Adhesion of conidia of the fungus Dilophospora alopecuri to the cuticle of the nematode Anguina agrostis,the vector in annual ryegrass toxicity

The adhesion of conidia of the fungus Dilophospora alopecuri to the surface of the second stage dauer larva (DL₂) of the nematode Anguina agrostis (syn. A. funesta) was examined using both light and electron optics. The process of attachment does not lead to any apparent damage to the epicuticle of the nematode. Photographs of sections cut tangentially through the setulose appendages of the conidia show that a mucilagenous fibrillar material appears to be exuded from the highly convoluted surface of these appendages. This material adheres to the surface of the nematode cuticle and is deposited in the transverse annulations. The adhesion of these spores to DL₂ of A. agrostis was examined in 4822 nematodes from four galls. The mean percentage of DL₂ with spores adhering was 64% and ranged from 43 to 85%. This adhesion was compared with that of Corynebacterium rathayi from bacterial galls and was found to coincide. Thus, bacteria adhere to nematodes with D. alopecuri conidia attached and these conidia adhere to nematodes with C rathayi attached. Furthermore, DL₂ that are free from conidial adhesion appear to be free from bacterial adhesion and, in most instances, DL₂ that remain from from bacterial adhesion remain free from conidial adhesion. These observations draw attention to the potential of D. alopecuri as an agent for the biological control of annual ryegrass toxicity. Conidial adhesion to A. agrostis differs from bacterial adhesion to this nematode in that no visible damage to the cuticle takes place. The concept that adhesion of microorganisms to nematodes occurs in two phases, one involving site recognition and the other, if it occurs, involving physiological interaction and morphological change is discussed.     

Effect of attachment of Corynebacterium rathayi on movement of Anguina agrostis larvae

Bird A. F. and Riddle D. L. 1984. Effect of attachment of Corynebacterium rathayi on movement of Anguina agrostis larvae. International Journal for Parasitology 14: 503–511. The movement of freshly hatched larvae (FHL₂) and dauer larvae (DL₂) of Anguina agrostis was compared on ‘agarose’ plates. The DL₂S moved faster and over greater distances. They were not attracted to Corynebacterium rathayi on agar plates, but contact with this bacterium, in most instances, markedly reduced their speed of movement. This reduction was found to be approximately proportional to the concentration of the bacteria (from 8 × 10⁵ to 8 × 10⁸ per ml) to which the DL₂ were exposed prior to observation of their movement, as was the number of bacteria observed to be adhering when viewed under the light microscope. This type of bacterial attachment appeared to be largely stage specific as it was much more pronounced and characteristically different in the DL₂ from that in the FHL₂. This interaction between the DL₂ and the bacterium was similar in material from fresh and from rehydrated nematode galls so that it was apparently not dependent on any surface cuticular changes associated with anhydrobiosis. Furthermore, pretreatment of the nematodes with the surfactant SDS did not influence attachment. An electron microscope study of sections cut through DL₂ exposed to bacteria showed that this interaction was indeed not a surface phenomenon but that the bacteria exerted a pathological effect on the nematode. The bacterium's capsular material actually penetrated and broke down the nematode epicuticle and part of the cortical zone. These observations make it easier to understand the dramatic physiological responses of this nematode to these bacteria.     

The nature of the adhesion of Corynebacterium rathayi to the cuticle of the infective larva of Anguina agrostis

The relationship between Corynebacterium rathayi and the infective second stage ‘dauer” larva (DL₂) of Anguina agrostis has been studied with particular regard to the nature and extent of bacterial adhesion to the nematode. Viability of the 1-year-old DL₂ was high, the number of dead specimens per gall only ranging from 5.1% to 8.3%. The DL₂ were exposed to C. rathayi either from cultures or galls and the numbers with and without adhering bacteria were counted. A total of 22,917 DL₂ from 15 galls were examined, the mean number of DL₂ per gall being 1,528. The mean percentage per gall of DL₂ with bacteria adhering was 42%. However there was much variability (from 0 to 98%). The significance of galls with DL₂ that did not succumb to bacterial adhesion is discussed. The sequence of events associated with the process of bacterial adhesion to the nematode cuticle was examined with the aid of the electron microscope. The process involves fusion of the glycocalyx with the bacterial capsule followed by thickening and, at times, displacement of the epicuticle together with changes in the main body of the cuticle. The possible nature of the process of adhesion is discussed.     

