Ultrastructural study of anther parasitism of Ficus laevigata by Ficophagus laevigatus (Aphelenchoididae).

Transmission electron microscopy (TEM) was used to compare the ultrastructural differences between healthy male florets (anthers) and one floret parasitized by Ficophagus laevigatus in late phase C syconia of Ficus laevigata from southern Florida. Previous light-microscopic examination of paraffin-sectioned material showed that F. laevigatus-infested anthers of F. laevigata manifested as malformed, often with aberrant pollen and hypertrophied epidermal cells closest to regions containing propagating nematodes. Female florets or fig wasp-parasitized female florets were not observed to be parasitized by nematodes. Considering that plant-feeding in the Aphelenchoididae is purportedly much less specialized than in certain groups of the Tylenchomorpha, where specialized hypertrophied feeder cells are produced in response to nematode feeding, we examined the putative induced response in this unusual aphelenchoidid system with the higher resolution afforded by TEM. TEM confirmed the expression of significant epidermal cell hypertrophy of the anther and anther filament in the presence of propagating nematodes, which was expressed as cell enlargement (2-5X), fractionation of large electron-dense stores into smaller aggregates, irregularly shaped nuclei enclosed by an elongated nuclear envelope, nucleolus enlargement, increased organelle production, and apparent metabolism with increased numbers of mitochondria, pro-plastids, and endoplasmic reticulum, as well as increased thickening of the cell walls. Pathological effects were observed in adjacent cells/tissue (e.g., anther and anther filament parenchymal cells, pollen tubes, pollen, and endothecium) with apparent diminishment as the distance from propagating nematodes increased (which was also probably affected by number of nematodes). Some TEM sections captured previously undocumented ultrastructural highlights of propagating individuals of F. laevigatus.

The compact genome of Caenorhabditis niphades n. sp., isolated from a wood-boring weevil, Niphades variegatus.

BACKGROUND: The first metazoan genome sequenced, that of Caenorhabditis elegans, has motivated animal genome evolution studies. To date > 50 species from the genus Caenorhabditis have been sequenced, allowing research on genome variation. RESULTS: In the present study, we describe a new gonochoristic species, Caenorhabditis niphades n. sp., previously referred as C. sp. 36, isolated from adult weevils (Niphades variegatus), with whom they appear to be tightly associated during its life cycle. Along with a species description, we sequenced the genome of C. niphades n. sp. and produced a chromosome-level assembly. A genome comparison highlighted that C. niphades n. sp. has the smallest genome (59 Mbp) so far sequenced in the Elegans supergroup, despite being closely related to a species with an exceptionally large genome, C. japonica. CONCLUSIONS: The compact genome of C. niphades n. sp. can serve as a key resource for comparative evolutionary studies of genome and gene number expansions in Caenorhabditis species.

Tokorhabditis Tauri n. Sp. and T. Atripennis n. Sp. (Rhabditida: Rhabditidae), Isolated from Onthophagus Dung Beetles (Coleoptera: Scarabaeidae) from the Eastern USA and Japan.

Two new species of Tokorhabditis, T. tauri n. sp. and T. atripennis n. sp., which were isolated from multiple Onthophagus species in North America and from O. atripennis in Japan, respectively, are described. The new species are each diagnosed by characters of the male tail and genitalia, in addition to molecular barcode differences that were previously reported. The description of T. tauri n. sp. expands the suite of known nematode associates of O. taurus, promoting ecological studies using a beetle that is an experimental model for insect-nematode-microbiota interactions in a semi-natural setting. Furthermore, our description of a third Tokorhabditis species, T. atripennis n. sp., sets up a comparative model for such ecological interactions, as well as other phenomena as previously described for T. tufae, including maternal care through obligate vivipary, the evolution of reproductive mode, and extremophilic living.

De Novo Genome Assembly of Auanema Melissensis, a Trioecious Free-Living Nematode.

Nematodes of the genus Auanema are interesting models for studying sex determination mechanisms because their populations consist of three sexual morphs (males, females, and hermaphrodites) and produce skewed sex ratios. Here, we introduce a new undescribed species of this genus, Auanema melissensis n. sp., together with its draft nuclear genome. This species is also trioecious and does not cross with the other described species A. rhodensis or A. freiburgensis. Similar to A. freiburgensis, A. melissensis' maternal environment influences the hermaphrodite versus female sex determination of the offspring. The genome of A. melissensis is ~60 Mb, containing 11,040 protein-coding genes and 8.07% of repeat sequences. Using the estimated ancestral chromosomal gene content (Nigon elements), it was possible to identify putative X chromosome scaffolds.

