Morphology, complete mitochondrial genome, and molecular phylogeny of Rhabdias macrocephalum n. sp. (Nematoda: Rhabdiasidae) from Diploderma splendidum (Reptilia: Agamidae).

Species of the genus Rhabdias Stiles & Hassall, 1905 are common parasitic nematodes occurring in the lungs of amphibians and reptiles worldwide. In the present study, Rhabdias macrocephalum n. sp. is described using integrated morphological methods (light and scanning electron microscopy) and molecular approaches (sequencing of the nuclear 28S and ITS regions, and mitochondrial cox1, cox2, and 12S genes) based on specimens collected from the green striped tree dragon Diploderma splendidum (Barbour & Dunn) (Reptilia: Agamidae) in China. The complete mitochondrial genome of R. macrocephalum n. sp. was sequenced and annotated: it is 14,819 bp in length, including 12 protein coding genes (missing atp8), 22 tRNA genes, 2 rRNA genes and three non-coding regions. The gene arrangement of R. macrocephalum n. sp. is different from all of the currently available mitogenomes of nematodes and represents a novel type of mitochondrial gene arrangement reported in Nematoda. Molecular phylogenetic results based on the ITS + 28S data support the monophyly of Entomelas, Pneumonema, Serpentirhabdias, and Rhabdias, and showed R. macrocephalum n. sp. forming a most basal lineage in Rhabdias.

Title: Morphologie, génome mitochondrial complet et phylogénie moléculaire de Rhabdias macrocephalum n. sp. (Nematoda : Rhabdiasidae) de Diploderma splendidum (Reptilia : Agamidae). Abstract: Les espèces du genre Rhabdias Stiles & Hassall, 1905 sont des nématodes parasites courants présents dans les poumons des amphibiens et des reptiles du monde entier. Dans cette étude, Rhabdias macrocephalum n. sp. est décrit à l'aide de méthodes morphologiques intégrées (microscopie optique et électronique à balayage) et d'approches moléculaires (séquençage des régions nucléaires 28S et ITS et des gènes mitochondriaux cox1, cox2 et 12S) basées sur des spécimens collectés chez le lézard Diploderma splendidum (Barbour & Dunn) (Reptilia : Agamidae) de Chine. Le génome mitochondrial complet de R. macrocephalum n. sp. a été séquencé et annoté : il a une longueur de 14 819 pb, dont 12 gènes codants pour des protéines (atp8 manquant), 22 gènes d'ARNt, 2 gènes d'ARNr et trois régions non codantes. L'arrangement génétique de R. macrocephalum n. sp. est différent de tous les mitogénomes de nématodes actuellement disponibles et représente un nouveau type d'arrangement de gènes mitochondriaux signalé chez les nématodes. Les résultats phylogénétiques moléculaires basés sur les données ITS + 28S ont soutenu la monophylie d'Entomelas, Pneumonema, Serpentirhabdias et Rhabdias, et ont montré que R. macrocephalum n. sp. forme la lignée la plus basale chez Rhabdias.

Phasmarhabditis zhejiangensis sp. nov. (Nematoda: Rhabditidae), a new rhabditid nematode from Zhejiang, China.

A new nematode species of the genus Phasmarhabditis was isolated from the body surface of a slug (Philomycus bilineatus Benson, PB). Morphological and molecular analyses confirmed this nematode as a new species. The nematode was named Phasmarhabditis zhejiangensis sp. nov. (Nematoda: Rhabditidae) and is dioecious. In males, the open bursa with genital papillae is characterized by the formula 1-1-1-2-1-3, and the spicule length is 58µm. In female, the vulva is located approximately in the middle of the body. The nematode belongs to papillosa group because of its tail shape pointed with filiform tip. The phasmids are rod-shaped. The posterior anus is slightly swollen. P. zhejiangensis was further characterized by internal transcribed spacer (ITS), 18S rDNA and 28S rDNA sequences. After the sequencing results were compared with sequences available from the National Center for Biotechnology Information (NCBI), the maximum similarities of ITS, 18S and 28S sequences were 89.81%, 96.22% and 95.28%, respectively. Phylogenetic analyses placed Phasmarhabditis zhejiangensis sp. nov. in the genus Phasmarhabditis.

