Absence of <Emphasis Type="Italic">Wolbachia</Emphasis> in Nonfilariid Worms Parasitizing Arthropods

Wolbachia are strictly intracellular maternally inherited α-proteobacteria, largely widespread among arthropods and filariids (i.e., filarial nematodes). Wolbachia capacities to infect new host species have been greatly evidenced and the transfer of Wolbachia between arthropods and filariids has probably occurred more than once. Interestingly, among nematode species, Wolbachia infection was found in filariids but not in closely related lineages. Their occurrence in filariids has been supposed a consequence of the parasitic lifestyle of worms within Wolbachia-infected arthropods, implying that nonfilariid worms parasitizing arthropods are also likely to be infected by some Wolbachia acquired from their hosts. To further investigate this hypothesis, we have examined seven species of nonfilariid worms of Nematoda and Nematomorpha phyla, all interacting intimately with arthropods. Wolbachia infection in nonfilariid parasitic worms was never detected by polymerase chain reaction assays of the 16S rDNA and wsp genes. By contrast, some arthropod hosts are well infected by Wolbachia of the B supergroup. Then the intimate contact with infected arthropods is not a sufficient condition to explain the Wolbachia occurrence in filariids and could underline a physiological singularity or a particular evolutionary event to acquire and maintain Wolbachia infection.     

An endosymbiotic bacterium in a plant-parasitic nematode: Member of a new Wolbachia supergroup

Wolbachia is an endosymbiotic bacterium widely present in arthropods and animal-parasitic nematodes. Despite previous efforts, it has never been identified in plant-parasitic nematodes. Random sequencing of genes expressed by the burrowing nematode Radopholus similis resulted in several sequences with similarity to Wolbachia genes. The presence of a Wolbachia-like endosymbiont in this plant-parasitic nematode was investigated using both morphological and molecular approaches. Transmission electron microscopy, fluorescent immunolocalisation and staining with DAPI confirmed the presence of the endosymbiont within the reproductive tract of female adults.16S rDNA, ftsZ and groEL gene sequences showed that the endosymbiont of R. similis is distantly related to the known Wolbachia supergroups. Finally, based on our initial success in finding sequences of this endosymbiont by screening an expressed sequence tag (EST) dataset, all nematode ESTs were mined for Wolbachia-like sequences. Although the retained sequences belonged to six different nematode species, R. similis was the only plant-parasitic nematode with traces of Wolbachia. Based on our phylogenetic study and the current literature we designate the endosymbiont of R. similis to a new supergroup (supergroup I) rather than considering it as a new species. Although its role remains unknown, the endosymbiont was found in all individuals tested, pointing towards an essential function of the bacteria.     

Supergroup C Wolbachia,mutualist symbionts of filarial nematodes,have a distinct genome structure

Wolbachia pipientis is possibly the most widespread endosymbiont of arthropods and nematodes. While all Wolbachia strains have historically been defined as a single species, 16 monophyletic clusters of diversity (called supergroups) have been described. Different supergroups have distinct host ranges and symbiotic relationships, ranging from mutualism to reproductive manipulation. In filarial nematodes, which include parasites responsible for major diseases of humans (such as Onchocerca volvulus, agent of river blindness) and companion animals (Dirofilaria immitis, the dog heartworm), Wolbachia has an obligate mutualist role and is the target of new treatment regimens. Here, we compare the genomes of eight Wolbachia strains, spanning the diversity of the major supergroups (A–F), analysing synteny, transposable element content, GC skew and gene loss or gain. We detected genomic features that differ between Wolbachia supergroups, most notably in the C and D clades from filarial nematodes. In particular, strains from supergroup C (symbionts of O. volvulus and D. immitis) present a pattern of GC skew, conserved synteny and lack of transposable elements, unique in the Wolbachia genus. These features could be the consequence of a distinct symbiotic relationship between C Wolbachia strains and their hosts, highlighting underappreciated differences between the mutualistic supergroups found within filarial nematodes.     

The Root-Knot Nematode Producing Galls on Spartina alterniflora Belongs to the Genus Meloidogyne: Rejection of Hypsoperine and Spartonema spp.

Root-knot nematodes are a major group of plant-parasitic nematodes, but their sister group within the Tylenchida remains to be identified. To find the sister group and for any investigation of the evolutionary biology of the genus Meloidogyne, it would be useful to identify the most basal species within Meloidogyninae. Meloidogyne spartinae, a root-knot nematode parasitic on cordgrass (Spartina spp.), constitutes a potentially interesting early diverging (or at least highly divergent) root-knot nematode because it was originally described in a different genus, Hypsoperine (and later Spartonema), due to its unique anatomy and biology (although it was later put in synonymy by some, but not all, taxonomists). We have sequenced the whole 18S rDNA of this species and compared it to other sequences of this region that are available in GenBank for numerous Meloidogyne species. Phylogenetic analysis unambiguously locates the branch corresponding to M. spartinae as a lately diverging species, more closely related to M. maritima, M. duytsi or the M. ardenensis-hapla group. Thus, the distinction of a separate genus (Hypsoperine or Spartonema) for this species is not justified.     

