Single Nematode Research Articles

Spread and transmission of bacterial pathogens in experimental populations of the nematode Caenorhabditis elegans.

Caenorhabditis elegans is frequently used as a model species for the study of bacterial virulence and innate immunity. In recent years, diverse mechanisms contributing to the nematode's immune response to bacterial infection have been discovered. Yet despite growing interest in the biochemical and molecular basis of nematode-bacterium associations, many questions remain about their ecology. Although recent studies have demonstrated that free-living nematodes could act as vectors of opportunistic pathogens in soil, the extent to which worms may contribute to the persistence and spread of these bacteria has not been quantified. We conducted a series of experiments to test whether colonization of and transmission between C. elegans nematodes could enable two opportunistic pathogens (Salmonella enterica and Pseudomonas aeruginosa) to spread on agar plates occupied by Escherichia coli. We monitored the transmission of S. enterica and P. aeruginosa from single infected nematodes to their progeny and measured bacterial loads both within worms and on the plates. In particular, we analyzed three factors affecting the dynamics of bacteria: (i) initial source of the bacteria, (ii) bacterial species, and (iii) feeding behavior of the host. Results demonstrate that worms increased the spread of bacteria through shedding and transmission. Furthermore, we found that despite P. aeruginosa's relatively high transmission rate among worms, its pathogenic effects reduced the overall number of worms colonized. This study opens new avenues to understand the role of nematodes in the epidemiology and evolution of pathogenic bacteria in the environment.

On the Helminthofauna of Moschus Moschiferus

A helminthological study of the digestive tracts of 15 Far Eastern musk deer (Moschus moschiferus turovi) of Primorsky Krai detected single nematodes, which were assigned to the following taxons: Spiculopteragia spiculoptera, Nematodirus filicolis, Pygarginema skrjabini, and Trichuris sp. The species S. spiculoptera was registered for musk deer for the first time. Analyses of the literature data on the species composition of musk deer helminths concluded that there was a necessity for further helminthological studies examining these ruminants.

Distribution and evolution of glycoside hydrolase family 45 cellulases in nematodes and fungi.

BackgroundHorizontal gene transfer (HGT) has been suggested as the mechanism by which various plant parasitic nematode species have obtained genes important in parasitism. In particular, cellulase genes have been acquired by plant parasitic nematodes that allow them to digest plant cell walls. Unlike the typical glycoside hydrolase (GH) family 5 cellulase genes which are found in several nematode species from the order Tylenchida, members of the GH45 cellulase have only been identified in a cluster including the families Parasitaphelenchidae (with the pinewood nematode Bursaphelenchus xylophilus) and Aphelenchoididae, and their origins remain unknown.ResultsIn order to investigate the distribution and evolution of GH45 cellulase genes in nematodes and fungi we performed a wide ranging screen for novel putative GH45 sequences. This revealed that the sequences are widespread mainly in Ascomycetous fungi and have so far been found in a single major nematode lineage. Close relationships between the sequences from nematodes and fungi were found through our phylogenetic analyses. An intron position is shared by sequences from Bursaphelenchus nematodes and several Ascomycetous fungal species.ConclusionsThe close phylogenetic relationships and conserved gene structure between the sequences from nematodes and fungi strongly supports the hypothesis that nematode GH45 cellulase genes were acquired via HGT from fungi. The rapid duplication and turnover of these genes within Bursaphelenchus genomes demonstrate that useful sequences acquired via HGT can become established in the genomes of recipient organisms and may open novel niches for these organisms to exploit.

Exploitation of FTA Cartridges for the Sampling, Long-Term Storage, and DNA-Based Analyses of Plant-Parasitic Nematodes

