Integrative taxonomy of Mesocriconema onoense (Tylenchida: Criconematidae) from Vietnam highly suggests the synonymization of Mesocriconema brevistylus and related species.

The genus Mesocriconema is one of the most diverse genera within the family Criconematidae, known as ring nematodes, with more than 90 species. Although species in this genus usually show distinct morphological characterizations, the identification based only on morphology can lead to misidentification in many studies resulted in a number of synonymizations in the genus over time. In this study, an integrated approach has been applied in characterizing Mesocriconema onoense from Vietnam. The molecular data of 28S rRNA, ITS, 18S rRNA regions were analyzed and discussed to confirm the correct names on GenBank. Besides, phylogenetic analyses of 28S rRNA, ITS, and 18S rRNA regions of Mesocriconema species revealed that Mesocriconema brevistylus should be considered as a junior synonym of M. onoense. Consequently, M. helicus, M. onostris, and M. paronostris should also be considered as the synonyms of M. onoense.

First Morphological and Molecular Report of Aphelenchoides blastophthorus on Strawberry Plants in Switzerland.

Foliar nematodes represent a minor feeding group within the genus Aphelenchoides Fischer, 1894. The facultative plant parasitic species A. blastophthorus can cause crinkling of leaves, reduced vigor, and stunting of agricultural and ornamental plants. Here we report the first finding of A. blastophthorus in leaves, crowns, and roots of strawberry plants collected in Switzerland in 2018. Species identification was confirmed by morphological and morphometric characterization supported by molecular barcoding of 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), and cytochrome c oxidase I (COI) gene fragment analyses. Phylogenetic analysis of 18S indicated that A. blastophthorus was grouped within close distance to A. fragariae, a well-known foliar nematode affecting strawberry plants. Furthermore, the newly generated molecular barcodes of the partial 28S and COI of A. blastophthorus will support species identification in the future.

The first report of Xenorhabdus indica from Steinernema pakistanense: co-phylogenetic study suggests co-speciation between X. indica and its steinernematid nematodes.

During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode-bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the 'bicornutum' group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.

Nematode parasites of Duttaphrynus stomaticus (Lutken, 1864) (Amphibia: Anura) with description of a new species of Rhabdias Stiles and Hassall, 1905 (Nematoda: Rhabdiasidae) from Dehradun (Uttarakhand), India.

Rhabdias stomatica sp. nov. from the lungs of Duttaphrynus stomaticus (Lutken, 1864) from Dehradun, Uttarakhand, India is described and illustrated. Rhabdias stomatica sp. nov. is the 16th species described from the Oriental biogeographical region and the 8th species from India. The new species is differentiated from the closely related Oriental species in having 4 weakly developed lips, a trapezoidal shaped buccal cavity, different position of nerve ring and in the esophagus/body length ratio. In addition, to the new species found in the lungs, mature specimens of Aplectana macintoshii (Stewart, 1914) Travassos, 1931 and larvae representing two unidentified species of nematode were found in the large intestine of the D. stomaticus.

Molecular and morphological characterisation of Rhabdias picardiae Junker, Lhermitte-Vallarino et Bain, 2010 (Nematoda: Rhabdiasidae) from Delaland's River Frog, Amietia delalandii (Duméril et Bibron, 1841) (Amphibia: Pyxicephalidae) in South Africa.

Rhabdias picardiae previously known from the Guttural Toad, Sclerophrys gutturalis (Bufonidae) is described from a new host, Delaland's River Frog, Amietia delalandii (Pyxicephalidae). The species identification is confirmed by analysis of the COI gene sequence and morphological comparison with the original description. Morphological details of apical structures in adult parasitic R. picardiae, namely, the shape and position of lips, the number and position of external labial papillae, and the buccal capsule morphology are described using light and scanning electron microscopy. The buccal capsule width and length, the distance from anterior end to nerve ring and excretory pore, and ratios of distance to vulva to body length and body length to width were identified as less variable metrical characters in the studied sample of 30 specimens. Adult specimens demonstrated differences in the position of lips in relation to the oral opening and position of the buccal capsule in relation to the oesophagus depending on the size (age) of worms. The infective larvae of R. picardiae are described for the first time and characterised by two lateral alae consisting of two ridges, triangular pseudolabia with rounded tops, and presence of ornamentation on the rounded tail tip.

