Ancylostoma caninum: the finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm

Bhopale, V. M., Kupprion, E. K., Ashton, F. T., Boston, R., and Schad, G. A. 2001. Ancylostoma caninum: The finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm. Experimental Parasitology 97, 70-76. In the amphids (anteriorly positioned, paired sensilla) of the free-living nematode Caenorhabditis elegans, the so-called finger cells (AFD), a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors. A similar neuron pair in the amphids of the parasitic nematode Haemonchus contortus is also known to be thermoreceptive. The hookworm of dogs, Ancylostoma caninum, has apparent structural homologs of finger cells in its amphids. The neuroanatomy of the amphids of A. caninum and H. contortus is strikingly similar, and the amphidial cell bodies in the lateral ganglia of the latter nematode have been identified and mapped. When the lateral ganglia of first-stage larvae (L1) of A. caninum are examined with differential interference contrast microscopy, positional homologs of the recognized amphidial cell bodies in the lateral ganglia of H. contortus L1 are readily identified in A. caninum. The amphidial neurons in A. caninum were consequently given the same names as those of their apparent homologs in H. contortus. It was hypothesized that the finger cell neurons (AFD) might mediate thermotaxis by the skin-penetrating infective larvae (L3) of A. caninum. Laser microbeam ablation experiments with A. caninum were conducted, using the H. contortus L1 neuronal map as a guide. A. caninum L1 were anesthetized and the paired AFD class neurons were ablated. The larvae were then cultured to L3 and assayed for thermotaxis on a thermal gradient. L3 with ablated AFD-class neuron pairs showed significantly reduced thermotaxis compared to control groups. The thermoreceptive function of the AFD-class neurons associates this neuron pair with the host-finding process of the A. caninum infective larva and shows functional homology with the neurons of class AFD in C. elegans and in H. contortus.     

Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi

RNA interference (RNAi) has been used widely as a tool for examining gene function and a method that allows its use with plant-parasitic nematodes recently has been described. Here, we use a modified method to analyze the function of secreted beta-1,4, endoglucanases of the potato cyst nematode Globodera rostochiensis, the first in vivo functional analysis of a pathogenicity protein of a plant-parasitic nematode. Knockout of the beta-1,4, endoglucanases reduced the ability of the nematodes to invade roots. We also use RNAi to show that gr-ams-1, a secreted protein of the main sense organs (the amphids), is essential for host location.     

The plant apoplasm is an important recipient compartment for nematode secreted proteins

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode.     

The neurons of class ALD mediate thermotaxis in the parasitic nematode, Strongyloides stercoralis

Strongyloides stercoralis, a skin-penetrating nematode parasite of homeotherms, migrates to warmth. In nematodes, the amphids, anteriorly positioned, paired sensilla, each contain a bundle of sensory neurons. In the amphids of the free-living nematode Caenorhabditis elegans, a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors, and have been named the finger cells (class AFD). A similar neuron pair in the amphids of the parasite Haemonchus contortus is also known to be thermosensory. Strongyloides stercoralis lacks finger cells but, in its amphids, it has a pair of neurons whose dendrites end in a multi-layered complex of lamellae, the so-called lamellar cells (class ALD). Consequently, it was hypothesised that these lamellar cells might mediate thermotaxis by the skin-penetrating infective larva of this species. To investigate this, first stage S. stercoralis larvae were anaesthetised and the paired ALD class neurons were ablated with a laser microbeam. The larvae were then cultured to the infective third stage (L3) and assayed for thermotaxis on a thermal gradient. L3 with ablated ALD class neuron pairs showed significantly reduced thermotaxis compared with control groups. The thermoreceptive function of the ALD class neurons (i) associates this neuron pair with the host-finding process of S. stercoralis and (ii) demonstrates a functional similarity with the neurons of class AFD in C. elegans. The structural and positional characteristics of the ALD neurons suggest that these neurons may, in fact, be homologous with one pair of flattened dendritic processes known as wing cells (AWC) in C. elegans, while their florid development and thermosensory function suggest homology with the finger cells (AFD) of that nematode.     