Responses of Anguina agrostis to Detergent and Anesthetic Treatment

The infective dauer juvenile (DJ2) of Anguina agrostis, a stage capable of surviving desiccation, is up to sixfold more resistant to the detergent sodium dodecyl sulfate than are freshly hatched juveniles or adult males, and twofold more resistant to the anesthetic phenoxypropanol. Thus, the DJ2, like dauer stages of other species, may also be more resistant to various types of environmental stress in its natural habitat. In A. agrostis, however, resistance appears to be acquired gradually during development of the second juvenile stage, rather than during a molt.     

Wheat Germ Agglutinin Bound to the Outer Cuticle of the Seed Gall Nematodes Anguina agrostis and A. tritici

The presence of wheat germ agglutinin (WGA) on the cuticular surface of the seed gall nematodes Anguina agrostis and Anguina tritici was demonstrated, and the nature of its binding was examined. Crude extracts from the cuticles of A. tritici agglutinated human red blood cells, and only N-acetylglucosamine (GlucNAc) inhibited the agglutination. Distribution of the lectin was visualized by treating live infective juveniles (J2) with rabbit anti-WGA antibody and staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. The lectin bound to the outer cuticular surface of the whole body wall. Pretreatment with GlucNAc oligomers did not reduce the fluorescence created by the anti-WGA-WGA binding, indicating at least a partial nonspeciflc adhesion of the WGA to the nematode surface. Proteolytic enzyme pretreatments diminished the fluorescence, whereas lipase and periodate pretreatments increased the fluorescence. Adult females and males were labeled only on the head and tail, whereas eggs were not labeled at all. It was concluded that the WGA on the J2 cuticle originates from the host.     

The Influence of Environmental Factors on the Respiration of Plant-Parasitic Nematodes

Respiration of selected nematode species was measured relative to CO₂ level, temperature, osmotic pressure, humidity, glucose utilization and high ionic concentrations of sodium and potassium.In general, respiration was stimulated most by the dominant environmental factors at levels near those expected in the nematode''s "natural" habitat. Soil-inhabiting nematodes utilized O₂, most rapidly with high (1-2%) CO₂ whereas a foliar nematode (Aphelenchoides ritzemabosi) did so with 0.03% CO₂, the concentration typically found in air. Temperature optima for respiration corresponded closely to those for other activities. Ditylenchus dipsaci and Pratylenchus penetrans adults and Anguina tritici and A. agrostis second-stage larvae respired within the range of osmotic pressures from 0 to 44.8 arm and respiration of their drought-resistant stages was stimulated by increasing osmotic pressure which accompanies the onset of drought. Rehydration of A. tritici and A. agrostis larvae with RH as low as 5% stimulated measurable respiration. Glucose utilization from liquid medium by A. tritici larvae or A. ritzembosi was not detectable. Supplemental Na⁺ stimulated respiration of Anguina tritici, K⁺ did not.     

Stage-specific Differences in Lectin Binding to the Surface of Anguina tritici and Meloidogyne incognita

The occurrence and distribution of several lectin binding sites on the outer surfaces of eggs, preparasitic second-stage juveniles (J2), parasitic second-stage juveniles (PJ2), females, and males of two tylenchid nematodes, Anguina tritici and Meloidogyne incognita race 3, were compared. In both species, a greater variety of lectins bound to the eggs than to other life stages; lectin binding to eggs was also more intense than it was to other life stages. Species-specific differences also occurred. More lectins bound to the amphids or amphidial secretions of M. incognita J2 than to the amphids or amphidial secretions of A. tritici J2. Lectins also bound to the amphids or amphidial secretions of adult male and female A. tritici, but binding to the cuticle occurred only at the head and tail and was not consistent in all specimens. Canavalia ensiformis and Ulex europaeus lectins bound specifically to the outer cuticle of M. incognita. Several other lectins bound nonspecifically. Oxidation of the cuticle with periodate under mild conditions, as well as pretreatment of the nematodes with lipase, markedly increased the binding of lectins to the cuticle of A. tritici J2 but not, in most cases, to M. incognita J2 or eggs of either species.     

