A biological invasion modifies the dynamics of a host-parasite arms race.

By imposing novel selection pressures on both participants, biological invasions can modify evolutionary 'arms races' between hosts and parasites. A spatially replicated cross-infection experiment reveals strong spatial divergence in the ability of lungworms (Rhabdias pseudosphaerocephala) to infect invasive cane toads (Rhinella marina) in Australia. In areas colonized for longer than 20 years, toads are more resistant to infection by local strains of parasites than by allopatric strains. The situation reverses at the invasion front, where super-infective parasites have evolved. Invasion-induced shifts in genetic diversity and selective pressures may explain why hosts gain advantage over parasites in long-colonized areas, whereas parasites gain advantage at the invasion front.

In an arms race between host and parasite, a lungworm's ability to infect a toad is determined by host susceptibility not parasite preference.

Evolutionary arms races can alter both parasite infectivity and host resistance, and it is difficult to separate the effects of these twin determinants of infection outcomes. We used a co-introduced, invasive host-parasite system (the lungworm Rhabdias pseudosphaerocephala and cane toads Rhinella marina), where rapid adaptation and dispersal have led to population differences in infection resistance. We quantified behavioural responses of parasite larvae to skin-chemical cues of toads from different invasive populations, and rates at which juvenile hosts became infected following standardized exposure to lungworms. Chemical cues from toad skin altered host-seeking behaviour by parasites, similarly among populations. The number of infection attempts (parasite larvae entering the host's body) also did not differ between populations, but rates of successful infection (establishment of adult worm in host lungs) were higher for range-edge toads than for range-core conspecifics. Thus, lower resistance to parasite infection in range-edge juvenile toads appears to be due to less effective immune defences of the host rather than differential behavioural responses of the parasite. In this ongoing host-parasite arms race, changing outcomes appear to be driven by shifts in host immunocompetence.

A core set of venom proteins is released by entomopathogenic nematodes in the genus Steinernema.

Parasitic helminths release molecular effectors into their hosts and these effectors can directly damage host tissue and modulate host immunity. Excreted/secreted proteins (ESPs) are one category of parasite molecular effectors that are critical to their success within the host. However, most studies of nematode ESPs rely on in vitro stimulation or culture conditions to collect the ESPs, operating under the assumption that in vitro conditions mimic actual in vivo infection. This assumption is rarely if ever validated. Entomopathogenic nematodes (EPNs) are lethal parasites of insects that produce and release toxins into their insect hosts and are a powerful model parasite system. We compared transcriptional profiles of individual Steinernema feltiae nematodes at different time points of activation under in vitro and in vivo conditions and found that some but not all time points during in vitro parasite activation have similar transcriptional profiles with nematodes from in vivo infections. These findings highlight the importance of experimental validation of ESP collection conditions. Additionally, we found that a suite of genes in the neuropeptide pathway were downregulated as nematodes activated and infection progressed in vivo, suggesting that these genes are involved in host-seeking behavior and are less important during active infection. We then characterized the ESPs of activated S. feltiae infective juveniles (IJs) using mass spectrometry and identified 266 proteins that are released by these nematodes. In comparing these ESPs with those previously identified in activated S. carpocapsae IJs, we identified a core set of 52 proteins that are conserved and present in the ESPs of activated IJs of both species. These core venom proteins include both tissue-damaging and immune-modulating proteins, suggesting that the ESPs of these parasites include both a core set of effectors as well as a specialized set, more adapted to the particular hosts they infect.

A new species of Rhabdias (Nematoda: Rhabdiasidae), a lung parasite of Pseudopaludicola pocoto (Anura: Leptodactylidae) from north-eastern Brazil: description and phylogenetic analyses.

