The evolution of tissue migration in parasitic nematodes (Nematoda: Strongylida) inferred from a protein-coding mitochondrial gene

Polymerase chain reaction was used to amplify and sequence a 710-bp region of the mtDNA protein-encoding gene, cytochrome c oxidase subunit I, in nine nematodes belonging to the order Strongylida, one nematode from the order Rhabditida and two nematodes from the order Ascaridida. This study tested the hypothesis that in the Strongylida, tissue migration in orally infecting nematodes reflects a legacy of skin penetrating strategies of host infection that were retained after an oral route of infection became established (percutaneous transmission model). Phylogenetic analyses (NJ, MP, ML) consistently produced a single tree topology with two clades within the Strongylida, one containing nematodes that incorporated tissue migration as part of their life cycles, and the other containing nematodes with no tissue migration. Our analysis concluded that the ancestral Strongylida possessed skin penetrating and tissue migrating characters that were modified as members of the group progressed along one of two evolutionary pathways. In one pathway, skin penetration was lost but tissue migration was retained as the nematodes evolved an oral infection route. In the other, both skin penetration and tissue migration characters were lost as the nematodes evolved an oral infection route. These data support the hypothesis of percutaneous transmission as being ancestral in the Strongylida.     

Parasitism of Molluscs by Nematodes: Types of Associations and Evolutionary Trends

Although there are no confirmed fossil records of mollusc parasitic nematodes, diverse associations of more than 108 described nematode species with slugs and snails provide a fertile ground for speculation of how mollusc parasitism evolved in nematodes. Current phylogenic resolution suggests that molluscs have been independently acquired as hosts on a number of occasions. However, molluscs are significant as hosts for only two major groups of nematodes: as intermediate hosts for metastrongyloids and as definitive hosts for a number of rhabditids. Of the 61 species of nematodes known to use molluscs as intermediate hosts, 49 belong to Metastrongyloidea (Order Strongylida); of the 47 species of nematodes that use molluscs as definitive hosts, 33 belong to the Order Rhabditida. Recent phylogenetic hypotheses have been unable to resolve whether metastrongyloids are sister taxa to those rhabditids that use molluscs as definitive hosts. Although most rhabditid nematodes have been reported not to kill their mollusc hosts prior to their reproduction, some species are pathogenic. In fact, infective juveniles of Phasmarhabditis hermaphrodita vector a lethal bacterium into the slug host in which they reproduce. This life cycle is remarkably similar to the entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae. Also, the discoveries of Alloionema and Pellioditis in slugs are interesting, as these species have been speculated to represent the ancestral forms of the entomopathogenic nematodes. Development of the infective stage appears to be an important step toward the acquisition of molluscs as definitive hosts, and the association with specific bacteria may have arisen in conjunction with the evolution of necromeny.     

Conservation of large foci formation in arrested oocytes of <Emphasis Type="Italic">Caenorhabditis</Emphasis> nematodes

Within the rhabditid phylogeny of nematodes, the great majority of species are gonochoristic, having evolved as obligate male/female species. In contrast, the well-studied nematode model system, Caenorhabditis elegans, is androdioecious, utilizing a hermaphroditic/male reproductive system. We have previously determined that in the arrested oocytes of old-aged C. elegans hermaphrodites with depleted sperm, large cytoplasmic ribonucleoprotein foci form. The formation of these foci is reversible, as they dissociate within 3 h after a male mates with the hermaphrodite, resupplying it with sperm. The functional significance of these oocyte foci is not known and previously has not been clear for a hermaphroditic species in which oocytes of young adults wait only approximately 23 min to be fertilized. One hypothesis is that the foci function to maintain maternal mRNAs in oocytes while fertilization is delayed. In this paper, we examine four gonochoristic rhabditid species: Caenorhabditis remanei, Caenorhabditis sp. CB5161, Caenorhabditis sp. PS1010, and Rhabditella axei DF5006. We demonstrate that in three of these four species, ovulation arrests in unmated females until mating occurs and large cytoplasmic foci develop in arrested oocytes. The oocyte foci contain nuclear pore proteins and, in C. remanei at least, the RNA-binding protein MEX-3 as well as RNA. We speculate that these foci maintain the integrity of ooctyes, possibly maintaining the stability or translational repression of maternal mRNAs in unmated females. We further speculate that their presence in oocytes of old-aged C. elegans hermaphrodites is due to conservation from an ancestral gonochoristic state.     