Description of two new species of Seinura Fuchs, 1931 (Nematoda: Aphelenchoididae) from Iran.

Two new species of Seinura are described and illustrated using both morphological and molecular characters. Seinura persica n. sp. is characterized by having females with a distinct cephalic region, stylet 19.5 (18-20) µm long, four incisures in the lateral fields, position of excretory pore anterior to the base of metacorpus, post-uterine sac 54.8 (50-60) µm long, and an elongate-conoid tail with a shallow depression on the dorsal side ending in a sharply pointed tip. Males have body length of 467 and 592 µm, spicules 15 and 16 µm long with a prominent triangular-shaped rostrum, developed condylus and no cucullus. Seinura hyrcania n. sp. is characterized by having an offset lip region, stylet 21.5 (18-24) µm long, three incisures in the lateral fields, excretory pore anterior to the base of the metacorpus, post-uterine sac 19 (12-25) µm long and conical tail ending in a filiform terminus. Morphological and morphometric diagnostic characters together with molecular data based on partial sequences of SSU and LSU of the ribosomal DNA gene are provided for these new species.

The effect of two fixation methods (TAF and DESS) on morphometric parameters of Aphelenchoides ritzemabosi.

Identification of nematode species by using conventional methods requires fixation of the isolated material and a suitable preparation for further analyses. Tentative identification using microscopic methods should also be performed prior to initiating molecular studies. In the literature, various methods are described for the preparation of nematodes from the genus Aphelenchoides for identification and microscopic studies. The most commonly used fixatives are formalin (Timm 1969; Szczygiel & Cid del Prado Vera 1981, Crozzoli et al. 2008, Khan et al. 2008), FAA (Wasilewska 1969; Vovlas et al. 2005, Khan et al. 2007) and TAF (Hooper 1958, Chizhov et al. 2006, Jagdale & Grewal 2006).

Species discovery and diversity in Lobocriconema (Criconematidae: Nematoda) and related plant-parasitic nematodes from North American ecoregions.

There are many nematode species that, following formal description, are seldom mentioned again in the scientific literature. Lobocriconema thornei and L. incrassatum are two such species, described from North American forests, respectively 37 and 49 years ago. In the course of a 3-year nematode biodiversity survey of North American ecoregions, specimens resembling Lobocriconema species appeared in soil samples from both grassland and forested sites. Using a combination of molecular and morphological analyses, together with a set of species delimitation approaches, we have expanded the known range of these species, added to the species descriptions, and discovered a related group of species that form a monophyletic group with the two described species. In this study, 148 specimens potentially belonging to the genus Lobocriconema were isolated from soil, individually measured, digitally imaged, and DNA barcoded using a 721 bp region of cytochrome oxidase subunit 1 (COI). One-third of the specimens were also analyzed using amplified DNA from the 3' region of the small subunit ribosomal RNA gene (18SrDNA) and the adjacent first internal transcribed spacer (ITS1). Eighteen mitochondrial haplotype groups, falling into four major clades, were identified by well-supported nodes in Bayesian and maximum likelihood trees and recognized as distinct lineages by species delimitation metrics. Discriminant function analysis of a set of morphological characters indicated that the major clades in the dataset possessed a strong morphological signal that decreased in comparisons of haplotype groups within clades. Evidence of biogeographic and phylogeographic patterns was apparent in the dataset. COI haplotype diversity was high in the southern Appalachian Mountains and Gulf Coast states and lessened in northern temperate forests. Lobocriconema distribution suggests the existence of phylogeographic patterns associated with recolonization of formerly glaciated regions by eastern deciduous forest, but definitive glacial refugia for this group of plant parasitic nematodes have yet to be identified. Unlike agricultural pest species of plant-parasitic nematodes, there is little evidence of long-distance dispersal in Lobocriconema as revealed by haplotype distribution. Most haplotype groups were characterized by low levels of intragroup genetic variation and large genetic distances between haplotype groups. The localization of nematode haplotypes together with their characteristic plant communities could provide insight into the historical formation of these belowground biotic communities.