Pristionchus trametes n. sp. (Diplogastridae) isolated from the mushroom Trametes orientalis in Kyoto, Japan.

A new species of Pristionchus was isolated from fruiting bodies of the wood-decaying fungus Trametes orientalis collected from Kyoto, Japan. Attempts to culture it using bacteria, yeast, and freeze-killed wax moth larvae as food or substrate failed. The eurystomatous form of the species was not found in the collected material, and the species is typologically characterized by: its 'small' stoma with thin, membrane-like cheilostomatal plates, a small triangular right subventral tooth, thorn-like dorsal tooth, and small left subventral denticles; a short, blunt male tail spike; and a short, conical female tail. Although the posterior probability support was not high (66%), phylogenetic analysis of both small and large ribosomal RNA gene subunits suggests that the species is closely related to P. elegans and P. bucculentus. The new species can be distinguished from those two by its diagnostic characters comprising the stomatal morphology and male and female tail characters.

Four Pristionchus species associated with two mass-occurring Parafontaria laminata populations.

Phoretic nematodes associated with two mass-occurring populations of the millipede Parafontaria laminata were examined, focusing on Pristionchus spp. The nematodes that propagated on dissected millipedes were genotyped using the D2-D3 expansion segments of the 28S ribosomal RNA gene. Four Pristionchus spp. were detected: P. degawai, P. laevicollis, P. fukushimae, and P. entomophagus. Of the four, P. degawai dominated and it was isolated from more than 90% of the millipedes examined. The haplotypes of partial sequences of mitochondrial cytochrome oxidase subunit I examined for Pristionchus spp. and P. degawai showed high haplotype diversity.

Two new Species of Pristionchus (Nematoda: Diplogastridae) include the Gonochoristic Sister Species of P. fissidentatus.

The genus Pristionchus (Kreis, 1932) consists of more than 30 soil nematode species that are often found in association with scarab beetles. Three major radiations have resulted in the "maupasi species group" in America, the "pacificus species group" in Asia, and the "lheritieri species group," which contains species from Europe and Asia. Phylogenetic analysis indicates that a group of three species, including the gonochorists P. elegans and P. bucculentus and the hermaphrodite P. fissidentatus, is basal to the above-mentioned radiations. Two novel species are described here: Pristionchus paulseni sp. n. from Taiwan and P. yamagatae sp. n. from Japan by means of morphology, morphometrics and genome-wide transcriptome sequence analysis. Previous phylotranscriptomic analysis of the complete Pristionchus genus recognized P. paulseni sp. n. as the sister species of P. fissidentatus, and thus its importance for macro-evolutionary studies. Specifically, the gonochorist P. paulseni sp. n. and the hermaphrodite P. fissidentatus form a species pair that is the sister group to all other described Pristionchus species. P. paulseni sp. n. has two distinct mouth forms, supporting the notion that the mouth dimorphism is ancestral in the genus Pristionchus.The genus Pristionchus (Kreis, 1932) consists of more than 30 soil nematode species that are often found in association with scarab beetles. Three major radiations have resulted in the "maupasi species group" in America, the "pacificus species group" in Asia, and the "lheritieri species group," which contains species from Europe and Asia. Phylogenetic analysis indicates that a group of three species, including the gonochorists P. elegans and P. bucculentus and the hermaphrodite P. fissidentatus, is basal to the above-mentioned radiations. Two novel species are described here: Pristionchus paulseni sp. n. from Taiwan and P. yamagatae sp. n. from Japan by means of morphology, morphometrics and genome-wide transcriptome sequence analysis. Previous phylotranscriptomic analysis of the complete Pristionchus genus recognized P. paulseni sp. n. as the sister species of P. fissidentatus, and thus its importance for macro-evolutionary studies. Specifically, the gonochorist P. paulseni sp. n. and the hermaphrodite P. fissidentatus form a species pair that is the sister group to all other described Pristionchus species. P. paulseni sp. n. has two distinct mouth forms, supporting the notion that the mouth dimorphism is ancestral in the genus Pristionchus.

Pine wilt disease causes cavitation around the resin canals and irrecoverable xylem conduit dysfunction.