The complete mitochondrial genome of Anisakis simplex (Ascaridida: Nematoda) and phylogenetic implications

We determined the nucleotide sequence of the complete mitochondrial genome of the nematode species Anisakis simplex. The genome is circular, 13,916 bp in size and conforms to the general characteristics of nematode mitochondrial DNAs. The gene arrangement of A. simplex is the same as that of Ascaris suum and almost identical to those of rhabditid species with a minor exception concerning the relative position of the AT-rich and non-coding regions and radically different from those of spirurid species. Along with comparisons of gene arrangement, phylogenetic analyses (maximum parsimony, neighbour joining and maximum likelihood methods) based on concatenated amino acid sequences of 12 protein-coding genes from 13 nematode species provided strong support for the sister-group relationship between Ascaridida and Rhabditida. The Shimodaira-Hasegawa and Templeton's tests both rejected the alternative hypothesis of a closer relationship between Ascaridida and Spirurida. These results contradicted the traditional view of nematode classification and a recent molecular phylogenetic study of 18S rDNA data that assigned Ascaridida and Spirurida as being a sister-group. Mapping of gene arrangement across the phylogenetic tree lead to the assumption that the conserved gene arrangement found in Ascaridida-Rhabditida members might have been acquired after the most recent common ancestor of ascaridid/rhabditid members branched off from the basal stock of the rhabditid lineage.     

Robustness and flexibility in nematode vulva development

The Caenorhabditis elegans vulva has served as a paradigm for how conserved developmental pathways, such as EGF-Ras-MAPK, Notch and Wnt signaling, participate in networks driving animal organogenesis. Here, we discuss an emerging direction in the field, which places vulva research in a quantitative and microevolutionary framework. The final vulval cell fate pattern is known to be robust to change, but only recently has the variation of vulval traits been measured under stochastic, environmental or genetic variation. Whereas the resulting cell fate pattern is invariant among rhabditid nematodes, recent studies indicate that the developmental system has accumulated cryptic variation, even among wild C. elegans isolates. Quantitative differences in the signaling network have emerged through experiments and modeling as the driving force behind cryptic variation in Caenorhabditis species. On a wider evolutionary scale, the establishment of new model species has informed about the presence of qualitative variation in vulval signaling pathways.     

Molecular phylogeny of beetle associated diplogastrid nematodes suggests host switching rather than nematode-beetle coevolution

Background  

Nematodes are putatively the most species-rich animal phylum. They have various life styles and occur in a variety of habitats, ranging from free-living nematodes in aquatic or terrestrial environments to parasites of animals and plants. The rhabditid nematode Caenorhabditis elegans is one of the most important model organisms in modern biology. Pristionchus pacificus of the family of the Diplogastridae has been developed as a satellite model for comparison to C. elegans. The Diplogastridae, a monophyletic clade within the rhabditid nematodes, are frequently associated with beetles. How this beetle-association evolved and whether beetle-nematode coevolution occurred is still elusive. As a prerequisite to answering this question a robust phylogeny of beetle-associated Diplogastridae is needed.     

The mitochondrial genome sequence of Enterobius vermicularis (Nematoda: Oxyurida)--an idiosyncratic gene order and phylogenetic information for chromadorean nematodes

The complete mitochondrial genome sequence was determined for the human pinworm Enterobius vermicularis (Oxyurida: Nematoda) and used to infer its phylogenetic relationship to other major groups of chromadorean nematodes. The E. vermicularis genome is a 14,010-bp circular DNA molecule that encodes 36 genes (12 proteins, 22 tRNAs, and 2 rRNAs). This mtDNA genome lacks atp8, as reported for almost all other nematode species investigated. Phylogenetic analyses (maximum parsimony, maximum likelihood, neighbor joining, and Bayesian inference) of nucleotide sequences for the 12 protein-coding genes of 25 nematode species placed E. vermicularis, a representative of the order Oxyurida, as sister to the main Ascaridida+Rhabditida group. Tree topology comparisons using statistical tests rejected an alternative hypothesis favoring a closer relationship among Ascaridida, Spirurida, and Oxyurida, which has been supported from most studies based on nuclear ribosomal DNA sequences. Unlike the relatively conserved gene arrangement found for most chromadorean taxa, E. vermicularis mtDNA gene order is very unique, not sharing similarity to any other nematode species reported to date. This lack of gene order similarity may represent idiosyncratic gene rearrangements unique to this specific lineage of the oxyurids. To more fully understand the extent of gene rearrangement and its evolutionary significance within the nematode phylogenetic framework, additional mitochondrial genomes representing a greater evolutionary diversity of species must be characterized.     