The life cycle of Anguina agrostis: Embryogenesis

Bpird A. F. and Sptynes B. A. 1981. The life cycle of Anguina agrostis: embryogenesis. International Journal for Parasitology11: 23–33. Egg development, from laying to hatching, of two widely separated populations of Anguina agrostis, has been followed over a range of temperatures. Development rates for these two populations have been shown to be identical with a thermal optimum between 18 and 20°C. The minimum time recorded for embryogenesis through to hatching was 9–10 days.Embryogenesis was inhibited by temperatures of 27°C and above and hatching by temperatures greater than 23°C.No significant differences were detected in the dimensions of eggs from either population. These eggs have an average length of 95 μm and an average width of 38 μm.Electron microscope studies of sections through eggs undergoing synchronous development show that the first and apparently only moult of the larva in the egg commences about 7 days after the start of embryogenesis under optimal conditions. The sequence of morphological events that occur throughout embryogenesis are described and recorded for whole specimens observed at low resolution and the moulting sequence is described from high resolution electron micrographs of the cuticles of synchronously developing embryos and larvae.     

The life cycle of Anguina agrostis: Post-embryonic growth of the second stage larva

The growth and development of the freshly hatched second stage larva (FHL₂) into the infective second stage “dauer” larva (DL₂) has been followed under field conditions and in callus tissue culture. A comparison of this development has been made between specimens originating from widely separated geographic regions. The FHL₂'s from Narrogin (Western Australia) are slightly shorter than those from Murray Bridge (South Australia) and have a mean length of 543 ± 55 μm compared with 580±51 μm. However, the lengths of the DL₂s from these areas are similar, having mean lengths of 841 ± 35 μm and 849 ±26 μm respectively. Furthermore stylet lengths in all these larvae are similar and are approx. 10 μm. Morphological changes associated with the transition from FHL₂ to DL₂ include thickening of the cuticle, a change in shape of the lateral alae associated with stretching and the synthesis of numerous lipid storage granules. Physiological changes include a marked increase in swimming activity and the ability to enter into an anhydrobiotic state. Growth from FHL₂ to DL₂ in Lolium multiflorum callus tissue culture took place within 9 days at 20°C. No moulting was observed and growth did not take place beyond the DL₂ stage under these conditions.     

Population dynamics of the parasitic stages of Ostertagia spp. in sheep

Callinan A. P. L. and Arundel J. H. 1982. Population dynamics of the parasitic stages of Ostertagia spp. in sheep. International Journal for Parasitology12: 531–535. The development and survival of continuing infections of Ostertagia spp. in weaner sheep were studied in order to develop a general model of the parasitic stages of the life cycle of these sheep nematodes. After 10 days, 13.8% of infective larvae (L₃) given at the rate of 1 dose of 1000 L₃ twice per week (group 1) and 20.8% of L₃ given at the rate of 10,000 L₃ twice per week (group 2) were recovered in the first of the serial nematode counts. In the final counts at 137 days, 7.7 and 0.7% were recovered in these groups. The build up and maintenance of nematode populations was regulated and related to the level of infection. A model in which the death rate of the parasitic stages was a function of the time of exposure to infection and rate of infection was used to describe the serial total nematode counts. During the experiment there was no noticeable trend in numbers of fourth stage larvae (L₄) in nematode counts, the size of adult nematodes, nematode egg counts (EPG) and egg output per female nematode (EPF). After 112 days, liveweight gains were significantly reduced in group 2 only, but increases in wool lengths were significantly reduced in both groups.     

Characterization of Sialyl and Galactosyl Residues on the Body Wall of Different Plant Parasitic Nematodes

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-β-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.     

Anguina plantaginis n. sp. Parasitic on Plantago aristata with a Description of Its Developmental Stages

Anguina plantaginis n. sp., parasitic on Plantago aristata, is described and illustrated. This new species is most closely related to A. klebahni, A. millefolii, A. mobilis, and A. moxae and is characterized as follows: moderate body size for the genus; absence of esophageal "storage organ"; postvulval uterine sac extending about 45% of vulva-anus distance; crustaformeria of young females longer than spermatotheca or uterus proper; spicules with 2 sclerotized thickenings; long, conical tail in both sexes, narrowing at about 1/6 of its length to peg-like tip; parasitic only on P. aristata. Two nematode generations that are morphologically similar but differ in body size develop in one plant gall. The postembryogenesis, studied with respect to morphological development of the larval stages, is similar to that of Ditylenchus. The sexes can be differentiated from the second molt on. The infective larva is the third stage, which is morphologically distinct from the regularly developing third-stage larva.     