Effects of crowding and temperature on Wolbachia infection density among life cycle stages of Aedes albopictus

Species of the genus Wolbachia are a group of Rickettsia-like, maternally-inherited bacteria (gram negative), which cause various reproductive alterations in their arthropod and nematode hosts including cytoplasmic incompatibility (CI), male-killing, parthenogenesis and feminization. They can be divided into supergroups such as A and B based on phylogenetic analysis of 16S rDNA sequences. In this study, we examined the relative infection densities of Wolbachia strains among life cycle stages in the mosquito, Aedes albopictus in terms of crowding effect and temperature effect. A. albopictus is known to be superinfected with both A- and B-supergroup Wolbachia which cause CI. The relative Wolbachia densities within each individual mosquito were determined and quantified by using real-time quantitative PCR assay based on the wsp gene. We found that B-supergroup Wolbachia strain densities in this host species were consistently and significantly higher than in the A-supergroup. Larval crowding also reduced adult size of mosquitoes. Our results show clearly that the higher densities of mosquito larvae cause lower densities of Wolbachia strains. Examination of the effect of temperature on Wolbachia density in each stage of the mosquito clearly revealed a significant decrease in bacterial density following exposure to elevated temperature (37 °C) in both males and females.     

A Cell-Based Screen Reveals that the Albendazole Metabolite,Albendazole Sulfone,Targets Wolbachia

Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts.     

Diagnostic Probes Targeting the Major Sperm Protein Gene That May Be Useful in the Molecular Identification of Nematodes

Discrimination of closely related nematode species is typically problematic when traditional identification characteristics are prone to intraspecific variation. In this study, a molecular approach that can distinguish Pratylenchus penetrans and P. scribneri is described. The approach uses universal primers in conjunction with polymerase chain reaction (PCR) to amplify equivalent fragments of the major sperm protein (msp) gene from any nematode. This gene fragment typically includes an intron of variable sequence. The presence of this highly variable segment in an otherwise conserved gene sequence allows P. penetrans and P. scribneri to be distinguished by either a species-specific amplification or by dot-blot hybridization. The approach is potentially of general utility in species-specific identification of nematodes.     

Nonsusceptibility of the Honey Bee,Apis mellifera (Hymenoptera: Apidae), to Steinernematid and Heterorhabditid Nematodes

We exposed honey bee workers and brood to four entomopathogenic nematode species under conditions normally encountered in the hive by spraying nematodes onto combs. Mortality of adult bees exposed to any of the nematode species was less than 10%, and there was no evidence of nematode infection when dead adults were dissected. To assess the impact of nematodes on brood, we used smaller-size honey combs placed in the second story (super) of a hive and large brood combs placed in the main section of the hive. Our results were inconsistent between these two experimental designs. The smaller honey combs sprayed with Steinernema carpocapsae contained the largest number of uncapped ceils, those sprayed with Heterorhabditis baeteriophora or S. riobravis contained an intermediate number of uncapped cells, and control combs and those sprayed with S. glaseri contained the fewest nmnber of uncapped cells. Large combs sprayed with S. riobravis contained more uncapped ceils than controls or those sprayed with S. carpocapsae, although the differences were not significant. Our results do not support the hypothesis that high-temperature-tolerant species of nematodes are necessarily more infective to honey bees.     

Heterorhabditidoides rugaoensis n. sp. (Rhabditida: Rhabditidae), a Novel Highly Pathogenic Entomopathogenic Nematode Member of Rhabditidae

A novel entomopathogenic nematode species, Heterorhabditidoides rugaoensis n. sp. RG081015, collected from Rugao, China, is described. The new species is morphologically very similar to H. chongmingensis but can be distinguished from it on the basis of some morphological characteristics, combined with molecular data and a cross-hybridization test. Males of the new species can be recognized on the basis of body length averaging 1396.2 μm; lateral field with one ridge; metastome isoglottoid with one hemispherical swellings comprised of two to three well-developed warts; asymmetric spicules; peloderan bursa. In IJs, EP = 134.5 μm; ES = 149.3 μm; tail length = 82.5 μm; and a = 20.5. Hermaphroditic females have four to five lateral ridges. The 18S rDNA and ITS sequences of the two nematodes share 99% and 98% identity, respectively. Phylogenetic trees of 18S rDNA and ITS indicate that the new species is most closely related to H. chongmingensis; thus, the two nematodes belong to the same genus. Failure of cross-hybridization between them indicates that nematode strain RG081015 is a novel species and is described herein as H. rugaoensis n. sp. The LC₅₀ of the novel species against Galleria mellonella were 24.35 IJs / ml within 48 hours of infection. Morphological characteristics, genetic similarity analyses, and phylogenetic relationships provide strong evidence that some species of Oscheius/Insectivora-group should be reassigned to the genus Heterorhabditidoides.     