Description of Cryptaphelenchus iranicus n. sp. (Nematoda: Ektaphelenchinae) recovered from bark samples of Pinus nigra from Iran.

Cryptaphelenchus iranicus n. sp., recovered from bark and wood samples of a weakened Pinus nigra in Kermanshah Province, western Iran, is described. The new species has females with body length of 250-330 µm and males 230-275 µm long, lip region set-off from body contour, 7-8 µm long stylet with small basal swellings, excretory pore located at 1.5-2.0 body diam. posterior to median bulb, post-vulval uterine sac short, 7-10 µm long and conical female posterior body end (tail) ending to a pointed tip. Males of the new species have seven (1+2+2+2) caudal papillae and a short mucro at tail tip. Based on morphological characters, the new species is close to C. cirrus, C. latus and C. leptocaudus. Phylogenetic analyses using the D2/D3 fragment of 28S rDNA show the new species forming a clade with other Cryptaphelenchus species with maximal (1.00) Bayesian posterior probability (BPP) in Bayesian inference and 100% bootstrap value (BS) in the maximum likelihood method. The Cryptaphelenchus clade forms a monophyletic group with members of subfamily Ektaphelenchinae.

Double-stranded RNA-mediated interference of dumpy genes in Bursaphelenchus xylophilus by feeding on filamentous fungal transformants.

RNA interference (RNAi) is a valuable tool for studying gene function in vivo and provides a functional genomics platform in a wide variety of organisms. The pinewood nematode, Bursaphelenchus xylophilus, is a prominent invasive plant-parasitic nematode and has become a serious worldwide threat to forest ecosystems. Presently, the complete genome sequence of B. xylophilus has been published, and research involving genome-wide functional analyses is likely to increase. In this study, we describe the construction of an effective silencing vector, pDH-RH, which contains a transcriptional unit for a hairpin loop structure. Utilising this vector, double-stranded (ds)RNAs with sequences homologous to the target genes can be expressed in a transformed filamentous fungus via Agrobacterium tumefaciens-mediated transformation technology, and can subsequently induce the knockdown of target gene mRNA expression in B. xylophilus by allowing the nematode to feed on the fungal transformants. Four dumpy genes (Bx-dpy-2, 4, 10 and 11) were used as targets to detect RNAi efficiency. By allowing the nematode to feed on target gene-transformed Fusarium oxysporum strains, target transcripts were knocked down 34-87% compared with those feeding on the wild-type strain as determined by real-time quantitative PCR (RT-qPCR). Morphological RNAi phenotypes were observed, displaying obviously reduced body length; weak dumpy or small (short and thin) body size; or general abnormalities. Moreover, compensatory regulation and non-specific silencing of dpy genes were found in B. xylophilus. Our results indicate that RNAi delivery by feeding in B. xylophilus is a successful technique. This platform may provide a new opportunity for undertaking RNAi-based, genome-wide gene functional studies in vitro in B. xylophilus. Moreover, as B. xylophilus feeds on endophytic fungi when a host has died, RNAi feeding technology will offer the prospect for developing a novel control strategy for the nematode. Furthermore, this platform may also be applicable to other parasitic nematodes that have a facultative, fungivorous habit.

Transcriptional and morphological changes in the transition from mycetophagous to phytophagous phase in the plant-parasitic nematode Bursaphelenchus xylophilus.

