession ofnonreactive surfaces, or (4) molecular mimicry. Evidence of a protective coat, albeit from insect midgut, may explain the reduction of humoral encapsulation of Brugia pahangi mic ofilariae in mosquitoes (Sutherland et al., 1984; Lafond et al., 1985). Entomophilic nematodes are not known to use molecular mimicry, although it may be used by cestodes and ac nthocephala (Lackie and Lackie, 1979) in some insects. The DD136 strain of Steinernema feltiae evades the hemolymph resistance systems of the gr ater wax moth, Galleria mellonella (Lepidoptera) larvae by avoiding nonself recognition (Dunphy and Webster, 1985), but the means by which this is achieved are unknown. The present study examines some of the epicuticular components of sheathed dauer juvenile S. feltiae DD136, and assesses the contribution of these components in evasion of encapsulation by the nematodes in G. mellonella. MATERIALS AND METHODS

Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode, Steinernema riobrave (Rhabditida: Steinernematidae).

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.

Virulence mechanisms of Heterorhabditis heliothidis and its bacterial associate, Xenorhabdus luminescens, in non-immune larvae of the greater wax moth, Galleria mellonella

Abstract Short-term (4 h) exposure of monoxenically and axenically cultured, dauer, juvenile Heterorhabditis heliothidis to the haemolymph of non-immune Galleria mellonella neither stimulated nor inhibited haemocyte function or lysozyme activity. It is proposed that the nematodes are not recognized as foreign by these non-self defences and that they allow the host to maintain a sterile, non-competitive environment for X. luminescens . By 6 h post-injection with monoxenic nematodes the number of damaged haemocytes increased as the number of X. luminescens and endotoxin activity increased. Similar results occurred with the injection of X. luminescens into G. mellonella . The bacteria were not harmed by the non-self defences of the haemolymph. Haemocyte damage was attributed to the fatty acids of the lipid A moiety associated with lipopolysaccharide released from X. luminescens in the haemolymph. The amino sugars d -glucosamine and N -acetyl- d -glucosamine decreased [ 32 P]lipopolysaccharide binding to insect granular cells at low concentration and enhanced binding at higher concentrations. A mechanism for lectin-mediated binding of lipopolysaccharide to haemocytes is discussed.

The use of amino acid fungal auxotrophs to study the predisposition phenomena in the root-knot-wilt fungus disease complex of tomato

Abstract Root-knot nematode ( Meloidogyne incognita ) infections of tomato plants resistant to the wilt fungus ( Fusarium oxysporum f. sp. lycopersici ) often predispose the tomato to this fungus. The role of free amino acids, which are abundant in nematode-galled tissue, in the predisposition of a resistant host to the fungus was investigated. Amino acid levels in the exudates of healthy and nematode-galled tomato cultivars were determined. Single step amino acid mutants (auxotrophs) were induced in the wild type (race 1) wilt fungus. Those auxotrophs that lost their relative pathogenicity as compared to the wild type were selected for the study of the influence of increased or decreased levels of the corresponding free amino acids in the exudates of host cultivars. Auxotrophs requiring threonine, proline, methionine, histidine and glycine were pathogenic only when the relevant amino acid was present above a certain threshold level in the exudates of genetically susceptible, nematode-galled cultivars. However, these caused negligible pathogenicity in nematode-inoculated cultivars that are genetically fungal resistant. A similar trend in pathogenicity was observed when threonine, proline, methionine and histidine were applied exogenously to the tomato cultivars before inoculation with the fungal auxotrophs. The role of these and other amino acid auxotrophs are discussed in the light of the nutritional hypotheses of Lewis and Garber [ 12, 18 ]. These experiments show that amino acids may play a significant role in predisposing fungal-resistant plants in a disease complex.