Predation of entomopathogenic nematodes by <Emphasis Type="Italic">Sancassania</Emphasis> sp. (Acari: Acaridae)

Predation of the entomopathogenic nematode, Steinernema feltiae (Rhabditida: Steinernematidae), by Sancassania sp. (Acari: Acaridae) isolated from field-collected scarab larvae was examined under laboratory conditions. Adult female mites consumed more than 80% of the infective juvenile (IJ) stage of S. feltiae within 24 h. When S. feltiae IJs were exposed to the mites for 24 h and then exposed to Galleria mellonella (Lepidoptera: Pyralidae) larvae, the number of nematodes penetrating into the larvae was significantly lower compared to S. feltiae IJs that were not exposed to mites (control). Soil type significantly affected the predation rate of IJs by the mites. Mites preyed more on nematodes in sandy soil than in loamy soil. We also observed that the mites consumed more S. feltiae IJs than Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae). No phoretic relationship was observed between mites and nematodes and the nematodes did not infect the mites.     

Tissue treatment for whole mount internal lectin staining in the nematodes Caenorhabditis elegans,Panagrolaimus superbus and Acrobeloides maximus

Four different fixation schemes, using ten fluorescent-labelled lectins, were investigated for whole mount internal staining of three rhabditid nematodes: Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus. Acetone-only fixation was found to give strong and reproducible staining, which could be prevented either by periodate treatment of the organisms or by specific inhibitory sugars of the lectins under investigation. Whereas the use of either phosphate or TRIS buffers had no effect on the staining pattern or the fluorescence intensity, the incubation time as well as the incubation temperature affected the staining reaction. The best results were obtained upon overnight incubation at 4° C: the lectin staining could be inhibited in all cases, except for the intestinal brush border of C. elegans by the lectin of Lens culinaris.     

Evaluation of hydraulic,pneumatic and mechanical agitation for the spray application of Steinernema carpocapsae (Rhabditida: Steinernematidae)

The application of entomopathogenic nematodes (EPN) is generally done using standard spray application techniques. However, in contrast to chemical pesticides, these biological antagonists must remain viable during and after the application process. For the application of EPN, a good agitation system is indispensable as the nematodes tend to sediment fast in a spray tank without agitation. Three agitation systems, viz. mechanical, pneumatic and hydraulic agitation were tested for their ability to keep Steinernema carpocapsae (Rhabditida: Steinernematidae) suspended in an undamaged way. Hydraulic agitation was tested using a centrifugal and a diaphragm pump. Nematode damage was quantified based on viability and infectivity of the EPN. The ability of the agitation system to keep the nematodes in suspension was examined by comparing the nematode concentration observed in the samples taken at different agitation times. Only the hydraulic agitation using the centrifugal pump damaged the nematodes. After 120 min of recirculation, only 19.3% of the nematodes survived. Infectivity was even reduced to 0%. An additional experiment revealed that the temperature rise, from 21.7 to 45.4°C, was responsible for the observed nematode damage. The concentration measurements showed that the pneumatic agitation was unstable. Agitation during 120 min using the other agitation systems resulted in a significant loss of nematodes at 15 cm above the spray tank bottom. In conclusion, mechanical and hydraulic agitation using a diaphragm pump can be recommended when S. carpocapsae is applied, although attention should be paid to possible nematode loss during application.     