Monoclonal antibodies reactive with secreted-excreted products from the amphids and the cuticle surface of Globodera pallida affect nematode movement and delay invasion of potato roots

This paper describes Excreted-secreted proteins (ES) proteins that were immunolocalised in the cuticle, amphids and subventral glands of second stage juveniles of the two species of potato cyst nematodes (Globodera pallida and Globodera rostochiensis). Monoclonal antibodies reactive with these ES proteins were used in a bioassay to investigate their effect on nematode movement and on their ability to invade potato roots. Antibodies recognising the nematode cuticle surface and the amphids affected nematode movement and delayed nematode penetration of roots. These effects were temporary, since the nematodes were able to recover and infect potato roots. Movement of second stage juveniles treated with the antibodies was impaired for the first 30 min after inoculation: the juveniles remained close to the point of introduction and moved slowly and abnormally. They recovered normal movement after 1–2 h, possibly because the turnover rate of the secreted proteins meant that they were no longer blocked by the monoclonal antibodies. No effect was observed on second stage juveniles treated with an antibody reactive with secretions from the oesophageal glands. Nematodes treated with antibodies reactive with the nematode cuticle surface were notably more affected than those treated with other antibodies; nematodes failed to recover movement when in continuous contact with the antibodies. It is possible that the physical presence of the antibodies on the nematode surface affected their motility. Nematodes treated with antibodies reactive with secretions from the amphids were temporarily unable to move towards potato roots and their exploratory behaviour was greatly affected by the antibody treatment. Whether these antibodies were able to inhibit temporarily the function of the amphids or this effect was due to physical presence of the antibodies blocking the amphidial pore remains to be determined.     

Some ultrastructural observations on the microfilaria of Breinlia sergenti--nervous system

The amphids, cephalic papillae, phasmids, excretory complex, anal vesicle, rectal cells, G cell and the nerve ring are described in the microfilaria of Breinlia sergenti. The significance of these various structures in playing a nervous role in this organism is discussed, and a working hypothesis presented to explain the nervous co-ordination in this parasitic stage.     

Amphids: the neuronal ultrastructure of macrocyclic-lactone-resistant Haemonchus contortus

The development of anthelmintic resistance by nematode parasites is a growing problem for veterinarians and producers. The intensive use of the macrocyclic lactones for the treatment of a variety of parasitic diseases has hastened the development of resistance to this family of parasiticides among sheep, goats and cattle. As a result, resistance to ivermectin, moxidectin and doramectin by Haemonchus contortus has been documented throughout the world. While the exact sites of action of the macrocyclic lactones remain incompletely known, a critical point of entry for these drugs may be the terminally exposed sensory major neurons located in the cephalic end of the worms. These neurons, called amphidial neurons, are located in a pair of channels, the amphids, on either side of the pharynx and are exposed to the external environment via pores at the anterior tip of the worm. Through these neurons, important chemical and thermal cues are gathered by the parasite. Examination of serial electron micrographs of ivermectin-susceptible and ivermectin-resistant H. contortus allows for comparison of neuronal structure, arrangement of neurons within the amphidial channel, and distance of the tip of the dendritic processes to the amphidial pore. The latter of these characteristics provides a useful means by which to compare the association between the neurons and the external environment of the worm. Comparison of parental laboratory strains of ivermectin-susceptible H. contortus with related selected, ivermectin-resistant strains and with a wild-type ivermectin-susceptible field strain of H. contortus from Louisiana reveals that the ivermectin-resistant worms examined have markedly shorter sensory cilia than their ivermectin-susceptible parental counterparts. Additionally, the amphidial neurons of ivermectin-resistant worms are characterized by generalized degeneration and loss of detail, whereas other neurons outside of the channels, such as the labial and cephalic neurons, are normal in structure. Similar degeneration was also observed in field strains of doramectin-resistant H. contortus collected from a New Jersey alpaca heard. These findings raise a number of questions regarding the relationship between amphidial structure and macrocyclic lactone resistance as well as the role of amphids as a means of entry for these molecules. While shortened amphidial sensilla are associated with ivermectin resistance, it remains unclear if such a structural modification facilitates survival of nematodes exposed to the macrocyclic lactones.     

Strongyloides ratti: chemokinesis of glycolytic enzyme- and lectin-treated third-stage infective larvae in vitro

The infective third-stage larvae (L3s) of Strongyloides ratti, a parasitic nematode in rodents, showed two types of chemokinesis on a gradient of sodium chloride (NaCl) in an in vitro agarose tracking assay. The types were a consistent directional avoidance behavior under unfavorable environmental conditions and a reduced avoidance behavior under favorable conditions. We examined the effects of treatments with glycolytic enzymes and lectins by analyzing the avoidance behavior. L-Fucose dehydrogenase, hyaluronidase, beta-glucosidase, alpha-mannosidase, beta-galactosidase, concanavalin A, wheat germ agglutinin and soybean agglutinin exhibited inhibitory or enhancive effects on chemokinesis. We also confirmed the sites of the amphids of L3s aside from the mouth at the anterior end by scanning electron microscopy, and that concanavalin A-binding sites existed in the vicinity of the amphids using lectin-histochemistry. The carbohydrate moieties in the amphids of S. ratti L3s may play an important role as chemosensors in perceiving environmental cues.     

