Insect Immune Evasion by Dauer and Nondauer Entomopathogenic Nematodes.

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.

New species of Macropostrongyloides Yamaguti, 1961 (Nematoda: Strongylida) and the redescription of Ma. baylisi (Wood, 1930) from Australian macropodid marsupials.

Specimens of four genetically distinct groups of Macropostrongyloides baylisi Wood, 1930 were analysed morphologically. Each genotype was found to represent a morphologically distinct species: Ma. baylisi from Osphranter robustus woodwardi (Thomas) and Osphranter robustus erubescens (Sclater); Ma. spearei n. sp. from Osphranter robustus robustus (Gould) and O. r. erubescens; Ma. mawsonae n. sp. from Macropus giganteus Shaw and Ma. woodi n. sp. from Osphranter rufus (Desmarest). The new species described here are differentiated primarily by several male-specific features that have been overlooked in previous taxonomic revisions. These features include striations on the terminal part of the spicule ala, the papillae surrounding the genital cone and the bursal striations. Furthermore, scanning electron photomicrographs have revealed greater details of previously undefined structures within the buccal cavity that warrant further investigations.

Two new species of Dorcopsistrongylus (Strongylida: Strongyloidea) from Dorcopsis muelleri (Marsupialia: Macropodidae) from Papua Indonesia.

In a survey of the brown dorcopsis, Dorcopsis muelleri , on Salawati Island, Papua, Indonesia, 2 new species were found in the stomach. Dorcopsistrongylus ewini n. sp. can be differentiated from its congeners in having the proximal end of the spicule twisted and from Dorcopsistrongylus labiacarinatus in having shorter intestinal diverticula and in the proportions of the elements of the ovejector. Dorcopsistrongylus salawatiensis n. sp. can be differentiated from its congeners in having bilobed lateral lip-like elements and an elongated terminal bulb of the esophagus. A survey of other Dorcopsis and Dorcopsulus spp. may reveal more species of Dorcopsistrongylus .

In vitro development of cyathostomin larvae from the third stage larvae to the fourth stage: morphologic characterization, effects of refrigeration, and species-specific patterns.

A mixed population of equine cyathostomin (Nematoda, Strongyloidea) infective third stage larvae (L3) was cultured in vitro using a cell-free medium. Some L3 were cultured immediately after Baermann collection from fecal cultures, while others were kept in water at 4 degrees C for 7 days before initiating the in vitro cultures. Cultures were examined daily for viability. At days 2, 7, 14 and 21 larvae were collected for identification of developmental stage and morphological changes, using both light and scanning electron microscopy. Larvae were classified as early L3 (EL3), developing L3 (DL3), late L3 (LL3) and fourth stage larvae (L4) on the basis of morphological features. Viability remained high throughout the entire study period in cultures of both non-refrigerated (84.7%) and refrigerated (77.4%) larvae. However, viability of the non-refrigerated was significantly greater from 7 through 21 days of culture. Significant differences were also observed in the percentage of DL3 between the non-refrigerated and refrigerated larval cultures by day 7. The highest percentage of DL3 larvae (22.5%) was reached at the end of study in those larvae that were not previously refrigerated. The data suggests that prior refrigeration decreases viability and slows L3 development. At day 21 LL3 larvae were only a small percentage of the DL3: 6.9 and 5% in non-refrigerated and refrigerated cultures, respectively. Few of these larvae freed themselves from the L3 cuticle and moulted to L4 stage. Characteristics of individual species in vitro developmental patterns were determined by the molecular identification of individual larvae in pools of larvae randomly collected at days 0 and 21. Seven species (Coronocyclus coronatus, Cylicostephanus goldi, Cylicostephanus longibursatus, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus ashworthi, Petrovinema poculatum) were identified in the day 0 pool. The greatest tendency to develop in vitro was shown by the genus Cylicostephanus with the species C. goldi and C. longibursatus that developed to the LL3-L4 stages. C. nassatus, C. ashworthi and C. coronatus did not progress in their development beyond the EL3 stage, while no apparent signs of development were registered for C. catinatum.