Physiological mechanisms of irreversible hydraulic dysfunction in seedlings infected with pine wilt disease (PWD) are still unclear. We employed cryo-scanning electron microscopy (cryo-SEM) to investigate the temporal and spatial changes in water distribution within the xylem of the main stem of 2-year-old Japanese black pine seedlings infested by pine wood nematodes (PWNs). Our experiment was specifically designed to compare the water relations among seedlings subjected to the following water treatment and PWN combinations: (i) well-watered versus prolonged drought (no PWNs); and (ii) well-watered with PWNs versus water-stressed with PWNs (four treatments in total). Cryo-SEM imaging observations chronicled the development of patchy cavitations in the xylem tracheids of the seedlings influenced by PWD. With the progression of drought, many pit membranes of bordered pits in the xylem of the main stem were aspirated with the decrease in water potential without xylem cavitation, indicating that hydraulic segmentation may exist between tracheids. This is the first study to demonstrate conclusively that explosive and irreversible cavitations occurred around the hydraulically vulnerable resin canals with the progression of PWD. Our findings provide a more comprehensive understanding of stressors on plant-water relations that may eventually better protect trees from PWD and assist with the breeding of trees more tolerant to PWD.

Two New Species of Pristionchus (Nematoda: Diplogastridae) from Taiwan and the Definition of the pacificus Species-Complex Sensu Stricto.

Pristionchus pacificus Sommer, Carta, Kim, and Sternberg, 1996 is an important model organism in evolutionary biology that aims to integrate developmental biology and evo-devo with population genetics and ecology. Functional studies in P. pacificus are supported by a well-established phylogenetic framework of around 30 species of the genus Pristionchus that have been described in the last decade based on their entomophilic and necromenic association with scarab beetles. Biogeographically, East Asia has emerged as a hotspot of Pristionchus speciation and recent samplings have therefore focused on Islands and mainland settings in East Asia. Here, we describe in a series of three publications the results of our sampling efforts in Taiwan, Japan, and Hongkong in 2016 and 2017. We describe a total of nine new species that cover different phylogenetic species-complexes of the Pristionchus genus. In this first publication, we describe two new species, Pristionchus sikae sp. n. and Pristionchus kurosawai sp. n. that are closely related to P. pacificus . Together with five previously described species they form the " pacificus species-complex sensu stricto" that is characterized by all species forming viable, but sterile F1 hybrids indicating reproductive isolation. P. sikae sp. n. and P. kurosawai sp. n. have a gonochorist mode of reproduction and they are described using morphology, morphometrics, mating experiments, and genome-wide sequence analysis. We discuss the extreme diversification in the pacificus species-complex sensu stricto in East Asia and its potential power to study speciation processes.

Samplings of Millipedes in Japan and Scarab Beetles in Hong Kong result in five new Species of Pristionchus (Nematoda: Diplogastridae).

The authors describe five new species of Pristionchus from Japan and Hongkong. Scarab beetle samplings in Hongkong identified P. hongkongensis sp. n. and P. neolucani sp. n., representing the first beetle-associated Pristionchus species from China. Surprisingly, samplings of millipedes in Japan revealed a previously unknown association of Pristionchus nematodes with these arthropods. Specifically, the authors found three previously known Pristionchus species, P. arcanus, P. entomophagus, and P. fukushimae on Japanese millipedes. In addition, the authors found three new Pristionchus species on millipedes, which are described as P. riukiariae sp. n., P. degawai sp. n., and P. laevicollis, sp. n., the latter of which was also found on stag beetles. These species are most closely related to P. maxplancki, P. japonicus, and P. quartusdecimus and belong to the pacificus species-complex. The authors describe all species based on morphology, morphometrics, and genome-wide sequence analysis. Mating experiments indicated that all species are reproductively isolated from each other and in contrast to the species of the "pacificus species-complex sensu stricto" they do not form F1 hybrids.The authors describe five new species of Pristionchus from Japan and Hongkong. Scarab beetle samplings in Hongkong identified P. hongkongensis sp. n. and P. neolucani sp. n., representing the first beetle-associated Pristionchus species from China. Surprisingly, samplings of millipedes in Japan revealed a previously unknown association of Pristionchus nematodes with these arthropods. Specifically, the authors found three previously known Pristionchus species, P. arcanus, P. entomophagus, and P. fukushimae on Japanese millipedes. In addition, the authors found three new Pristionchus species on millipedes, which are described as P. riukiariae sp. n., P. degawai sp. n., and P. laevicollis, sp. n., the latter of which was also found on stag beetles. These species are most closely related to P. maxplancki, P. japonicus, and P. quartusdecimus and belong to the pacificus species-complex. The authors describe all species based on morphology, morphometrics, and genome-wide sequence analysis. Mating experiments indicated that all species are reproductively isolated from each other and in contrast to the species of the "pacificus species-complex sensu stricto" they do not form F1 hybrids.