Rhabdias pocoto n. sp. is herein described from the lungs of the swamp frog Pseudopaludicola pocoto Magalhães, Loebmann, Nogueira, Kokubum, Baptista, Haddad & Garda, 2014, from the Caatinga biome in the state of Ceará, in north-eastern Brazil. The new species is characterized by a body that dilates posteriorly, six small lips (protuberances) and two rounded lateral expansions of cuticular inflation on the anterior end, each containing an amorphous gland-like structure inside and a short and conical tail. Additionally, molecular analysis and comparison of the partial mitochondrial cytochrome c oxidase I sequence of R. pocoto n. sp. revealed genetic divergence between the new species and the sequences of Rhabdias spp. previously deposited in GenBank. Phylogenetic analysis grouped the new taxon into the R. pseudosphaerocephala species complex + R. glaurungi clade. The new discovery represents the 19th species of Rhabdias spp. described in the Neotropical region, the ninth in Brazil and the first species of Rhabdias found parasitizing South American frogs of the genus Pseudopaludicola, as well as the first Caatinga biome species of Rhabdias.

Transcriptomic analysis of Baltic cod (Gadus morhua) liver infected with Contracaecum osculatum third stage larvae indicates parasitic effects on growth and immune response.

High infection levels due to third-stage larvae of the anisakid nematode Contracaecum osculatum have been documented in cod from the eastern part of the Baltic sea during the latest decades. The nematode larvae mainly infect the liver of Baltic cod and prevalence of infection has reached 100% with a mean intensity up to 80 parasites per host in certain areas and size classes. Low condition factors of the cod have been observed concomitant with the rise in parasite abundance suggesting a parasitic effect on growth parameters. To investigate any association between parasite infection and physiological status of the host we performed a comparative transcriptomic analysis of liver obtained from C. osculatum infected and non-infected cod. A total of 47,025 predicted gene models showed expression in cod liver and sequences corresponding to 2084 (4.43%) unigenes were differentially expressed in infected liver when compared to non-infected liver. Of the differentially expressed unigenes (DEGs) 1240 unigenes were up-regulated while 844 unigenes were down-regulated. The Gene Ontology (GO) enrichment analysis showed that 1304 DEGs were represented in cellular process and single-organism process, cell and cell part, binding and catalytic activity. As determined by the Kyoto Encyclopedia of Gene and Genomes (KEGG) Pathways analysis, 454 DEGs were involved in 138 pathways. Ninety-seven genes were related to metabolic pathways including carbohydrate, lipid, and amino acid metabolism. Thirteen regulated genes were playing a role in immune response such as Toll-like receptor signaling, NOD-like receptor signaling, RIG-I-like receptor signalling and thirty-six genes were associated with growth processes. This indicates that the nematode infection in Baltic cod may affect on molecular mechanisms involving metabolism, immune function and growth.

Molecular characterization of human isolates of Strongyloides stercoralis and Rhabditis spp. based on mitochondrial cytochrome c oxidase subunit 1 (cox1).

BACKGROUND: Due to the similarity of Strongyloides stercoralis with free-living nematodes of Rhabditis species they might be miss-diagnosed with each other in microscopical examination of stool samples. The aim of this study was molecular characterization and differentiation of human derived isolates of S. stercoralis and Rhabditis species based on the mitochondrial gene of cytochrome c oxidase subunit 1 (cox1) amplification. METHODS: Using parasitological methods, ten isolates of S. stercoralis and three isolates of Rhabditis spp. were obtained from fresh stool samples of patients and the genomic DNA of the samples were extracted. PCR amplification of cox1 gene was carried out for all the isolates and the products were sequenced. RESULTS: The phylogenetic analysis illustrated that S. stercoralis and Rhabditis spp. isolates were placed in two distinguishable separate clades. Inter-species genetic variation between isolates of S. stercoralis and Rhabditis spp. were ranged from 13.5 to 14.5%. CONCLUSIONS: Cox1 gene was a suitable marker for discrimination of S. stercoralis from Rhabditis spp. retrieved from human in the current study. The availability of gene sequence information will be helpful in the future development and validation of discriminatory PCR-based assays of these nematodes.

Pheromone extracts act as boosters for entomopathogenic nematodes efficacy.