Illustrated identification keys to strongylid parasites (Strongylidae: Nematoda) of horses, zebras and asses (Equidae).

The Equidae (the horse, Equus caballus, the ass, Equus asinus, zebras and their hybrids) are hosts to a great variety of nematode parasites, some of which can cause significant morbidity or mortality if individual hosts are untreated. Worldwide the nematode parasites of horses belong to 7 suborders, 12 families, 29 genera and 83 species. The great majority (19 of 29 genera and 64 of 83 species) are members of the family Strongylidae, which includes the most common and pathogenic nematode parasites of horses. Only the Strongylidae are included in this treatise. The Strongylidae (common name strongylids) of horses--nematodes with a well-developed buccal capsule, a mouth collar with two leaf-crowns, and a strongyloid (common name of superfamily Strongyloidea) copulatory bursa--can be separated into two subfamilies: Strongylinae (common name strongylins), usually large or medium-sized with a globular or funnel-shaped buccal capsule; and Cyathostominae (common name cyathostomins), usually small to medium-sized with a cylindrical buccal capsule. The increased attention to strongylid nematode parasites of horses has resulted in the need for updated diagnostic keys to these parasites using readily recognizable characters and the most recent literature on their systematics. Because the cyathostomins have been historically difficult to identify, and because they have emerged as the most significant nematode pathogens of horses, we provide a brief nomenclatural and taxonomic history and an introduction to the morphology of this group. This treatise is intended to serve as a basic working tool--providing easy identifications to genus and species of adult strongylid nematodes of equids. All strongylid nematodes normally parasitic in horses, the ass (and their hybrids), and zebras are included. The keys are illustrated with line drawings and halftone photomicrographs of each species. A short discussion of the systematics of the genus and species is provided for each genus following the species descriptions. Species diagnoses and a synonymy of each species is provided. Geographic distribution, prevalence, and location in host are also given for each species.

A redescription of Cylicocyclus adersi Boulenger, 1920 (Nematoda: Strongyloidea: Cyathostominae) parasitic in equids.

As part of a revision of the systematics of the Cyathostominea of equids, we determined that the single type-specimen of Cylicocyclus adersi Boulenger, 1920 is missing and a modern description of this rare species parasitic in donkeys Equus asinus L. and zebras E. burchelli (Gray) and E. zebra L. is not available. Systematists at a workshop on the systematics of the Cyathostominea of horses, convened on August 11, 1997 in Sun City, South Africa, listed C. adersi as a species inquirenda . Herein, we provide a redescription of C. adersi and propose a neotype for the species. The distinctive shape of the buccal capsule of C. adersi , with a relatively tiny 'hoop-like thickening' at its base, a relatively small oesophageal funnel with a thick cuticular lining, and a short, but distinct, dorsal gutter readily distinguishes this species from other large species of Cylicocyclus Ihle, 1922.

Review of Papillostrongylus Johnston & Mawson, 1939 (Nematoda: Strongyloidea) from wallabies and kangaroos (Marsupialia: Macropodidae) using morphological and molecular techniques, with the description of P. barbatus n. sp.

The strongyloid nematode genus Papillostrongylus Johnston & Mawson, 1939, from kangaroos and wallabies, is reviewed using morphological and molecular methods. P. labiatus Johnston & Mawson, 1939 is re-described from material from the type-host, the black-striped wallaby Macropus dorsalis, from eastern Queensland, Australia, in which it is a relatively common parasite. Additional records from M. parryi and Thylogale thetis are confirmed and considered to represent examples of host-switching. A geographically disjunct population of the nematode species occurs in M. bernardus and Petrogale brachyotis in Arnhem Land, Northern Territory, but assessment of its status requires additional material. Nematodes from M. rufus, M. giganteus, M. fuliginosus and M. robustus from inland regions of Australia, formerly attributed to P. labiatus, are here assigned to a new species, P. barbatus, distinguished by the presence of an external leaf-crown, larger size, by greater spicule length in the male and by a sinuous vagina in the female. Additional hosts of P. barbatus n. sp. are Petrogale assimilis and Pet. lateralis purpureicollis. Sequence analyses of the second internal transcribed spacer of ribosomal DNA (ITS-2) also showed that P. barbatus n. sp. differed at 40 (16.7%) of the 240 alignment positions when compared with P. labiatus. Most of these interspecific sequence differences occurred in loops or bulges of the predicted precursor rRNA secondary structure, or represented partial or total compenstory base pair changes in stems.