The use of DNA-based analyses in molecular plant nematology research has dramatically increased over recent decades. Therefore, the development and adaptation of simple, robust, and cost-effective DNA purification procedures are required to address these contemporary challenges. The solid-phase-based approach developed by Flinders Technology Associates (FTA) has been shown to be a powerful technology for the preparation of DNA from different biological materials, including blood, saliva, plant tissues, and various human and plant microbial pathogens. In this work, we demonstrate, for the first time, that this FTA-based technology is a valuable, low-cost, and time-saving approach for the sampling, long-term archiving, and molecular analysis of plant-parasitic nematodes. Despite the complex structure and anatomical organization of the multicellular bodies of nematodes, we report the successful and reliable DNA-based analysis of nematode high-copy and low-copy genes using the FTA technology. This was achieved by applying nematodes to the FTA cards either in the form of a suspension of individuals, as intact or pestle-crushed nematodes, or by the direct mechanical printing of nematode-infested plant tissues. We further demonstrate that the FTA method is also suitable for the so-called "one-nematode-assay", in which the target DNA is typically analyzed from a single individual nematode. More surprisingly, a time-course experiment showed that nematode DNA can be detected specifically in the FTA-captured samples many years after initial sampling occurs. Collectively, our data clearly demonstrate the applicability and the robustness of this FTA-based approach for molecular research and diagnostics concerning phytonematodes; this research includes economically important species such as the stem nematode (Ditylenchus dipsaci), the sugar beet nematode (Heterodera schachtii), and the Northern root-knot nematode (Meloidogyne hapla).

Adult of Dirofilaria repens in a dog with recurrent multiple subcutaneous nodular lesions

Subcutaneous dirofilariosis is a zoonosis parasitizing the dog, transmitted by Culicidae mosquitoes. An increasing number of new cases have been reported both in dogs and in humans all over the world. The aim of this work was to describe an uncommon case of recurrent multiple subcutaneous nodules by Dirofilaria repens in a 2-year-old dog. Surgical excision of two nodules on both hind limbs revealed a single thread-like live nematode and multiple parasite fragments, respectively. The parasite was morphologically identified as an adult female of D. repens. Haematoxylin-eosin tissue sections of nodules revealed a diffuse pyogranulomatous angiocentric dermatitis with an intense eosinophilic infiltrate throughout the dermis. One month post-surgery, a new nodular lesion was detected on the right thigh and resolved spontaneously in a few weeks. During the following 3 months, two other nodules were briefly noted both on the right hip and on the left side of the chest. The dog was periodically treated with milbemycin oxime and praziquantel, starting a week before the appearance of the first nodule. Microfilariae were not detected neither in the bloodstream nor in fine-needle aspirates at any time, and the diagnosis was made only after surgery. Practitioners should consider dirofilariosis in the differential diagnosis of subcutaneous swellings, especially if they have a recurrent behaviour, previously described in humans but not in dogs.

Molecular Characterization and Development of Real-Time PCR Assay for Pine-Wood Nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

Bursaphelenchus xylophilus, the pine-wood nematode (PWN), is the causal agent of pine wilt disease, one of the most damaging emerging pest problems to forests around the world. It is native to North America where it causes relatively minor damage to native conifers but is labeled an EPPO-A-2 pest and a quarantine nematode for many countries outside of the United States because of its potential for destruction to their native conifers. Exports of wood logs and commodities involving softwood packaging materials now require a lab test for the presence/absence of this regulated nematode species. We characterized the DNA sequences on the ribosomal DNA small subunit, large subunit D2/D3, internal transcribed spacer (ITS) and mitochondrial DNA cytochrome oxidase subunit one on the aphelenchid species and described the development of a real-time-PCR method for rapid and accurate identification of PWN targeting the ITS-1. A total of 97 nematode populations were used to evaluate the specificity and sensitivity of this assay, including 45 populations of B. xylophilus and 36 populations of 21 other species of Bursaphelenchus which belong to the abietinus, cocophilus, eggersi, fungivorus, hofmanni, kevini, leoni, sexdentati, and xylophilus groups and one unassigned group from a total of 13 groups in the genus Bursaphelenchus; 15 populations of Aphelenchoides besseyi, A. fragariae, Aphelenchoides species and Aphelenchus avenae; and one population of mixed nematode species from a soil sample. This assay proved to be specific to B. xylophilus only and was sensitive to a single nematode specimen regardless of the life stages present. This approach provides rapid species identification necessary to comply with the zero-tolerance export regulations.