Survey of slug-parasitic nematodes in East and West Flanders, Belgium and description of Angiostoma gandavensis n. sp. (Nematoda: Angiostomidae) from arionid slugs.

A survey for slug-associated nematodes in five locations of East and West Flanders in Belgium revealed the presence of one new and six known slug-parasitic nematodes, Agfa flexilis (Dujardin, 1845), Alloionema appendiculatum (Schneider, 1859), Angiostoma dentiferum (Mengert, 1953), Angiostoma limacis (Dujardin, 1845), Angiostoma norvegicum (Ross et al., 2017) and Phasmarhabditis hermaphrodita (Schneider, 1859). Angiostoma norvegicum and P. hermaphrodita are recorded for the first time in Belgium. The six known species are documented by light microscopy (LM) microphotographs and informative DNA sequences. Angiostoma gandavensis n. sp. (Angiostomatidae), discovered from arionid slugs, is described based on light microscopy, scanning electron microscopy (SEM) and molecular data. Based on analyses of D2D3 expansion segment of 28S and 18S rDNA sequences, this new species is found to be related to A. limacis, A. norvegicum, A. margaretae (Ross et al., 2011) and A. milacis (Ivanova and Wilson, 2009). The new species can be distinguished from these others based on morphological characters such as the distinctive mucronate structures at the tail tip of both sexes, presence of lateral ala, reflexed female ovaries and the number and arrangement pattern of male genital papillae.

Atomic force microscopy for imaging and nanomechanical characterisation of live nematode epicuticle: A comparative Caenorhabditis elegans and Turbatrix aceti study.

Atomic force microscopy (AFM), a powerful tool in interdisciplinary biomedical research, has been applied here to investigate the surface of live nematodes epicuticle. We have used AFM in PeakForce Tapping non-resonant imaging and nanomechanical characterisation mode to investigate and compare the surface features of epicuticle of two free-living microscopic nematodes, Caenorhabditis elegans and Turbatrix aceti. We have successfully immobilised live anesthetized adult nematodes on glass supports using either layer-by-layer-deposited polyelectrolyte films or bioadhesive coatings, which allowed for imaging the living nematodes in native environment. We have obtained AFM images and corresponding nanomechanical maps of annular rings and furrows, demonstrating the differences in topography and structure between the species. Our results demonstrate that AFM in PeakForce Tapping mode can be used to image and characterise surfaces of relatively-large live immobilised multicellular organisms, which can be further applied to a number of invertebrates.

Rhabdias paraensis sp. nov.: a parasite of the lungs of Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia.

The nematode parasites of Rhinella marina include species of the genus Rhabdias (Rhabdiasidae: Rhabditoidea). The present study describes Rhabdias paraensis sp. nov., which parasitizes the lungs of R. marina in Brazilian Amazonia. Of the more than 70 known species of this genus, 18 are parasites of bufonids, of which, eight are Neotropical. The new species described here is similar to Rhabdias alabialis in the absence of lips is different by the presence of conspicuous cephalic papillae. We describe details of the four rows of pores, which are distributed equally along the whole of the length of the body and connected with hypodermal cells, using histology and scanning electron microscopy. Other histological aspects of the internal structure of this nematode are also described.

Rhabdias paraensis sp. nov.: a parasite of the lungs of Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

The nematode parasites of Rhinella marina include species of the genus Rhabdias (Rhabdiasidae: Rhabditoidea). The present study describes Rhabdias paraensis sp. nov., which parasitizes the lungs of R. marina in Brazilian Amazonia. Of the more than 70 known species of this genus, 18 are parasites of bufonids, of which, eight are Neotropical. The new species described here is similar to Rhabdias alabialis in the absence of lips is different by the presence of conspicuous cephalic papillae. We describe details of the four rows of pores, which are distributed equally along the whole of the length of the body and connected with hypodermal cells, using histology and scanning electron microscopy. Other histological aspects of the internal structure of this nematode are also described.