Morphological and Molecular Identification of Globodera pallida Associated with Potato in Idaho

The identity of a newly discovered population of pale potato cyst nematode Globodera pallida associated with potato in eastern Idaho was established by morphological and molecular methods. Morphometrics of cysts and second-stage juveniles were generally within the expected ranges for G. pallida with some variations noted. The Idaho population and paratype material from Epworth, Lincolnshire, England, both showed variations in tail shape, with bluntly rounded to finely pointed tail termini. Compared to literature values for the paratypes, second-stage juveniles of the Idaho population had a somewhat shorter mean body length, and cysts had a slightly higher mean distance from the anus to the nearest edge of the fenestra. PCR-RFLP of the rDNA ITS region, sequence-specific multiplex PCR and DNA sequence comparisons all confirmed the identity of the Idaho population as G. pallida. The ITS rDNA sequence of the Idaho isolate was identical to those from York, England, and the Netherlands. Species-specific primers that can positively identify the tobacco cyst nematode Globodera tabacum were also developed, providing a new assay for distinguishing this species from G. pallida and the golden potato cyst nematode Globodera rostochiensis.     

Current Research on the Major Nematode Problems in Japan

Among important nematode species occurring in Japan, current research achievements with the following four nematodes are reviewed: 1) Soybean cyst nematode (SCN), Heterodera glycines - breeding for resistance, race determination, association with Cephalosporium gregatum in azuki bean disease, and isolation of hatching stimulant. 2) Potato-cyst nematode (PCN), Globodera rostochiensis - pathotype determination (Ro 1), breeding for resistance, and control recommendations. 3) Pinewood nematode (PWN), Bursaphelenchus xylophilus - primary pathogen in pine wilt disease, life cycle exhibiting a typical symbiosis with Japanese pine sawyer, Monochamus alternatus, and project for control. 4) Rice root nematodes (RRN), Hirschmanniella imamuri and H. oryzae - distribution of species, population levels in roots, and role of these nematodes in rice culture.     

Nonhost Root Penetration by Soybean Cyst Nematode

A total of 66 plants in 50 species were inoculated with eggs and juveniles of soybean cyst nematode, Heterodera glycines. Roots were stained and observed for penetration and development of the nematode. Twenty-six plants were not penetrated; twenty-three were penetrated, but there was no development of the nematode; eight were penetrated with some nematode development; two were penetrated and had considerable nematode development, but few nematodes, if any, matured; and seven were penetrated with many nematodes maturing. The penetration of nonhosts may imply some susceptibility and that populations eventually would build up on the penetrated plants. Plants not penetrated may be useful as rotation plants because no reproduction would occur.     

Newly Discovered Transmission Pathway of Bursaphelenchus xylophilus from Males of the Beetle Monochamus alternatus to Pinus densiflora Trees via Oviposition Wounds

The transmission of Bursaphelenchus xylophilus from Monochamus alternatus males to Pinus densiflora trees via oviposition wounds has been determined. Nematode-infested males, with mandibles fixed experimentally to prevent feeding, were placed for 48 hours with pine bolts containing oviposition wounds that had been made by nematode-free females. After removal of the nematode-infested males, the pine bolts were held for 1 month and then examined for the presence of nematodes. Reproducing nematode populations were recovered from pine bolts that were exposed to male beetles carrying a high number of nematodes. No reproducing nematode population could be recovered from pine bolts exposed to beetles with a small number of nematodes. Nematode reproduction in the pine bolts was not related to the number of oviposition wounds per bolt. Fourth-stage dispersal B. xylophilus juveniles, collected from beetle body surfaces, were inoculated on pine bolt bark 0, 5, 10, and 15 cm away from a single artificial, small hole. These dauer juveniles successfully entered some bolts. The probability of successful nematode reproduction decreased with increased distance between inoculation point and artificial hole. The results indicated that B. xylophilus can move a significant distance to oviposition wounds along the bark surface and enter a tree via the wounds. The new transmission pathway is considered important for the nematode to persist in pine forests such as in North America where pine wilt disease does not occur.     