Drastic physiological and morphological changes in parasites are crucial for the establishment of a successful infection. The nematode Bursaphelenchus xylophilus is the pathogenic agent of pine wilt disease, and little is known about the physiology and morphology in this nematode at the initial stage of infection. In this study, we devised an infection system using pine stem cuttings that allowed us to observe transcriptional and morphological changes in the host-infecting phytophagous phase. We found that 60 genes enriched in xenobiotic detoxification were up-regulated in two independent post-inoculation events, whereas down-regulation was observed in multiple members of collagen gene families. After 48 h of inoculation, the tails in some of the adult females exposed to the host changed in morphology. These results suggest that B. xylophilus may change its physiology and morphology to protect itself and to adapt to the host pine wood environment.

Molecular and morphological characterization of Veleshkinema iranicum n. gen., n. sp. (Nematoda: Hexatylina, Sphaerularioidea) from Iran.

Veleshkinema  iranicum n. gen., n. sp. is described and illustrated based on morphological, morphometric and molecular characters. The new genus is characterized by having slender females and males, stylet with asymmetrical knobs, dorsal gland orifice just posterior to subventral knob, lip region with flattened apex and eight sectors, pharynx with a non-muscular and non-valvular median bulb, pharyngeal glands slightly overlapping intestine dorsally, visible cellular cardia, female with a single gonad having a quadricolumellate crustaformeria with 8-10 cells in each column, no postvulval uterine sac and rounded and offset spermatheca containing spheroid sperm cells, males with arcuate tylenchoid spicules and subterminal bursa. The new genus is morphologically compared with four genera: Abursanema, Deladenus, Prothallonema and Sphaerularia. Molecular phylogenetic studies of the new genus using 808 bp partial sequences of SSU ribosomal RNA gene placed the new genus in a clade with Sphaerularia spp. In phylogenetic analyses using 756 bp partial sequences of the 28S ribosomal RNA gene (D2-D3 segments), the new genus formed a monophyletic group with Abursanema iranicum and Sphaerularia spp.

An updated list of the plants associated with plant-parasitic Aphelenchoides (Nematoda: Aphelenchoididae) and its implications for plant-parasitism within this genus.

Few Aphelenchoides spp. are facultative plant-parasites (foliar and bulb nematodes); three of them are well known in agricultural systems, namely Aphelenchoides besseyi, A. fragariae and A. ritzemabosi. Ten other plant-parasitic species, A. arachidis, A. bicaudatus, A. blastophthorus, A. dalianensis, A. ensete, A. nechaleos, A. paranechaleos, A. saprophilus, A. sphaerocephalus and A. subtenuis, have been reported from a limited number of plant species. We compiled a new database of the associated plants for these thirteen species, a comprehensive list that includes 1104 reports from 126 botanical families. A. besseyi, A. fragariae and A. ritzemabosi represent 94% of the reports, circa 83% and 16% of the total reports correspond to flowering plants and ferns, respectively, with three records on conifers and two from other botanical groups also listed. Most plant-parasitic Aphelenchoides show a remarkably broad diversity of associated plants. Most species appear to have no specific plant hosts (i.e. are generalists). The broad host ranges of these species and absence of more intimate interactions with the associated plants highlights the primitive mode of parasitism in Aphelenchoides species, making them potentially interesting in the study of the evolution of plant parasitism. Even though the compiled list of associated plants is long, it probably only represents a fraction of the potential range. The complete compilation has been uploaded to http://nematodes.myspecies.info/.

Proparasitylenchus californicus n. sp. (Tylenchida: Allantonematidae), parasitic in the intertidal rove beetle Tarphiota geniculata (Mäklin) (Coleoptera: Staphylinidae) in California, USA.

A new nematode species, Proparasitylenchus californicus n. sp., is described from the intertidal rove beetle Tarphiota geniculata (Mäklin) (Coleoptera: Staphylinidae) in California, USA. The new species differs from European representatives of the genus by possessing a cleft stylet in both sexes. The parasitic female is ovoviviparous and produces numerous juveniles that moult twice in the beetle host, then exit and moult twice to the adult stage in the environment. After mating, the free-living fertilised females enter a new host. Heavy infections sterilise the beetles. This is the first record of the genus Proparasitylenchus Wachek, 1955 in the New World and the first allantonematid parasite of a marine, intertidal beetle.