Description of a new species of Kurilonema (Nematoda: Rhabdiasidae) from lungs of the skink Sphenomorphus abdictus aquilonius (Reptilia: Squamata: Scincidae) in the Philippines.

Kurilonema browni n. sp. is described on the basis of specimens found in the lungs of the scincid lizard Sphenomorphus abdictus aquilonius from Aurora Province, Luzon Island, Philippines. The new species differs from Kurilonema markovi, the only previously known species in the genus, by the presence of 2 prominent lateral pseudolabia, larger body size, shorter tail length relative to total body length, and more numerous eggs in the uteri, containing fully developed larvae. The inner surface of the buccal capsule in the new species is densely covered with rounded scales that have not been observed in K. markovi or any other rhabdiasid species. This is the first record of the genus from the Philippines.

Two new species of Angiostoma Dujardin, 1845 (Nematoda: Angiostomatidae) from British terrestrial molluscs.

Two new species of Angiostoma Dujardin, 1845 taken from the intestines of terrestrial molluscs (Gastropoda: Stylommatophora) collected in the UK are described and illustrated. Angiostoma milacis n. sp. is parasitic in milacid and agriolimacid slugs and is closely related to A. asamati Spiridonov, 1985. Angiostoma zonitidis n. sp. was found in a zonitid snail and is most closely related to A. kimmeriense Korol & Spiridonov, 1992 (emend.), a species also from zonitid snails. An amended generic diagnosis is proposed. The distribution of A. limacis and A. milacis sp. n. in hosts is discussed and a key to the species of Angiostoma is presented.

Cell Invasion and Matricide during Photorhabdus luminescens Transmission by Heterorhabditis bacteriophora Nematodes.

Many animals and plants have symbiotic relationships with beneficial bacteria. Experimentally tractable models are necessary to understand the processes involved in the selective transmission of symbiotic bacteria. One such model is the transmission of the insect-pathogenic bacterial symbionts Photorhabdus spp. by Heterorhabditis bacteriophora infective juvenile (IJ)-stage nematodes. By observing egg-laying behavior and IJ development, it was determined that IJs develop exclusively via intrauterine hatching and matricide (i.e., endotokia matricida). By transiently exposing nematodes to fluorescently labeled symbionts, it was determined that symbionts infect the maternal intestine as a biofilm and then invade and breach the rectal gland epithelium, becoming available to the IJ offspring developing in the pseudocoelom. Cell- and stage-specific infection occurs again in the pre-IJ pharyngeal intestinal valve cells, which helps symbionts to persist as IJs develop and move to a new host. Synchronous with nematode development are changes in symbiont and host behavior (e.g., adherence versus invasion). Thus, Photorhabdus symbionts are maternally transmitted by an elaborate infectious process involving multiple selective steps in order to achieve symbiont-specific transmission.

Two new species of Rhabdias (Nematoda: Rhabdiasidae) from the marine toad, Bufo marinus (L.) (Lissamphibia: Anura: Bufonidae), in Central America.

Two new Rhabdias species are described from the lungs of the cane toad Bufo marinus (L.) from Costa Rica and Nicaragua. Rhabdias alabialis n. sp. differs from other known species of the genus by the remarkable morphology of its head end, i.e., the absence of lips or pseudolabia, the slitlike oral opening, and the triangular shape of the buccal capsule in apical view. Rhabdias pseudosphaerocephala n. sp. is identified as a form previously known in Central and South America as Rhabdias sphaerocephala Goodey, 1924, a species initially described from toads in Europe. The new species is differentiated from R. sphaerocephala based on head-end morphology and sequences of nuclear rDNA.

New species of Rhabdias (Nematoda: Rhabdiasidae) from Bufo occidentalis (Anura: Bufonidae) from Sierra Madre del Sur, Mexico.