Immunolocalization of a putative cuticular collagen protein in several developmental stages of Meloidogyne arenaria,Globodera pallida and G. rostochiensis

The monoclonal antibody IACR-CCNj.3d has previously been used to isolate a gene (gp-col-8) with strong similarity to cuticular collagen from a mixed stage Globodera pallida cDNA expression library. The antibody has also been shown to label specifically the amphidial canal of pre-parasitic second stage juveniles (J2) of several plant nematode species without any reactivity on the cuticular surface, indicating that this protein is either not present or is inaccessible on the cuticular surface. This paper investigates the cross-reactivity of Mab IACR-CCNj.3d with Meloidogyne arenaria and the localization of the putative collagen protein on the cuticular surface of parasitic stages in planta and on the cuticular surface of juveniles inside eggs. The antigen was shown to be present in all developmental stages of the two species of potato cyst nematodes and M. arenaria. The antibody bound strongly to the amphidial canal and hypodermis of pre-parasitic J2 and adult females. The antigen was present on the cuticular surface of the sausage-shaped J2 in planta and of first stage juveniles (J1) inside the eggs. The presence of collagen on the surface of the cuticle of moulting stages of plant parasitic nematodes has been observed for the first time. It is clear that this protein has a role in the construction of the cuticle of the first stage juveniles and parasitic second stage juveniles, during moulting inside the eggs and in the root tissue, respectively.     

A First Report of Anguina pacificae in Ireland

Anguina pacificae is a significant pest of Poa annua golf course greens in northern California. This study presents the first confirmed case of an A. pacificae infestation outside of North America, where the nematode’s distribution is further restricted to a relatively limited coastal region. Species confirmation was made by morphometric and molecular methods and comparisons to closely related species including the European species, Anguina agropyri. The A. pacificae population detected on an Irish golf course was monitored over a 2-yr period and the life cycle compared with Californian population dynamics. A. pacificae was assessed for the potential risk of spreading to the local agricultural sector, in addition, the biosecurity risks from A. pacificae and plant parasitic nematodes in general were reviewed for northwest Europe.     

Carbonic acid as the host signal for the development of parasitic stages of nematodes

Petronijevic T., Rogers W. P. and Sommerville R. I. 1985. Carbonic acid as the host signal for the development of parasitic stages of nematodes. International Journal for Parasitology15: 661–667. This paper gives results on which may be based an identification of the component of the system CO₂ + H₂O ai H₂CO₃ ai H⁺ HCO₃^? which acts as the stimulus from the animal host for some nematodes. Using infective juveniles of Nematospiroides dubius and Haemonchus contortus, the effects on exsheathment of (1) low pCO₂ values, (2) the presence of carbonic anhydrase in the stimulating medium, and (3) the inhibition of carbonic anhydrase within the juveniles have been examined. The results lead to the suggestion that it is the “readily available” undissociated H₂CO₃, or H₂CO₃ + HCO₃^? which is the critical factor in the stimulus for development. The wide range of [H⁺]s over which “readily available” H₂CO₃ is present in physiological environments suggests that this host signal may be important for infection with many species.     

Undissociated bases as the stimulus for the development of early parasitic stages of nematodes

Petronijevic T. and Rogers W. P. 1987. Undissociated bases as the stimulus for the development of early parasitic stages of nematodes. International Journal for Parasitology 17 :911–915. The effects of NH₄Cl and NH₂CH₃ on infective stages of Haemonchus contortus, Nematospiroides dubius and Ascaris suum have been compared with the action of H₂CO₃. Detailed experiments were carried out with H. contortus. NH₄Cl at pH 8 under air was less toxic to infective stages than it was to free-living juveniles of Panagrellus redivivus. The induction of exsheathment or hatching by bases was slow (20–30 h) though the time of development of 4th stage H. contortus was not proportionally increased. Activity at pH 6 was less than that at pH 8. In contrast to the action of H₂CO₃ as the stimulus, NH₄Cl was not Ca²⁺-dependent. Prolonged exposure to anoxia at pH 8 was toxic, but in the presence of NH₄Cl or H₂CO₃ toxicity was decreased. The inhibition of exsheathment due to Dnp at pH 7 was greater when NH₄Cl was the stimulus.The process whereby host signals induce development of parasitic stages is discussed.     