Effect of Soybean Tip Removal on Penetration and Development of Heterodera glycines

On a few occasions, soybeans with broken root tips were included in tests to evaluate resistance to Heterodera glycines. Although females developed on these plants, the numbers tended to be lower than on similarly treated intact roots. To test the possibility that removal of the root meristem affected nematode development, a culture system using pruned soybeans was devised that permitted access to the roots without disturbing the plants. Treatments included removal of 2 mm of root tip at various times ranging from 24 hours before to 10 days after inoculation, or roots left intact. In each experiment, all roots were inoculated at the same time with equal numbers of freshly hatched second-stage juveniles of Heterodera glycines. No differences in nematode development were detected in plants with root tips removed after inoculation compared to the control. When tips were removed at or before inoculation, fewer juveniles entered roots and relatively fewer nematodes developed. Penetration levels and development correlated with root tip removal such that progressively fewer nematodes entered roots and relatively greater numbers of nematodes remained undeveloped as the time interval between root tip removal and inoculation was increased.     

Longicorn beetle that vectors pinewood nematode carries many Wolbachia genes on an autosome

Monochamus alternatus is the longicorn beetle notorious as a vector of the pinewood nematode that causes the pine wilt disease. When two populations of M. alternatus were subjected to diagnostic polymerase chain reaction (PCR) detection of four Wolbachia genes, only the ftsZ gene was detected from one of the populations. The Wolbachia ftsZ gene persisted even after larvae were fed with a tetracycline-containing diet for six weeks. The inheritance of the ftsZ gene was not maternal but biparental, exhibiting a typical Mendelian pattern. The ftsZ gene titres in homozygotic ftsZ⁺ insects were nearly twice as high as those in heterozygotic ftsZ⁺ insects. Exhaustive PCR surveys revealed that 31 and 30 of 214 Wolbachia genes examined were detected from the two insect populations, respectively. Many of these Wolbachia genes contained stop codon(s) and/or frame shift(s). Fluorescent in situ hybridization confirmed the location of the Wolbachia genes on an autosome. On the basis of these results, we conclude that a large Wolbachia genomic region has been transferred to and located on an autosome of M. alternatus. The discovery of massive gene transfer from Wolbachia to M. alternatus would provide further insights into the evolution and fate of laterally transferred endosymbiont genes in multicellular host organisms.     

Susceptibility of the Carrot Weevil (Coleoptera: Curculionidae) to Steinernema feltiae,S. bibionis,and Heterorhabditis heliothidis

Larvae, pupae, and adults of the carrot weevil (Listronotus oregonensis) were infected and killed by the three entomophagous nematodes (Steinernema feltiae, S. bibionis, and Heterorhabditis heliothidis) under controlled conditions. Third-stage larvae were more susceptible than pupae or adults. S. feltiae and S. bibionis were the most aggressive nematode species, causing larval mortality after 24-48 hours in both continuous and 2-hour contact with nematode suspension. The nematodes multiplied sufficiently in all insects at all stages of development; however, production of infective-stage larvae per host cadaver was variable.     

Plant-parasitic Nematode Acetylcholinesterase Inhibition by Carbamate and Organophosphate Nematicides

The sensitivity of acetylcholinesterases (ACHE) isolated from the plant-parasitic nematodes Meloidogyne arenaria, M. incognita, and Heterodera glycines and the free-living nematode Caenorhabditis elegans to carbamate and organophosphate nematicides was examined. The AChE from plant-parasitic nematode species were more sensitive to carbamate inhibitors than was AChE from C. elegans, but response to the organophosphates was approximately equivalent. The sulfur-containing phosphate nematicides were poor inhibitors of nematode acetylcholinesterase, but treatment with an oxidizing agent greatly improved inhibition. Behavioral bioassays with living nematodes revealed a poor relationship between enzyme inhibition and expression of symptoms in live nematodes.     

Ultrastructure and Development of Pasteuria sp. (S-1 strain), an Obligate Endoparasite of Belonolaimus longicaudatus (Nemata: Tylenchida)

Pasteuria sp., strain S-1, is a gram-positive, obligate endoparasitic bacterium that uses the phytoparasitic sting nematode, Belonolaimus longicaudatus, as its host in Florida. The host attachment of S-1 appears to be specific to the genus Belonolaimus with development occurring only in juveniles and adults of B. longicaudatus. This bacterium is characterized from other described species of Pasteuria using ultrastructure of the mature endospore. Penetration, development, and sporogenesis were elucidated with TEM, LTSEM, and SEM and are similar to other nematode-specific Pasteuria. Recent analysis of 16S rDNA sequence homology confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans, and corroborates ultrastructural, morphological, morphometric, and host-range evidence suggesting separate species status.