Rhabditophanes schneideri (Rhabditida) phoretic on a cave pseudoscorpion

Information of phoretic nematode-pseudoscorpion associations and cases of parasitism on five European species of pseudoscorpions was summarized by Cur?ic et al. [Curci?, B.P.M., Dimitrijevi?, R.N., Makarov, S.E., Luci?, L.R., Curci?, S.B., 1996. Further report on nematode-pseudoscorpion associations. Acta arachnol. 45, 43-46; Curci?, B.P.M., Sudhaus, W., Dimitrijevi?, R.N., Tomi?, V.T., Curci?, S.B., 2004. Phoresy of Rhabditophanes schneideri (Bütschli) (Rhabditida: Alloionematidae) on pseudoscorpions (Arachnida: Pseudoscorpiones). Nematology 6 (3), 313-317]. An examination of a sample of the cavernicolous pseudoscorpion Neobisium rajkodimitrijevici (Curci? and Tomi?, 2006) (comprising a holotype male and a paratype tritonymph) from a cave in Eastern Serbia revealed this false scorpion to be a nematode carrier; the present paper reports this finding and extends our knowledge of phoresy of Rhabditophanes on pseudoscorpions. This is the first time that the rhabditid R. schneideri (Bütschli, 1873) has been noted in association with a cavernicolous pseudoscorpion. There must be some patchily distributed micro-habitats in caves where saprobiotic nematodes and small arthropods can complete their life-cycles, for example something like deposits of bat guano. The transportation of Rhabditophanes J3 by pseudoscorpions indicate that Neobisium specimens often visit these micro-habitats to find their prey.     

British and European Legislation Regulating Rhabditid Nematodes

Rhabditid nematodes are invertebrates that introduce pathogenic bacteria to the insects and molluscs they parasitise. Marketed as biopesticides, they provide safe and effective alternatives to chemical pesticides in several important sectors of horticulture. In Great Britain, the introduction of non-indigenous species of nematodes is strictly controlled, as is the contained use and deliberate release of genetically modified nematodes and their bacterial symbionts. Currently, indigenous, unmodified nematodes do not have to be registered for use as biopesticides in the UK, but in some other European countries registration is mandatory. The European Commission is seeking to harmonize procedures for the authorization of plant protection products, including rhabditid nematodes, under the provisions of Council Directive 91/414/EEC.     

Synergistic effect of the herbicides glyphosate and MCPA on survival of entomopathogenic nematodes

The survival and infectivity of the infective juveniles of two species of entomopathogenic nematodes, Steinernema feltiae (Rhabditida: Steinernematidae) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), were determined after exposure for 72 h to two concentrations of the herbicides glyphosate and MCPA, as well as to the combination of the two herbicides (glyphosate + MCPA). For all herbicide treatments, concentrations and exposure times, S. feltiae was more tolerant to the herbicides than H. bacteriophora. The exposure of entomopathogenic nematodes to glyphosate + MCPA caused significantly higher mortality (26.33–57.33%) than glyphosate (0.67–15%) or MCPA (2.33–19%) alone. These results confirm the synergistic effect of the glyphosate + MCPA combination on the mortality in these nematodes. Nematode infectivity of Galleria mellonella larvae in response to the herbicides presence was evaluated in Petri dish assays containing sterile sand. Nematode infectivity was not significantly reduced by exposure to herbicides in S. feltiae but H. bacteriophora was less tolerant. Synergistic effect was obtained in the nematode mortality test but no synergistic effect was observed in the nematode infectivity assay. Our results suggest that possible synergistic effects of agrochemicals on survival of nematodes should be tested before mixing with entomopathogenic nematodes.     

Susceptibility of shore fly <Emphasis Type="Italic">Scatella stagnalis</Emphasis> to five entomopathogenic nematode strains in bioassays

The shore fly, Scatella stagnalis (Fallén) (Diptera: Ephydridae) is an important insect pest of greenhouse crops. We evaluated two different Spanish isolates of entomopathogenic nematodes, Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) and Steinernema arenarium (Artyukhovsky) (Rhabditida: Steinernematidae), and two commercially available strains, Steinernema feltiae (Nemaplus^®) and Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae) (Nematop^®) against shore flies. In tests conducted in 24-well plate filter paper applied at 5, 11, 22, 44 and 88 nematodes per larva, all nematodes produced significant shore fly larval mortality. The lowest concentration tested was enough to obtain high larval mortality (65.2–87.0%). The nematodes Steinernema feltiae and Steinernema arenarium, which parasitized the shore fly larvae faster, also penetrated in higher number in the shore fly larva (4.6–8.8% penetration rate). In bioassays conducted in algae, Steinernema feltiae, applied at 50 nematodes/cm², caused highest (100%) and Steinernema arenarium lowest shore fly mortality (94%). Our results suggest that entomopathogenic nematodes appear feasible for controlling shore flies but further tests are needed to determine their efficacy in the field.     