The cuticular proteins from free-living and parasitic stages of Haemonchus contortus--I. Isolation and partial characterization

1. Cuticles were isolated from the adult males, adult females, the second molt (2M) sheath from the infective larvae (L3(2M)), and the parasitic third stage (L3) of the sheep parasite Haemonchus contortus by a combination of mechanical disruption and detergent treatment. 2. The 2ME soluble cuticular proteins from adult males contained 4 or 5 major protein bands with molecular weights ranging from 100 to 56 kD with the most prominent band at 56 kD. The cuticular proteins from adult females were similar to the male. 3. Cuticular proteins from the larval stages, 2M cuticle, and L3 cuticle, differed from the adults and from each other. The most prominent protein bands were observed with molecular weights on 78 and 39 kD for the L3 cuticle and 100, 91 and 46 kD for the 2M cuticle. The 2ME soluble cuticular proteins from all developmental stages were at least partially digested by bacterial collagenase. 4. The amino acid composition of cuticular proteins was similar for the L3 and 2M, but adults had lesser amounts of glycine and greater amounts of basic amino acids than the larval stages. The amount of the isolated cuticle solubilized by the 2ME treatment was greatest in adults (80%) compared to the L3 (64%) and the 2M (22%). 5. These results support a hypothesis that there are quantitative and qualitative stage specific differences in the cuticular proteins of H. contortus.     

Granulosis virus in the intestinal lumen of Neoaplectana carpocapsae: Retention of infectivity after treatment with formaldehyde or high pH

High pH values (>11.0) cause the dissolution of occlusion bodies of the granulosis virus (GV) of Pseudaletia unipuncta and subsequent inactivation of the virus within 24 hr. The GV is also inactivated within 48 hr by 0.04% formaldehyde. The GV is found in the intestinal lumen of infective third stage nematodes (dauer juveniles) of Neoaplectana carpocapsae when development occurs in GV-infected hosts. The GV in these dauer juveniles retains its infectivity even when the nematodes are placed into an alkaline solution with pH values of 11.1 or 12.1 or in 0.04% formaldehyde up to 336 hr. However, significant loss of infectivity of GV occurs when the nematodes are in formaldehyde but not at high pH values. The dauer juveniles are ensheathed by the second stage cuticle. This cuticle probably protects the GV in the intestinal lumen of the nematode from the high pH and formaldehyde.     

Epidemiology of Anguina agrostis on Highland Colonial Bentgrass

The epidemiology of Anguina agrostis was investigated in field plots of Colonial bentgrass (cv. Highland), Agrostis tenuis, near Corvallis, Oregon. Each October from 1990-92, nylon mesh pouches, each containing 10 galls, were buried in the field or placed on the soil surface in microplots. Pouches were collected monthly or bimonthly between December and June and nematodes per gall counted. Nematode egression from galls began in late March and was completed by mid-May, corresponding to the period of floral initiation in bentgrass. In 1991 and 1992, 0.09-m² plots were inoculated with 0, 1, 5, 15, 50, 120, or 200 galls/plot. The disease severity (number of galls) and disease incidence (% seed heads with galls) increased linearly at inoculum densities below 50 galls/ plot. At higher inoculum densities, disease increase approached an asymptote. In 1991, plots were established to determine the characteristics of disease spread. Disease foci were established by placing 0, 5, 50, or 500 galls along 30-cm sections of row in the fall. In July 1992, seed heads were harvested at 30 and 60 cm from each focus within and across plant rows. Most infestations were found within 30 cm of foci at all inoculum levels. At high inoculum densities, the distribution of galls was aggregated with the majority of galls located on less than 10% of the seed heads. These disease spread and incidence data suggest populations of A. agrostis increase slowly in bentgrass in Oregon.     

Differential Adhesion and Infection of Nematodes by the Endoparasitic Fungus Meria coniospora (Deuteromycetes)

The conidia of the endoparasitic fungus Meria coniospora (Deuteromycetes) had different patterns of adhesion to the cuticles of the several nematode species tested; adhesion in some species was only to the head and tail regions, on others over the entire cuticle, whereas on others there was a complete lack of adhesion. After adhesion, the fungus usually infected the nematode. However, adhesion to third-stage larvae of five animal parasitic nematodes, all of which carry the cast cuticle from the previous molt, did not result in infection. M. coniospora infected animal parasitic nematodes when this protective sheath was removed. Seven preparations of sialic acid (N-acetylneuraminic acid) gave three types of response in adhesion-infection of nematodes: (i) a significant reduction in conidial adhesions; (ii) no interference with adhesion, but a 10-day delay in infection; and (iii) a delay in infection by 2 to 3 days. The current results support previous findings indicating involvement of sialic acids localized on nematode cuticles in recognition of prey by M. coniospora.