A comparative study of the ultrastructure of intestine in Protostrongylus commutatus and Protostrongylus rufescens (Protostrongylidae: Nematoda).

A comparison of the ultrastructure of intestinal cells of two taxonomically very close nematode species, Protostrongylus commutatus and Protostrongylus rufescens, revealed many interesting facts. The concentration of mitochondria in the basal part of intestinal cells, which is conspicuous particularly in P. rufescens, indicates that the nutrients penetrate also through the cuticle. An enormous occurrence of granular endoplasmic reticulum in the intestinal cells of P. commutatus shows an excessive protein synthesis. It may be supposed that the proteins will be used mainly for the construction of sex products, which are extremely abundant in P. commutatus. This phenomenon will be related with the conditions of egg development and localization of the worm in the vicinity of trachea bifurcation in the lungs of hare.

Scanning electron microscopy of Strongylus spp. in zebra.

The external ultrastructure of the anterior and posterior extremities of the nematodes, Strongylus asini , Strongylus vulgaris, Strongylus equinus and Strongylus edentatus, was studied with scanning electron microscopy (SEM). Fresh specimens of S. asini were collected from the caecum, ventral colon and vena portae of Equus burchelli and Equus zebra hartmannae ; S. vulgaris from the caecum, colon and arteria ileocolica of E. burchelli ; S. equinus from the ventral colon of E. z. hartmannae and S. edentatus from the caecum and ventral colon of both zebras , during surveys of parasites in zebras in the Etosha Game Reserve, South West Africa/Namibia, and the Kruger National Park, Republic of South Africa. The worms were cleaned, fixed and mounted by standard methods and photographed in a JEOL JSM - 35C scanning electron microscope (SEM) operating at 12kV . The SEM showed the following differences: the tips of the external leaf-crowns varied and were fine and delicate in S. asini , coarse and broad in S. vulgaris and, in S. equinus and S. edentatus, closely adherent, separating into single elements for half their length. The excretory pores showed only slight variation, and the morphology of the copulatory bursae did not differ from those seen with light microscopy. The genital cones differed markedly: S. asini had a ventral triangular projection and laterally 2 finger-like projections: in S. vulgaris there were numerous bosses on the lateral and ventral aspects of the cone; in S. equinus 2 finger-like processes projected laterocaudally ; and in S. edentatus 2 pairs of papilla-like processes projected laterally on the ventral aspects, and a pair of rounded projections and a pair of hair-like structures adorned the dorsal aspects.(ABSTRACT TRUNCATED AT 250 WORDS)

SEM study of Strongylus vulgaris larva-induced arteritis in the pony.

This paper describes the histological and scanning electron microscopical examinations of the right colic artery of eight ponies. Lesions all had large thrombi surrounding a larva or larvae, with arterial wall thickening. Endothelial shape change, degeneration and loss were present. Fibrin-platelet red blood cell aggregates were present on endothelial surfaces as well as on the surface of thrombi. Damage to the intima appeared to produce the conditions for progressive thrombus formation.

Influence of the anthelmintic mebendazole on microtubules and intracellular organelle movement in nematode intestinal cells.

The ultrastructural changes in the intestines of Ascaris suum and Syngamus trachea, induced by in vivo treatment of the hosts with the anthelmintic mebendazole, are reported. The primary site of drug action seemed to be the organelles involved in the secretory mechanism of the intestinal cells. The block in the transport of secretory granules and in the movement of other subcellular organelles coincided clearly with the disappearance of cytoplasmic microtubules. On the other hand, the microtubular system of the host cells was unaffected by the treatment. Degenerative changes in the intestinal cells of the parasites observed afterwards were correlated with the primary deteriorative effect of the drug on cytoplasmic microtubules.