Inconsistency in entomopathogenic nematode (EPN) efficacy is still one of the biggest challenges for the wider adoption of EPNs as biocontrol agents. Previous studies demonstrated that extracts from EPN-infected hosts enhance dispersal and efficacy, two key factors in success of EPNs. Some active components in the insect host cadavers responsible for dispersal, ascarosides, have been identified as nematode pheromones. We hypothesized that pheromone extracts increase dispersal of EPN infective juveniles (IJs) leading to increased efficacy. First, we determined whether pheromone extracts improved IJ movement/dispersal in soil columns baited with Tenebrio molitor larvae. We found that pheromone extracts induced higher numbers of Steinernema carpocapsae and Steinernema feltiae IJs to move towards T. molitor larvae in the bottom of the column compared to IJs treated with infected cadaver macerate and water, positive and negative controls, respectively. Furthermore, the number of S. carpocapsae IJs that invaded T. molitor larvae was higher for the pheromone extract treatment than the controls. S. feltiae IJs that were pretreated with pheromone extracts and macerate (positive control) infected T. molitor at the same rate but invasion was superior to IJs that were treated with water. Consistent with the soil column tests, both S. carpocapsae and S. feltiae IJs treated with pheromone extracts performed better in killing larvae of two economically important insect larvae, pecan weevil, Curculio caryae, and black soldier fly, Hermetia illucens, in greenhouse tests compared to IJs treated with water. We demonstrated pheromone-mediated behavioral manipulation of a biological control agent to enhance pest control potential. Conceivably, nematodes can be exposed to efficacy-enhancing pheromones prior to field application.

Rhabdias glaurungi sp. nov. (Nematoda: Rhabdiasidae), parasite of Scinax gr. ruber (Laurenti, 1768) (Anura: Hylidae), from the Brazilian Amazon.

The genus Rhabdias Stiles & Hassal, 1905 includes about 83 species of nematodes parasitic in amphibians and reptiles worldwide. Herein, we describe Rhabdias glaurungi sp. nov. from the hylid frog Scinax gr. ruber (Laurenti, 1768) in the Gunma Ecological Park, Santa Bárbara municipality, state of Pará, Brazil. This species has six small lips, an inflated cuticle along the entire body and a cup-shaped buccal capsule with smooth internal surface of its anterior part and irregularly folded internal surface of its posterior part in apical view. From the 17 valid species recognized in the Neotropical realm, the new species can be distinguished by the number of lips, the morphology and size of its buccal capsule, as well as the extent and shape of its cuticular inflation; in addition, there are molecular differences. Sequences of the mitochondrial Cytochrome c Oxidase subunit I gene strongly support the status of this form as a separate species. Molecular phylogenetic analysis shows R. glaurungi sp. nov. nested within the R. pseudosphaerocephala Kuzmin, Tkach & Brooks, 2007 species complex. Rhabdias glaurungi sp. nov. is the second species of the genus described from hosts of the family Hylidae in the Neotropical realm. We conclude that the diversity of Rhabdias within the Neotropics is likely largely underestimated.

Development of Phasmarhabditis hermaphrodita (and members of the Phasmarhabditis genus) as new genetic model nematodes to study the genetic basis of parasitism.

The genetic mechanisms of how free-living nematodes evolved into parasites are unknown. Current genetic model nematodes (e.g. Caenorhabditis elegans) are not well suited to provide the answer, and mammalian parasites are expensive and logistically difficult to maintain. Here we propose the terrestrial gastropod parasite Phasmarhabditis hermaphrodita as a new alternative to study the evolution of parasitism, and outline the methodology of how to keep P. hermaphrodita in the lab for genetic experiments. We show that P. hermaphrodita (and several other Phasmarhabditis species) are easy to isolate and identify from slugs and snails from around the UK. We outline how to make isogenic lines using 'semi-natural' conditions to reduce in-lab evolution, and how to optimize growth using nematode growth media (NGM) agar and naturally isolated bacteria. We show that P. hermaphrodita is amenable to forward genetics and that unc and sma mutants can be generated using formaldehyde mutagenesis. We also detail the procedures needed to carry out genetic crosses. Furthermore, we show natural variation within our Phasmarhabditis collection, with isolates displaying differences in survival when exposed to high temperatures and pH, which facilitates micro and macro evolutionary studies. In summary, we believe that this genetically amenable parasite that shares many attributes with C. elegans as well as being in Clade 5, which contains many animal, plant and arthropod parasites, could be an excellent model to understand the genetic basis of parasitism in the Nematoda.

Polyparasitism of Rhabditis Axei and Enterobius Vermicularis in a Child from Beijing, China.