Rhabdias kuzmini n. sp., a parasite of the lungs of Bufo occidentalis, is described and illustrated. This Mexican taxon differs from the related species in the genus by the possession of 4 lips (2 subdorsal and 2 subventral) and 2 lateral pseudolabia, corpus not inflated, a larger barrel-shaped buccal capsule, equatorial vulva, and the presence of a slightly swollen cuticle in the anterior and posterior ends of the body. This is the 16th species described in the Neotropical Realm and the first species of Rhabdias described from endemic anurans in México.

Pelodera (syn. Rhabditis) strongyloides as a cause of dermatitis--a report of 11 dogs from Finland.

BACKGROUND: Pelodera (Rhabditis) strongyloides is a small saprophytic nematode that lives in decaying organic matter. On rare occasions, it can invade the mammalian skin, causing a pruritic, erythematous, alopecic and crusting dermatitis on skin sites that come into contact with the ground. Diagnosis of the disease is based on case history (a dog living outdoors on damp straw bedding) with characteristic skin lesions and on the demonstration of typical larvae in skin scrapings or biopsy. Pelodera (rhabditic) dermatitis cases have been reported mainly from Central European countries and the United States. CASE PRESENTATION: During 1975-1999, we verified 11 canine cases of Pelodera dermatitis in Finland. The cases were confirmed by identifying Pelodera larvae in scrapings. Biopsies for histopathology were obtained from three cases, and typical histopathological lesions (epidermal hyperplasia, epidermal and follicular hyperkeratosis, folliculitis and furunculosis with large numbers of nematode larvae of 25-40 microm of diameter within hair follicles) were present. The Pelodera strongyloides dermatitica strain from the first verified case in Finland has been maintained in ordinary blood agar in our laboratory since 1975. Light microscopy (LM) and scanning electron microscopy (SEM) studies were employed to obtain detailed morphological information about the causative agent. The rhabditiform oesophagus at all developmental stages, the morphology of the anterior end of the nematode, copulatory bursa and spicules of the male and the tail of the female were the most important morphological features for identifying P. strongyloides. CONCLUSION: These cases show that Pelodera dermatitis occurs in Finland, and also farther north than described earlier in the literature. This condition should be considered when a dog living outdoors has typical skin lesions situated at sites in contact with the ground as the main presenting clinical feature. The fastest and easiest way to confirm the diagnosis is to demonstrate typical larvae in skin scrapings. In uncertain cases, skin biopsy and culturing of the worms are recommended as supplementary diagnostic procedures.

A new rhabdiasid species from Norops megapholidotus (Sauria: Polychrotidae) from Mexico.

Rhabdias leonae n. sp., a parasite of the lungs of Norops megapholidotus, is described and illustrated. The Mexican taxon differs from the other species in the genus by a combination of characters: possession of 4 lips, corpus slightly inflated, slightly postequatorial vulva, and presence of swollen cuticle of anterior and posterior ends. This is the 12th species described in the Neotropical realm and the first species of Rhabdias described from an endemic Mexican lizard.

Immuno-gold-labelling of CUT-1, CUT-2 and cuticlin epitopes in Caenorhabditis elegans and Heterorhabditis sp. processed by high pressure freezing and freeze-substitution.

CUT-1 and CUT-2 are two distinct protein components of cuticlin, the insoluble residue of the cuticles of nematodes. In previous experiments of gold-immuno-labelling on sections of chemically fixed Caenorhabditis elegans, CUT-1 and CUT-2 epitopes were specifically lost. Cryo-immobilization of C. elegans under high pressure followed by freeze-substitution, however, resulted in a good preservation of these antigenic sites and of the ultrastructure of the worms. The entomopathogenic nematode Heterorhabditis sp. processed by the same cryopreparation protocol has shown a strong reactivity with anti-sera raised against CUT-1, CUT-2 and against the whole cuticlin residue of C. elegans. The localization of these epitopes was conserved across the two species.