Laimaphelenchus hyrcanus n. sp. (Nematoda: Aphelenchoididae), a new species from northern Iran.

Laimaphalenchus hyrcanus n. sp., from bark samples of cypress (Cupressus sp.) and jujube (Ziziphus jujube Miller) from Gorgan, Golestan province in northern Iran, is described and illustrated based on morphological and molecular characters. The new species is characterized by females with 670-870 µm body length, slender stylet of 11-12 µm, six-lobed cephalic region not divided by ribs, three incisures in the lateral field with slightly areolated outer lines in the posterior region, cuticular vulval flap, males with two pairs of caudal papillae, and subcylindrical tail in both sexes ending in a single stalk-like terminus with four pedunculate tubercles bearing 9-10 finger-like projections. The new species most closely resembles L. persicus and L. penardi but it can be differentiated by its having three incisures in the lateral field and two pairs of caudal papillae in males. Molecular analysis of the D2/D3 region of the 28S ribosomal RNA gene indicates a sister-taxon relationship with L. penardi (Steiner, 1914) Filipjev & Schuurmans Stekhoven, 1941 and L. deconincki Elmiligy & Geraert, 1972. 

Nematodes from galls on Myrtaceae. IX. Fergusobia rosettae n. sp. on Melaleuca quinquenervia and F. tolgaensis n. sp. on Syzygium luehmannii, from Queensland.

Two new species of Fergusobia, collected from 'rosette' shoot bud galls on Melaleuca quinquenervia, and from leaf, stem, leaf and flower bud galls on Syzygium luehmannii, both from the Cairns region of Queensland, Australia, are described. Fergusobia rosettae Davies n. sp. is characterised by the combination of a small, arcuate parthenogenetic female having a short conoid tail with a bluntly rounded tip, an arcuate, relatively slender, infective female with an almost hemispherical tail tip, and arcuate males with arcuate to angular (not heavily sclerotised) spicules and leptoderan bursa arising at 40-50% of body length from tail tip. Fergusobia tolgaensis Davies n. sp. is characterised by the combination of a small open C-shaped parthenogenetic female with a broadly conoid tail, an arcuate infective female with a broadly rounded tail tip, and arcuate males with angular (not heavily sclerotised) spicules and short to mid-length leptoderan bursa. These two species of nematodes are associated with fly larvae that have dorsal shields comprising bars of raised cuticular ridges and spicules, similar to that of fly larvae from the M. leucadendra species group. The shield morphologies of these fly larvae and their possible genetic relationships are discussed. Possible evolutionary relationships of the Fergusobia nematodes from these galls are discussed, considering their morphology, DNA sequences, and the relationships of the associated Fergusonina flies and host plants.

Nematodes from galls on Myrtaceae. X. Fergusobia from galls on narrow-leaved Melaleuca spp. in Australia, with descriptions of three new species .

Three new species of Fergusobia, respectively collected from shoot bud galls on narrow-leaved Melaleuca spp. in Australia, are described. Fergusobia armillarisae n. sp. Davies is characterised by the combination of an arcuate to open C-shaped parthenogenetic female with an extensile uterus and a short, conoid tail, an arcuate infective female with a broadly rounded tail tip, and an arcuate male with an angular spicule and bursa arising at 50-80% of body length. Fergusobia decorae n. sp. Davies has an arcuate parthenogenetic female with a non-extensile uterus and a broadly conoid tail, an arcuate infective female with most curvature behind the vulva and a short tail with a broadly rounded tip, and an arcuate male with an arcuate spicule and bursa arising at 40-50% of body length. Fergusobia linariifoliae n. sp. Davies is characterised by the combination of an arcuate parthenogenetic female with an extensile uterus and a short, conoid tail with a bluntly rounded tip, a barely arcuate infective female with a broadly rounded tail tip, and an arcuate male with an angular spicule and bursa arising at 40-50% of body length. Earlier molecular analyses inferred from DNA sequencing of 28S rDNA D2/D3 domains and a portion of mitochondrial DNA cytochrome oxidase subunit I (mtCOI) are further discussed. 