BACKGROUND: Rhabditis (Rhabditellae) axei is a common species in soil, which has been reported repeatedly in human urine and the digestive system. Humans exposed to sewage or mistakenly polluted sewage is the cause of larvae infecting the digestive tract or via the urethra. We reported a patient infected with Rhabditis axei and Enterobius Vermicularis. The migration of the nematodes caused true signs of hematuria, diarrhea, and high eosinophilia. METHODS: Stool and urine are collected to detect parasite eggs and genotype. Specimens are sent for polymerase chain reaction (PCR)-based species identification. Amplification of the 18S ribosomal RNA gene was performed by PCR as described [1]. RESULTS: Morphological features and PCR amplification of the 18S ribosomal RNA gene confirmed Rhabditis axei and Enterobius vermicularis as the pathogen of infection. CONCLUSIONS: Herein, we presented a case that confirmed Rhabditis axei and Enterobius vermicularis infection in humans can be associated with high eosinophilia.

A new species of Diomedenema (Nematoda, Rhabditida, Spiruromorpha) from Spheniscus magellanicus (Aves, Sphenisciformes) found on the southern coast of Brazil.

A new species of the genus Diomedenema, a spiruromorph nematode, collected from the lung of Spheniscus magellanicus (Sphenisciformes) found on the southern coast of the state of Rio Grande do Sul, Brazil, is described. The new species is differentiated from the only previously described species of the genus, D. diomedeae Johston & Mawson, 1952, by males possessing a set of caudal papillae with three pairs of precloacal, two pairs of adcloacal and one pair of postcloacal papillae; precloacal papillae with the papillae of the first two pairs being closer to each other than those of the third pair; a longer and pointed tail in males; and females with the vulva at mid-body. This is the first report of a nematode infecting the lung of a sphenisciforme host.

Identification of candidate infection genes from the model entomopathogenic nematode Heterorhabditis bacteriophora.

BACKGROUND: Despite important progress in the field of innate immunity, our understanding of host immune responses to parasitic nematode infections lags behind that of responses to microbes. A limiting factor has been the obligate requirement for a vertebrate host which has hindered investigation of the parasitic nematode infective process. The nematode parasite Heterorhabditis bacteriophora offers great potential as a model to genetically dissect the process of infection. With its mutualistic Photorhabdus luminescens bacteria, H. bacteriophora invades multiple species of insects, which it kills and exploits as a food source for the development of several nematode generations. The ability to culture the life cycle of H. bacteriophora on plates growing the bacterial symbiont makes it a very exciting model of parasitic infection that can be used to unlock the molecular events occurring during infection of a host that are inaccessible using vertebrate hosts. RESULTS: To profile the transcriptional response of an infective nematode during the early stage of infection, we performed next generation RNA sequencing on H. bacteriophora IJs incubated in Manduca sexta hemolymph plasma for 9 h. A subset of up-regulated and down-regulated genes were validated using qRT-PCR. Comparative analysis of the transcriptome with untreated controls found a number of differentially expressed genes (DEGs) which cover a number of different functional categories. A subset of DEGs is conserved across Clade V parasitic nematodes revealing an array of candidate parasitic genes. CONCLUSIONS: Our analysis reveals transcriptional changes in the regulation of a large number of genes, most of which have not been shown previously to play a role in the process of infection. A significant proportion of these genes are unique to parasitic nematodes, suggesting the identification of a group of parasitism factors within nematodes. Future studies using these candidates may provide functional insight into the process of nematode parasitism and also the molecular evolution of parasitism within nematodes.

Description and phylogenetic position of a new species of Rhabdias Stiles et Hassall, 1905 (Nematoda: Rhabdiasidae) from the banded rubber frog, Phrynomantis bifasciatus (Smith) (Amphibia: Microhylidae), in South Africa.