Diplogasteroides asiaticus n. sp. is Associated with Monochamus alternatus in Japan.

Diplogasteroides asiaticus n. sp. is described and illustrated, and its molecular profile and phylogenetic status within the family Diplogastridae are inferred. Morphologically, the new species is characterized by its stomatal structure, a tube-like stoma with three small, rod-like dorsal teeth and two subventral ridges; a spicule clearly ventrally bent at 1/3 from the anterior end; a gubernaculum with a rounded anterior end and sharply pointed distal end in lateral view; nine pairs of genital papillae with an arrangement of ; a short tail spike in males; and a well-developed receptaculum seminis, i.e., the antiparallel blind sacs of the uteri beyond the vulva region and elongated conical tail in females. This new species is morphologically similar to D. haslacheri, but it can be distinguished by the morphology of the somewhat shorter tail in females. D. asiaticus n. sp. shares high sequence conservation with D. andrassyi as there is only one base pair difference in the nearly full-length 18S rDNA and seven base pair differences in the D2-D3 expansion segments of the 28S rDNA. Despite this sequence conservation, the species status of D. asiaticus n. sp. was confirmed using the biological species concept, as D. asiaticus n. sp. and D. andrassyi failed to generate viable F2 progeny in hybridization tests.

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

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

Phylogenetic Status and Morphological Characters of Rhabditolaimus anoplophorae (Rhabditida: Diplogastridae).

Rhabditolaimus anoplophorae Kanzaki and Futai was re-isolated from its type host (carrier), the cerambycid beetle Anoplophora malasiaca, collected in an experimental field of the Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan. The nematode was cultured on nematode growth medium plates seeded with Escherichia coli OP50, and its morphological characters and molecular profile were examined to modernize the description. Scanning electron microscopic and light microscopy revealed the presence of four stomatal flaps, a very long gymnostom, a single ventral papilla in males, and a horizontal slit-like vulval opening in females. The positions of the deirids, hemizonids, phasmids, and rectal glands are additionally described, and the absence of a male bursa was confirmed. Phylogenetically, the genus forms a well-supported clade in the family Diplogastridae. Rhabditolaimus anoplophorae is a member of the monophyletic Rhabditolaimus clade and is closely related to R. leuckarti and several undescribed species.

Leptojacobus dorci n. gen., n. sp. (Nematoda: Diplogastridae), an Associate of Dorcus Stag Beetles (Coleoptera: Lucanidae).

A new species of diplogastrid nematode, Leptojacobus dorci n. gen., n. sp., was isolated from adults of the stag beetle Dorcus ritsemae (Coleoptera: Lucanidae) that were purchased from a pet shop in Japan. Leptojacobus n. gen. is circumscribed by a very thin, delicate body and by a small stoma with minute armature. A combination of other stomatal characters, namely the division of the cheilostom into adradial plates, the symmetry of the subventral stegostomatal sectors, and the presence of a thin, conical dorsal tooth, further distinguishes Leptojacobus n. gen. from other genera of Diplogastridae. Phylogenetic analysis of nearly full-length SSU rRNA sequences support the new species, together with an isolate identified previously as Koerneria luziae, to be excluded from a clade including all other molecularly characterized diplogastrids with teeth and stomatal dimorphism. Therefore, the new species will be of importance for reconstruction of ancestral character histories in Diplogastridae, a family circumscribed by a suite of feeding-related novelties.

Two new and two recharacterized species from a radiation of pristionchus (nematoda: diplogastridae) in europe.

Two new species of Pristionchus, P. lucani n. sp. and P. bulgaricus n. sp., are described from France and Bulgaria, respectively. Additionally, new morphological and morphometric data are provided for two previously described species from Europe, P. brevicauda (Kotlán, 1928) Paramonov, 1952 and P. clavus (von Linstow, 1901) Sudhaus and Fürst von Lieven, 2003. A phylogeny including these four species was inferred from a dataset including 26 ribosomal protein-coding genes, sequences of which are original for P. bulgaricus n. sp. and P. clavus. Relationships support a radiation of all sequenced European Pristionchus species from a single, gonochoristic common ancestor, and current knowledge of species ranges supports "western" and "eastern" clades. Similar diagnostic morphologies reflect the close relationships among the new and recharacterized species, especially P. bulgaricus n. sp., P. brevicauda, and P. clavus, although mating tests as well as genetic and phylogenetic separation support their identities as unique species. Our results show that Pristionchus species in Europe are more diverse than typological characters suggest, and thus biological and molecular profiling will be essential for future delimitation of Pristionchus species from the region.