Novel primers for the amplification of nuclear DNA introns in the entomopathogenic nematode Heterorhabditis bacteriophora and their cross-amplification in seven other Heterorhabditis species

We describe 24 novel primers that amplify intron regions in housekeeping and structural genes of Heterorhabditis bacteriophora. The cross-amplification potential of these primers in seven other Heterorhabditis species was determined. The results obtained showed interspecific nucleotide, length and splice site variability in the sequenced introns and for one gene, an intron gain was observed. These primers will be useful tools for studying population genetics, genetic diversity and intron DNA evolution within the genus Heterorhabditis and other genera of rhabditid nematodes.     

Comparative embryology of nematodes and the law of embryo similarity

Two types of embryonic development can be distinguished within nematodes, with a variable (Enoplia) or invariant (remaining species) cleavage. In the case of invariant cleavage two main variants of cell lineage are presented in nematodes, with the posterior (Rhabditea) or anterior (Dorylaimida) localization of endoderm material at the two-cell stage. This classification is in a good agreement with some modern nematode taxonomy and it is supported by molecular phylogeny studies. The variable cleavage is plesiomorphic. Traditional concept of "mosaic" cleavage is not applicable for nematodes as inductive interactions and a regulation of experimental interventions are usual attributes of any mode of nematode development. The representatives of order Rhabditida have almost identical cell lineage, but at the same time they have strong interspecific differences in mechanisms of ooplasmic segregation any early inductive interactions. The diversity of geometric patterns in the early cleavage, often at the level of individual random variations, is a usual characteristic of nematodes including species with the invariant cleavage. Thus, the early stages of nematode development are evolutionary very flexible, but at the course of embryonic development similarity of different species is progressively increased up to the uniform morphogenetic stages. The dynamics of variation in nematode development contradict to the von Baer's law but are in an agreement with the modern "hourglass model" (Doboul, 1994; Raff, 1986).     

Convergent evolution of sperm gigantism and the developmental origins of sperm size variability in Caenorhabditis nematodes

Sperm cells provide essential, if usually diminutive, ingredients to successful sexual reproduction. Despite this conserved function, sperm competition and coevolution with female traits can drive spectacular morphological change in these cells. Here, we characterize four repeated instances of convergent evolution of sperm gigantism in Caenorhabditis nematodes using phylogenetic comparative methods on 26 species. Species at the extreme end of the 50‐fold range of sperm‐cell volumes across the genus have sperm capable of comprising up to 5% of egg‐cell volume, representing severe attenuation of the magnitude of anisogamy. Furthermore, we uncover significant differences in mean and variance of sperm size among genotypes, between sexes, and within and between individuals of identical genotypes. We demonstrate that the developmental basis of sperm size variation, both within and between species, becomes established during an early stage of sperm development at the formation of primary spermatocytes, while subsequent meiotic divisions contribute little further sperm size variability. These findings provide first insights into the developmental determinants of inter‐ and intraspecific sperm size differences in Caenorhabditis. We hypothesize that life history and ecological differences among species favored the evolution of alternative sperm competition strategies toward either many smaller sperm or fewer larger sperm.     

Operon structure and trans-splicing in the nematode Pristionchus pacificus

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.     

Convergence, recurrence and diversification of complex sperm traits in diving beetles (Dytiscidae)

Sperm display remarkable morphological diversity among even closely related species, a pattern that is widely attributed to postcopulatory sexual selection. Surprisingly few studies have used phylogenetic analyses to discern the details of evolutionary diversification in ornaments and armaments subject to sexual selection, and the origins of novel sperm traits and their subsequent modification are particularly poorly understood. Here we investigate sperm evolution in diving beetles (Dytiscidae), revealing dramatic diversification in flagellum length, head shape, presence of sperm heteromorphism, and the presence/type of sperm conjugation, an unusual trait where two or more sperm unite for motility or transport. Sperm conjugation was found to be the ancestral condition in diving beetles, with subsequent diversification into three forms, each exhibiting varying degrees of evolutionary loss, convergence, and recurrence. Sperm head shape, but not length or heteromorphism, was found to evolve in a significantly correlated manner with conjugation, consistent with the different mechanisms of head alignment and binding required for the different forms of conjugation. Our study reveals that sperm morphological evolution is channeled along particular evolutionary pathways (i.e., conjugate form), yet subject to considerable diversification within those pathways through modification in sperm length, head shape, and heteromorphism.