The lung-dwelling nematode Rhabdias engelbrechti sp. n. was found in five of eight examined banded rubber frogs in Limpopo Province, South Africa. The species is differentiated from species of Rhabdias Stiles et Hassall, 1905 occurring in the Afrotropical Realm based on the presence of a globular cuticular inflation at the anterior end, the buccal capsule walls being distinctly divided into anterior and posterior parts, the buccal capsule size (6-9 µm × 16-18 µm), and the body length (3.8-6.1 mm). Rhabdias engelbrechti is the tenth species of the genus found in Afrotropical anurans. Our molecular phylogenetic analysis based on the complete sequences of the ITS region and partial sequences of large subunit (28S) gene of the nuclear ribosomal RNA demonstrates that the new species is more closely related to the Eurasian species Rhabdias bufonis (Schrank, 1788) than to two other species from sub-Saharan Africa represented in the tree. In addition, partial sequences of the mitochondrial protein coding cox1 and ribosomal 12S genes of the new species have shown significant differences from all previously published sequences of these genes from African species of Rhabdias.

Morphometric and phylogenetic analyses of Serpentirhabdias viperidicus n. sp. (Nematoda: Rhabdiasidae) from the lancehead snake Bothrops moojeni Hoge, 1966 (Reptilia: Serpentes: Viperidae) in Brazil.

Serpentirhabdias viperidicus n. sp. (Nematoda: Rhabdiasidae) is described from the lungs of the 'Brazilian lancehead' Bothrops moojeni (Hoge, 1966) from the savannah in São Paulo State, Brazil. The new species is the eighth species of Serpentirhabdias described in the Neotropical region, and differs from other species mainly by a combination of characters: lips slightly notable, presence of fine striations at posterior ends, presence of two parallel lines with intercalated pores, a pore-shaped phasmid situated at the level of the anal aperture and another two in the posterior half of the tail. It is the first species of Serpentirhabdias reported in this snake host and the second species of this genus found parasitizing South American viperidian snakes. Molecular phylogenetic analysis using ribosomal (ITS and 28S partial) genes confirms Serpentirhabdias viperidicus n. sp. as a new species that clustered in the Serpentirhabdias clade, sister taxon to Serpentirhabdias fuscovenosa and Serpentirhabdias elaphe. This is the first description of Serpentirhabdias species from Brazil using molecular approaches and morphological characters to confirm the monophyly of this recent genus.

Ecological factors responsible for the geographic distribution of Rhabdias joaquinensis: where do lungworms infect anurans in nature?

The lungworm Rhabdias joaquinensis (Nematoda: Rhabdiasidae) is a common parasite of anurans in eastern Nebraska, yet absent from the same hosts in western Nebraska. This study investigated the ecology of the lungworm's free-living stages that reside in host feces and surrounding soils to establish the worm's free-living niche in eastern Nebraska. Using a comparative method, this study also investigated the absence of lungworms in western Nebraska's Sandhills. Soil composition, moisture, and temperature were experimentally varied in the laboratory to assess their effects on juvenile development and survival. Field mesocosm and host defecation experiments were used to determine where in nature lungworms survive and eventually infect frogs and toads and to discover if vegetation facilitates lungworm transmission to hosts. The results found loam soils were amenable to lungworm development, whereas soils with high clay or sand content produced few infective lungworms. Soil moisture <50 % did not support lungworm development. Infective juveniles successfully developed between 5 and 35 °C, albeit at different rates, whereas only a limited number of non-infective worms developed at 40 °C. Field studies found that shoreline environments supported lungworm development, and the majority of lungworm infections appear to occur within a zone of infection adjacent to shorelines in eastern Nebraska. The prevalence in vegetation mesocosms was 100 %, and a significantly higher mean abundance was found in toads from containers with vegetation than without. Overall, these experiments suggest that the spatial distribution of R. joaquinensis in Nebraska is constrained by the worm's ability to survive and reproduce in a soil matrix.

First report of fatal systemic Halicephalobus gingivalis infection in two Lipizzaner horses from Romania: clinical, pathological, and molecular characterization.