First evidence of cryptic species diversity and significant population structure in a widespread freshwater nematode morphospecies (Tobrilus gracilis)

Free-living nematodes are ubiquitous and highly abundant in terrestrial and aquatic environments, where they sustain ecosystem functioning by mineralization processes and nutrient cycling. Nevertheless, very little is known about their true diversity and intraspecific population structure. Recent molecular studies on marine nematodes indicated cryptic diversity and strong genetic differentiation of distinct populations, but for freshwater nematode species, analogous studies are lacking. Here, we present the first extensive molecular study exploring cryptic species diversity and genetic population structure of a widespread freshwater nematode morphospecies, Tobrilus gracilis, from nine postglacially formed European lakes. Taxonomic species status of individuals, analysed for fragments of the mitochondrial COI gene and for the large (LSU) and small (SSU) ribosomal subunits, were determined by morphological characteristics. Mitochondrial and nuclear markers strongly supported the existence of three distinct genetic lineages (Tg I-III) within Tobrilus gracilis, suggesting that this morphospecies indeed represents a complex of highly differentiated biological species. High genetic diversity was also observed at the population level. Across the nine lakes, 19 mitochondrial, and seven (LSU) and four (SSU) nuclear haplotypes were determined. A phylogeographical analysis revealed remarkable genetic differentiation even among neighbouring lake populations for one cryptic lineage. Priority and persistent founder effects are possible explanations for the observed population structure in the postglacially colonized lakes, but ask for future studies providing direct estimates of freshwater nematode dispersal rates. Our study suggests therefore that overall diversity of limnetic nematodes has been so far drastically underestimated and challenges the assumed ubiquitous distribution of other, single freshwater nematode morphospecies.

Genetic diversity of the root‐knot nematode Meloidogyne ethiopica and development of a species‐specific SCAR marker for its diagnosis

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species‐specific SCAR‐PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.

Characterization of the Two Intra-Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode, Rotylenchulus reniformis

The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.

Molecular survey of parasites in introduced Pelophylax perezi (Ranidae) water frogs in the Azores

Water frogs, Pelophylax perezi, that are introduced in the Azores, were screened for parasites using PCR primers known to amplify Apicomplexa parasites, and using nematode-specific primers. With the former, three different organisms were detected: Hepatozoon, a trichodinid protozoan ciliate and a possible Stramenopile. Using the latter set of primers, a single unknown spirurid nematode was also detected. Phylogenetic analyses indicate that Hepatozoon detected within amphibian hosts appear to form a clade, although relationships of these parasites do not match the vertebrate intermediate host phylogeny. Regarding the possible Stramenopile, it is unclear whether this organism was actually present on the amphibian or in the water on the surface of the tissue sample. Our findings highlight that many different organisms can be detected with these primers and that they can be used to screen introduced host populations to detect parasites that have been brought with them.

ASCARIDIASIS IN PEAFOWLPAVO CRISTATUS(PHASIANIDAE) DUE TOASCARIDIA GALLISCHRANK, 1788

Twelve white peafowl (Pavo cristatus) affected by an outbreak of an intestinal disease were referred for more detailed examination at the Universidade Estadual de Santa Cruz-BA, Brazil. During the course of the disease, peachicks were severely affected, with enteric signs such as diarrhea plus dehydration, decreased feed intake and progressive weight loss. After examination, 8 of 12 samples (66.6%) presented single or mixed nematode infection and Ascarid eggs were the most frequent finding on fecal examination. Adult peafowl did not present clinical signs even when positive after fecal exam. Morphological analysis, clinical signs, fecal and gross examinations resulted in a diagnosis of ascaridiasis caused by Ascaridia galli Schrank (1788).

DNA preparation method in eggs, immature stages, and diapausing females of Tetranychus spider mites (Acari: Tetranychidae) for diagnostic PCR-RFLP

A DNA preparation method for diapausing females, immature stages, and eggs of spider mites for diagnostic analyses using restriction fragment length polymorphisms after polymerase chain reaction (PCR-RFLP analysis) of the internal transcribed spacer (ITS) region including 5.8 S of the nucleic ribosomal DNA was developed using Tetranychus urticae Koch as a typical example of tetranychid mites. In diapausing females and deutonymphs, the method of crushing an individual mite with a glass rod was eminently successful and a useful method to obtain the DNA template for PCR. For protonymphs, larvae, and eggs older than 48 h after oviposition at 24 °C, the DNA preparation method for a single nematode, crushing a tiny sample with a filter paper chip, was also successful and a useful method to obtain the DNA template from mite individuals (eggs) for PCR. Consequently, we found a single distinctive band of an amplified DNA fragment after electrophoresis in all developmental stages, and digestion of the PCR product by a restriction endonuclease, DraI, resulted in identical banding patterns being exhibited in all developmental stages. Our findings indicate that PCR-RFLP analysis of the ITS region can be used for species identification of diapausing females, immature stages, and eggs of Tetranychus species.