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Two new species of Pristionchus (Nematoda: Diplogastridae) support the biogeographic importance of Japan for the evolution of the genus Pristionchus and the model system P. pacificus.

Pristionchus pacificus Sommer, Carta, Kim, and Sternberg, 1996 is an important model organism in evolutionary biology that integrates developmental biology with ecology and population genetics. This species is part of a sub-complex of the genus Pristionchus that is considered to have originated in East Asia. Here, we describe two new species of Pristionchus, P. maxplancki and P. quartusdecimus, which were isolated from beetles in Japan, supporting the hypothesis that a region including Japan is the origin of diversification of the P. pacificus species complex. Phytogeny inferred from a partial small subunit rRNA gene and 25 ribosomal protein genes shows P. maxplancki to be the closest known outgroup to a triad of sibling species, including P. pacificus. Pristionchus quartusdecimus is a putative outgroup to the P. pacificus species complex, supporting a more ancient origin of Pristionchus species in the region. Species diagnoses are based on morphological and molecular characters, in addition to reproductive isolation for P. maxplancki. Members of the P. pacificus species complex as well as P. quartusdecimus are distinguished by stegostomatal structures, male genital papilla arrangement, and gubernaculum shape. The discovery of a new member of the P. pacificus species complex allows greater precision in polarizing and reconstructing ancestral states in the comparative model system centering on P. pacificus. Together with previous reports, these findings support an important biogeographic role of Japan in the evolution of the genus Pristionchus and the P. pacificus species complex, especially the associated phenotypic evolution of mouth morphology.

Pristionchus bucculentus n. sp. (Rhabditida: Diplogastridae) Isolated from a Shining Mushroom Beetle (Coleoptera: Scaphidiidae) in Hokkaido, Japan.

Pristionchus bucculentus n. sp. was isolated from a shining mushroom beetle, Episcapha gorhami, associated with white rot on a decaying log in Hokkaido, Japan. The new species is distinguished by its stomatal morphology, which includes three regularly shaped, conical left subventral denticles and a vacuolated cheilostom with weak internal sclerotization. Also distinguishing P. bucculentus n. sp. are male sexual characters, including arrangement of genital papillae, a rounded and ventrally skewed manubrium, and a gubernaculum with a large, deep posterior curvature and a short, shallow anterior curvature. Morphological and molecular evidence support the new species as being close to P. elegans, which was previously the most basal known species of the genus. Comparative morphology of basal Pristionchus species is supported by a molecular phylogeny inferred from a partial small subunit ribosomal rRNA gene and 25 ribosomal protein-coding genes. Description of P. bucculentus n. sp. provides a new point of comparison for reconstructing the evolution of stomatal characters in the comparative model system of Pristionchus.

Two androdioecious and one dioecious new species of pristionchus (nematoda: diplogastridae): new reference points for the evolution of reproductive mode.

Rhabditid nematodes are one of a few animal taxa in which androdioecious reproduction, involving hermaphrodites and males, is found. In the genus Pristionchus, several cases of androdioecy are known, including the model species P. pacificus. A comprehensive understanding of the evolution of reproductive mode depends on dense taxon sampling and careful morphological and phylogenetic reconstruction. In this article, two new androdioecious species, P. boliviae n. sp. and P. mayeri n. sp., and one gonochoristic outgroup, P. atlanticus n. sp., are described on morphological, molecular, and biological evidence. Their phylogenetic relationships are inferred from 26 ribosomal protein genes and a partial SSU rRNA gene. Based on current representation, the new androdioecious species are sister taxa, indicating either speciation from an androdioecious ancestor or rapid convergent evolution in closely related species. Male sexual characters distinguish the new species, and new characters for six closely related Pristionchus species are presented. Male papillae are unusually variable in P. boliviae n. sp. and P. mayeri n. sp., consistent with the predictions of "selfing syndrome." Description and phylogeny of new androdioecious species, supported by fuller outgroup representation, establish new reference points for mechanistic studies in the Pristionchus system by expanding its comparative context.