Larval nematodes (Spiruroidea: Cystidicolidae) in Octopus vulgaris (Mollusca: Cephalopoda: Octopodidae) from the northeastern Atlantic Ocean.

Larval nematode parasites (Spiruroidea: Cystidicolidae) are recorded for the first time in Octopus vulgaris Cuvier, 1797 in the northeastern Atlantic Ocean. Prevalence was 16% and mean intensity was 1.46 worms/host. Body length of larval nematodes ranges from 8.3 to 9.3 mm, with a distance from the anterior end to nerve ring from 187.5 to 200 microm, and to excretory pore 194.6-350 microm. Anatomical characteristics, such as deirid, nerve ring, cephalic alae, excretory pore, pseudolabia amphids, sclerotized protuberance, and anus, examined using light microscopy (LM) or scanning electron microscopy (SEM), are illustrated. The nematode was designed as a cystidicolid "Type A" larva. The hemocytic infiltration present in the host tissue around the nematode capsule and the mechanical compression in the infected organs denote the pathogenicity of this nematode. In the study area, O. vulgaris may play the role of an intermediate or paratenic host in the nematode life cycle.     

Proteins secreted by root-knot nematodes accumulate in the extracellular compartment during root infection

Root-knot nematodes are biotrophic parasites that invade the root apex of host plants and migrate towards the vascular cylinder where they induce the differentiation of root cells into hypertrophied multinucleated giant cells. Giant cells are part of the permanent feeding site required for nematode development into the adult stage. To date, a repertoire of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection has been identified. However, the precise role of these candidate effectors during root invasion or during giant cell induction and maintenance remains largely unknown. Primarily, the identification of the destination of nematode effectors within plant cell compartment(s) is crucial to decipher their actual functions. We analyzed the fine localization in root tissues of five nematode effectors throughout the migratory and sedentary phases of parasitism using an adapted immunocytochemical method that preserves host and pathogen tissues. We showed that secretion of effectors from the amphids or the oesophageal glands is tightly regulated during the course of infection. The analyzed effectors accumulated in the root tissues along the nematode migratory path and along the cell wall of giant cells, showing the apoplasm as an important destination compartment for these effectors during migration and feeding cell formation.Key words: plant pathogen, effector, immunocytochemistry, root-knot nematode, secretion, plant apoplasm     

RNAi in Haemonchus contortus: a potential method for target validation

RNA interference (RNAi) is a method for the functional analysis of specific genes, and is particularly well developed in the free-living nematode Caenorhabditis elegans. There have been several attempts to apply this method to parasitic nematodes. In a recent study undertaken in Haemonchus contortus, Geldhof and colleagues concluded that, although a mechanism for RNAi existed, the methods developed for RNAi in C. elegans had variable efficacy in this parasitic nematode. The potential benefits of RNAi are clear; however, further studies are required to characterize the mechanism present in parasitic nematodes, and to improve culture systems for these nematodes to monitor the long-term effects of RNAi. Only then could RNAi become a reliable assay of gene function.     

(Homo)glutathione deficiency impairs root-knot nematode development in Medicago truncatula

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes.     

The functions of Deoxyribonuclease II in immunity and development

Apoptosis, which is usually accompanied by DNA degradation, is important not only for the homeostasis of metazoans but also for mammalian development. If DNA is not properly degraded in these processes, it can cause diverse diseases, such as anemia, cataracts, and some autoimmune diseases. A large effort has been made to identify these nucleases that are responsible for these effects. In contrast to Deoxyribonuclease I (DNase I), Deoxyribonuclease II (DNase II) has been less well characterized in these processes. Additionally, enzymes of DNase II family in Trichinella spiralis, which is an intracellular parasitic nematode, are also considered involved in the development of the nematode. We have compiled information from studies on DNase II from various organisms and found some nonclassic features in these enzymes of T. spiralis. Here we have reviewed the characterization and functions of DNase II in these processes and predicted the functions of these enzymes in T. spiralis during host invasion and development.     

Mitochondrial genomes of parasitic nematodes--progress and perspectives

Mitochondria are subcellular organelles in which oxidative phosphorylation and other important biochemical functions take place within the cell. Within these organelles is a mitochondrial (mt) genome, which is distinct from, but cooperates with, the nuclear genome of the cell. Studying mt genomes has implications for various fundamental areas, including mt biochemistry, physiology and molecular biology. Importantly, the mt genome is a rich source of markers for population genetic and systematic studies. To date, more than 696 mt genomes have been sequenced for a range of metazoan organisms. However, few of these are from parasitic nematodes, despite their socioeconomic importance and the need for fundamental investigations into areas such as nematode genetics, systematics and ecology. In this article, we review knowledge and recent progress in mt genomics of parasitic nematodes, summarize applications of mt gene markers to the study of population genetics, systematics, epidemiology and evolution of key nematodes, and highlight some prospects and opportunities for future research.     