Mermithid nematodes found in adult Anopheles from southeastern Senegal

BackgroundOver two dozen mermithid nematodes have been described parasitizing mosquitoes worldwide, however, only two species were found in Africa. Mermithid nematodes kill their mosquito host upon emergence, which suggests that they could be developed as biological control agents of mosquitoes. Both Romanomermis culicivorax and Romanomermis iyengari have been reared for mass release to control numerous Anopheles species vector populations, and in one instance this may have led to reduced malaria prevalence in a human population.MethodsAnopheles mosquitoes were collected during a malaria study in southeastern Senegal. Two different adult blood fed mosquitoes had a single mermithid nematode emerge from their anus while they were being held post-capture. Primers from the 18 S rDNA were developed to sequence nematode DNA and screen mosquitoes for mermithid DNA. 18 S rDNA from the Senegalese mermithid and other mermithid entries in GenBank were used to create a Maximum Parsimony tree of the Mermithidae family.ResultsThe mermithid was present in 1.8% (10/551) of the sampled adult Anopheles species in our study area. The mermithid was found in An. gambiae s.s., An. funestus, and An. rufipes from the villages of Ndebou, Boundoucondi, and Damboucoye. Maximum parsimony analysis confirmed that the nematode parasites found in Anopheles were indeed mermithid parasites, and of the mermithid sequences available in GenBank, they are most closely related to Strelkovimermis spiculatus.ConclusionsTo our knowledge, this is the first report of mermithids from adult Anopheles mosquitoes in Senegal. The mermithid appears to infect Anopheles mosquitoes that develop in diverse larval habitats. Although maximum parsimony analysis determined the mermithid was closely related to Strelkovimermis spiculatus, several characteristics of the mermithid were more similar to the Empidomermis genus. Future mermithid isolations will hopefully allow: formal taxonomic identification, laboratory colonization, determination of life history traits and species specificity, and characterize its usefulness as a biological control agent.

Simple and quick methods for nematode DNA preparation

A DNA extraction kit, ISOHAIR® (Nippon Gene), which was originally developed for preparing DNA from hair and nail samples, was used to prepare nematode DNA for PCR and sequencing analyses. The methods provided here, which involved digesting (resolving) a single nematode individual in a tube containing the mixed enzyme solution, enabled the DNA to be prepared within 20 min. The prepared DNA was suitable for PCR amplification of near-full-length small subunit ribosomal RNA (ca 1.7 kb), of the D2/D3 expansion segments of large subunit RNA (ca. 0.7 kb), and of partial mitochondrial COI (ca. 0.7 kb) genes, followed by sequencing analysis. Furthermore, the prepared material could be preserved in a freezer (−30 °C) for at least 20 months, and more than 300 PCR reactions could be performed from a single individual nematode.

Rapid and highly sensitive detection of single nematode via direct MALDI Mass Spectrometry

For the first time, we demonstrate the feasibility for the use of MALDI-TOF MS for rapid, direct and sensitive detection of single adult root-knot nematode, Meloidogyne incognita and their second stage juvenile (J2). We have proposed simple pretreatment protocols and have demonstrated that the crushed and washed nematodes yielded better spectra. We also report the differentiation between the harmless and harmful stages of the nematode based on mass spectrometric profiling. Peaks at m/z 4350, 4692, 4933, 8725 were only present in the adult stages, while m/z 3220, 3433, 3485, 3830, 6540, 7444, 7770 were unique to the J2 infective stage. The only common peak to both the phases was at m/z 3277. Thus, we show that MALDI-TOF MS can be used to differentiate between the infective and non-infective stage of the nematode and the detection sensitivity of MALDI-MS could be applied to a single nematode analysis.