Nematodes from galls on Myrtaceae. VI. Fergusobia from galls on Angophora in Australia, with description of F. colbrani n. sp. and key.

Collection data and biological information is presented on the Fergusobia (Nematoda: Neotylenchidae)/ Fergusonina (Diptera: Fergusoninidae) mutualism inducing galls on Angophora in Australia. Three species and two morphospecies have been recognised. Fergusobia colbrani Davies n. sp. is described from soft spheroid leaf galls on Angophora floribunda. It is characterised by a combination of morphological characters including a small C-shaped parthenogenetic female with a short broadly conoid tail, an arcuate infective female with an almost hemispherical tail tip, and an arcuate to barely J-shaped male with an angular spicule having a notched tip and mid-length leptoderan bursa. A key to the species and morphospecies of nematodes collected from Angophora is presented. Possible relationships of these organisms are discussed based on evidence from the nematode morphology, gall forms, and the morphology of the dorsal shield of the associated Fergusonina fly larvae. 

Nematodes from galls on Myrtaceae. VII. Fergusobia from 'leafy' leaf bud galls in Australia, with re-description of Fergusobia tumifaciens (Currie 1937) Wachek 1955 and descriptions of Fergusobia planchonianae n. sp. and Fergusobia viminalisae n. sp.

Fergusobia tumifaciens (Currie 1937) Wachek 1955, the type species for the genus Fergusobia, is re-described from specimens collected from 'leafy' leaf bud galls on Eucalyptus bridgesiana near Albury in New South Wales, Australia. It is morphologically characterized by the combination of an open C-shaped parthenogenetic female with a small broadly conoid tail, a C-shaped infective female with a bluntly rounded tail tip, and an arcuate to J-shaped male with angular spicules, not heavily sclerotised, and short to mid-length peloderan bursa. Two new species of Fergusobia, collected from 'leafy' leaf bud galls on, respectively, Eucalyptus planchoniana in Queensland, and E. viminalis in South Australia, Australia, are described. Fergusobia planchonianae Davies n. sp. is characterised by the combination of a C-shaped parthenogenetic female with a conoid tail, an arcuate infective female with an hemispherical tail tip, and an almost straight to arcuate to C-shaped male with an angular spicule, a long peloderan bursa and a narrow tail. Fergusobia viminalisae Davies n. sp. is characterised by the combination of an open C-shaped parthenogenetic female with a broadly conoid tail, a C-shaped infective female with a bluntly rounded tail tip, and an arcuate to J-shaped male with an angular (not heavily sclerotised) spicule and short to mid-length peloderan bursa. The shield morphologies of the fly larvae associated with the 'leafy' leaf bud galls and their possible relationships are outlined. Possible evolutionary relationships of the Fergusobia nematodes from these galls are discussed, considering their morphology, DNA sequences, and the relationships of the associated Fergusonina flies and host plants. 

Contribution to the study of the genus Paratylenchus Micoletzky, 1922 sensu lato (Nematoda: Tylenchulidae).

Morphological and morphometric analyses were conducted on the genus Paratylenchus, based on documented data in the literature and characters of Iranian populations of twelve species, including P. arculatus, P. colinus, P. conicephalus, P. coronatus, P. neoamblycephalus, P. neoprojectus, P. obtusicaudatus, P. paraperaticus, P. similis, P. straeleni, P. variabilis and P. veruculatus. Moreover, males of P. obtusicaudatus are described for the first time and additional data provided for males of P. paraperaticus. The variability of the most important taxonomic characters for species identification in the genus was estimated by calculation of coefficient of variation (CV) for 49 species and the ratio of extremes in the range of measured females (max/min ratios) for 82 species of the genus, based on 66 and 170 populations, respectively. A list of 117 valid species of Paratylenchus is provided, along with a dichotomous identification key and a diagnostic compendium.