New oral linguiform projections and their associated neurons in the third-stage infective larva of the parasitic nematode Oesophagostomum dentatum

The infective larvae (L3i) of the nematode parasite of swine, Oesophagostomum dentatum, are passively ingested by their hosts. The L3i exhibit certain behaviors that are probably selected to increase the likelihood of ingestion, by strategic positioning in the environment. The larvae show positive geotactic behavior and respond to temperature variations in their environment, as shown by their behavior on a thermal gradient. To investigate neuronal control of this behavior, we initiated a study of the structure of the amphidial neurons of this parasite. The same number and types of neuronal dendritic processes are found in the amphids of the O. dentatum L3i as in those of its close relatives Haemonchus contortus and Ancylostoma caninum. Well-developed dendritic processes of wing cells are located in the amphidial sheath cells, these being similar to wing cells AWA in the free-living nematode Caenorhabditis elegans but actually more extensive. Similar to its close relatives just mentioned, and C. elegans as well, O. dentatum L3i has prominent finger cell processes, the finger cell neurons being the thermoreceptors in all 3 of the preceding species. However, unlike the arrangement seen in H. contortus and A. caninum, where the microvilli-like "fingers" of these neurons lie dorsal to the amphidial channel and occupy a very large portion (>50%) of the anterior end of the larva, the dendritic process of the finger cells in O. dentatum extends into unusual linguiform projections that, in turn, extend into the lumen of the mouth tube, a complex structural arrangement that has not been described for any other nematode.     

Morphology of Philometroides barbi (Nematoda: Philometridae), a rare tissue parasite of the Mediterranean barbel Barbus meridionalis (Osteichthyes)

A female specimen of the nematode Philometroides barbi Moravec, Simková, Hanzelová, Spakulová et Caki?, 2005, a little-known histozoic parasite of the Mediterranean Barbus meridionalis, was recorded from the fin of its fish host in Bulgaria. Scanning electron microscopical examination, used for the first time in this species, made it possible to determine the character of cephalic papillae (14 papillae arranged in 2 circles) and amphids and confirmed the presence of 4 large caudal lobes. These features distinctly distinguish P. barbi from Philometroides cyprini and other congeners parasitizing European fishes.     

The ultrastructure of the microfilaria of Brugia, Nematoda: Filarioidea

The microfilaria of Brugia pahangi is a differentiated nematode larva. The basic nematode body plan is present showing cuticle, hypodermis, dorsal, ventral, and lateral cords, muscle cells, longitudinal nerves, papillary nerves, amphids and phasmids. Secretory granules are present in ganglionic cells and in axons in the nerve ring. There is no differentiated pseudocoelom. There is only a single row of muscle cells between each pair of cords. The excretory cell complex is similar in structure to the hypodermal gland cells of other nematodes. The alimentary canal of the microfilaria is very much modified. The pharyngeal cells are attached to the pharyngeal thread which is circular in cross section and there is no pharyngeal musculature. The intestine is represented by the solid mass of the inner body within paired intestinal cells. The intestine is separated from the rectum. The three rectal cells form a syncytium of villi in the anal vesicle. The structure in Brugia is related to the ultrastructure of other microfilariae and it is concluded that the evolution of the modifications of the basic larval structure is due to the small size of these nematodes as a consequence of their adaptation to a parasitic mode of life in the capillaries of the vertebrate host with transmission through an intermediate arthropod vector.     

Signaling between nematodes and plants

After hatching in the soil, root-knot nematodes must locate and penetrate a root, migrate into the vascular cylinder, and establish a permanent feeding site. Presumably, these events are accompanied by extensive signaling between the nematode parasite and the host. Hence, much emphasis has been placed on identifying proteins that are secreted by the nematode during the migratory phase. Further progress in understanding the signaling events has been made recently by studying the host response. Striking parallels can be drawn between the nematode-plant interaction and plant symbioses with other microorganisms, and evidence is emerging to suggest that nematodes acquired components of their parasitic armory from those microbes.     

The N-glycosylation pattern of Caenorhabditis elegans

Determining the exact nature of N-glycosylation in Caenorhabditis elegans, a nematode worm and genetic model organism, has proved to have been an unexpected challenge in recent years; a wide range of modifications of its N-linked oligosaccharides have been proposed on the basis of structural and genomic analysis. Particularly mass spectrometric studies by a number of groups, as well as the characterisation of recombinant enzymes, have highlighted those aspects of N-glycosylation that are conserved in animals, those which are seemingly unique to this species and those which are shared with parasitic nematodes. These data, of importance for therapeutic developments, are reviewed.