Baylisascaris procyonisInfection in Elderly Person, British Columbia, Canada

<i>Baylisascaris procyonis</i>Infection in Elderly Person, British Columbia, Canada

Anisakis pegreffii experimental challenge in Dicentrarchus labrax: An endoscopic study

To validate the use of imaging techniques to evaluate tissue damage in vivo, ten European sea bass (Dicentrarchus labrax) were anesthetized with MS-222 and experimentally infected with nematode larvae collected from three Lepidopus caudatus. Live nematode larvae were introduced by gastroscopy into the stomach of ten fish. Another challenge was performed 15days later. After a 60day period, an explorative celioscopy was carried out. Fish were examined for Anisakis larvae and biopsy samples were collected. Parasites sampled from L. caudatus and D. labrax were morphologically identified at genus level. Biopsy samples were routinely processed for histopathology. Some larvae were sampled for PCR. At gastroscopic exam 15days after the first challenge some live nematode larvae were found within the lumen of the stomach in four fish. Two months later, at celioscopy, four sea bass showed hemorrhages and/or irregular neoformations within the celomic cavity. Within each of these changes a single nematode larva was detected. Histopathology showed hemorrhagic lesions with a cystic aspect. Parasite larvae were identified as L3 stages Anisakis Type I. PCR-RFLP analysis has allowed the identification of larvae isolated as Anisakis pegreffii. Endoscopy is a suitable noninvasive procedure for evaluation of experimental challenges in sea bass using live Anisakis larvae. The evidence of tissue change is an unusual occurrence in fish parasitized by Anisakis. The paper sets out to assess the damage anisakid nematodes induce in infected fish by imaging techniques.

Infection patterns ofParadollfusnema amphisbaenia(Nematoda: Cosmocercidae) in a population ofAmphisbaena wuchereri(Squamata: Amphisbaenidae) from Minas Gerais state, south-eastern Brazil, and its relations with host size, sex and fat body mass

Specimens (n= 41) of the amphisbaenid Amphisbaena wuchereri taken from a population in Minas Gerais state, south-eastern Brazil, were examined for gastrointestinal parasites. A single nematode species was found, Paradollfusnema amphisbaenia. This was a new host record for this nematode species. This parasite was encountered in the large intestine (prevalence of 100%), in the stomach (prevalence of 2%) and in the small intestine (prevalence of 7.3%). The intensity of infection ranged from 1 to 457 individual parasites per host and was positively correlated with body size of both male and female amphisbaenians. The discrepancy index (D) indicated that P. amphisbaenia tended to an even distribution in this host population. The nematode, which did not affect fat body mass, induced inflammatory infiltrations in the small intestine, indicating that the parasites might injure the host's organs.

Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. The international economic impact of the introduction of the PWN into new areas has highlighted the need for the development of accurate and reliable detection methods of B. xylophilus, which are essential to define aspects of its control and management. In the present study, a methodology was developed for the direct detection of PWN by conventional PCR assay, with a species specific set of primers based on PWN satellite DNA, using total DNA extracted directly from maritime pine, Pinus pinaster, wood and bark samples, and from the insect vector, Monochamus galloprovincialis. This methodology involves homogenisation of wood, bark and insects using liquid nitrogen, DNA extraction and one or two PCR amplification steps, which permit the rapid and direct detection of one single nematode present in 100 mg of wood and bark and in one entire insect without the preliminary steps of nematode extraction.

A Perimotor Framework Reveals Functional Segmentation in the Motoneuronal Network Controlling Locomotion inCaenorhabditis elegans

The neuronal connectivity dataset of the nematode Caenorhabditis elegans attracts wide attention from computational neuroscientists and experimentalists. However, the dataset is incomplete. The ventral and dorsal nerve cords of a single nematode were reconstructed halfway along the body and the posterior data are missing, leaving 21 of 75 motoneurons of the locomotor network with partial or no connectivity data. Using a new framework for network analysis, the perimotor space, we identified rules of connectivity that allowed us to approximate the missing data by extrapolation. Motoneurons were mapped into perimotor space in which each motoneuron is located according to the muscle cells it innervates. In this framework, a pattern of iterative connections emerges which includes most (0.90) of the connections. We identified a repeating unit consisting of 12 motoneurons and 12 muscle cells. The cell bodies of the motoneurons of such a unit are not necessarily anatomical neighbors and there is no obvious anatomical segmentation. A connectivity model, composed of six repeating units, is a description of the network that is both simplified (modular and without noniterative connections) and more complete (includes the posterior part) than the original dataset. The perimotor framework of observed connectivity and the segmented connectivity model give insights and advance the study of the neuronal infrastructure underlying locomotion in C. elegans. Furthermore, we suggest that the tools used herein may be useful to interpret, simplify, and represent connectivity data of other motor systems.