Halicephalobus gingivalis (H. gingivalis) causes a rare and fatal infection in horses and humans. Despite the zoonotic potential and severity of the disease, the epidemiology and pathogenesis of halicephalobiasis are still poorly understood. Several European cases of equine halicephalobiasis have been documented; however, in South-Eastern European countries, including Romania, equine neurohelminthiasis caused by H. gingivalis has not been previously described. Two Lipizzaner horses with a clinical history of progressive neurological signs were referred to the Pathology Department of the Cluj-Napoca (Romania) for necropsy. Both horses died with severe neurological signs. Gross examination and cytological, histological, and molecular analyses were performed. The stallions came from two different breeding farms. No history of traveling outside Romania was recorded. At necropsy, granulomatous and necrotizing lesions were observed in the kidneys, lymph nodes, brain, retroperitoneal adipose tissue, and lungs, indicating a systemic infection. Parasitological and histopathological analyses evidenced larval and adult forms of rhabditiform nematodes consistent with Halicephalobus species. Parasites were observed in both lymph and blood vessels of different organs and were also identified in urine samples. A subunit of the large-subunit ribosomal RNA gene (LSU rDNA) of H. gingivalis (673 bp) was amplified from lesions in both horses.To the authors' knowledge, this is the first report of equine systemic H. gingivalis infection in Romania and in South-Eastern Europe. Our findings provide new insights into the geographic distribution of specific genetic lineages of H. gingivalis, while also raising public health awareness, as the parasite is zoonotic.

Description of Serpentirhabdias atroxi n. sp. (Nematoda: Rhabdiasidae), a parasite of Bothrops atrox (Linnaeus) (Reptilia: Serpentes: Viperidae) in Brazilian Amazonia.

A new lung-dwelling nematode species is described from the common lancehead Bothrops atrox (Linnaeus) in the Brazilian Amazon Region. The species is assigned to the genus Serpentirhabdias Tkach, Kuzmin & Snyder, 2014 based on the presence of six lips arranged in two lateral groups, the absence of prominent cuticular inflations, and lung parasitism in snakes. Serpentirhabdias atroxi n. sp. differs from other species of the genus mainly by details of the morphology of the anterior end: cuticularised ring surrounding the anterior part of the buccal cavity and six minute onchia present in the oesophastome. Serpentirhabdias atroxi n. sp. is the seventh species of the genus known from the Neotropical Realm and the second species described from viperid snakes.

First human case of fatal Halicephalobus gingivalis meningoencephalitis in Australia.

Halicephalobus gingivalis (previously Micronema deletrix) is a free-living nematode known to cause opportunistic infections, mainly in horses. Human infections are very rare, but all cases described to date involved fatal meningoencephalitis. Here we report the first case of H. gingivalis infection in an Australian human patient, confirmed by nematode morphology and sequencing of ribosomal DNA. The implications of this case are discussed, particularly, the need to evaluate real-time PCR as a diagnostic tool.

Outer ear canal infection with Rhabditis sp. nematodes in a human.

Here we report the first human case of an outer ear canal infection with a free-living nematode of the genus Rhabditis. Otomicroscopy revealed viable worms in the outer ear canal of a patient suffering from chronic otorrhea and hearing loss. The nematode was identified by microscopy and internal transcribed spacer (ITS)-PCR.

Phylogenetic affinities and systematic position of Entomelas sylvestris Baker, 1982 (Nematoda: Rhabdiasidae), a parasite of Breviceps sylvestris FitzSimons (Amphibia: Brevicipitidae) in South Africa.

The genus Entomelas Travassos, 1930 currently includes nine species of rhabdiasid nematodes, eight of them parasitic in lizards and only one, Entomelas sylvestris Baker, 1982, parasitic in amphibians. Entomelas sylvestris was originally described from the Forest Rain Frog Breviceps sylvestris FitzSimons in South Africa and was not reported since. It was placed in the genus Entomelas without any specific arguments for this taxonomic decision, presumably mainly based on details of the buccal capsule morphology. We have found this species in the same host in Limpopo province, South Africa. Molecular phylogenetic analysis based on the newly-obtained sequence of complete ITS region and partial nuclear large ribosomal subunit (28S) gene of E. sylvestris and previously published sequences of a variety of other rhabdiasid taxa, has convincingly demonstrated that this species does not belong in Entomelas. Instead, it clustered together with the members of Rhabdias Stiles & Hassall, 1905 from amphibian hosts. Therefore, we transfer E. sylvestris into Rhabdias as Rhabdias sylvestris (Baker, 1982) n. comb. In our analysis E. sylvestris appears, albeit with weak support, as a basal/sister taxon to the rest of Rhabdias spp. which explains to some extent the differences in the buccal capsule morphology between this species and other Rhabdias spp.