Small Subunit Ribosomal DNA Sequences Research Articles

Fukuyoa paulensis gen. et sp. nov., a new genus for the globular species of the dinoflagellate Gambierdiscus (Dinophyceae).

The marine epiphytic dinoflagellate Gambierdiscus is a toxicologically important genus responsible for ciguatera fish poisoning, the principal cause of non-bacterial illness associated with fish consumption. The genus currently contains species exhibiting either globular or anterior-posteriorly compressed morphologies with marked differences in cell shape and plate arrangement. Here we report a third globular, epiphytic and tychoplanktonic species from the coasts of Ubatuba, Brazil. The new species can be distinguished from G. yasumotoi and G. ruetzleri by its broader first apical plate that occupies a larger portion of the epitheca. Accordingly, phylogenetic trees from small subunit (SSU) and large subunit (LSU) ribosomal DNA sequences also showed strongly supported separation of the new species from the G. yasumotoi / G. ruetzleri group albeit with short distance. The molecular phylogenies, which included new sequences of the planktonic species Goniodoma polyedricum, further indicated that the globular species of Gambierdiscus formed a tight clade, clearly separated (with strong bootstrap support) from the clade of lenticular species including the type for Gambierdiscus. The morphological and molecular data in concert support the split of Gambierdiscus sensu lato into two genera. Gambierdiscus sensu stricto should be reserved for the species with lenticular shapes, highly compressed anterioposteriorly, with short-shank fishhook apical pore plate, large 2' plate, low and ascending cingular displacement, and pouch-like sulcal morphology. The new genus name Fukuyoa gen. nov. should be applied to the globular species, slightly laterally compressed, with long-shank fishhook apical pore plate, large 1' plate, greater and descending cingular displacement, and not pouch-like vertically-oriented sulcal morphology. Fukuyoa contains the new species Fukuyoa paulensis gen. et sp. nov., and F. yasumotoi comb. nov. and F. ruetzleri comb. nov.

Myxobolus oralis sp. n. (Myxosporea: Bivalvulida) infecting the palate in the mouth of gibel carp Carassius auratus gibelio (Cypriniformes: Cyprinidae)

During a survey on the myxosporean fauna of gibel carp Carassius auratus gibelio (Bloch) in China, a species of Myxobolus Bütschli, 1882 that did not conform to any known species was found. The species is characterised by the presence of round to ellipsoidal plasmodia of 2.6-4.0 mm in diameter in the palate of host. Mature spores are obovate in frontal view and lemon-shaped in lateral view, with the following range, mean and standard deviation of dimensions: 10.8-12.8 μm (11.7 ± 0.4 μm) long, 8.2-9.9 μm (8.9 ± 0.4 μm) wide and 6.0-7.5 μm (6.8 ± 0.3 μm) thick. Two polar capsules are pyriform, 4.0-5.5 μm (4.8 ± 0.3 μm) long by 2.9-3.6 μm (3.0 ± 0.2 μm) wide. Polar filaments are coiled, with 5 to 6 turns. A small proportion of spores possesses a short caudal process. Scanning electron microscopy revealed discoid spores with a low sutural ridge and middle bulge. The small subunit ribosomal DNA sequence of this species did not match any available sequences in GenBank. Phylogenetically, this species is sister to M. nielii (Nie et Li, 1973) and M. hearti Chen, 1998 in a Henneguya-Myxobolus clade with robust support. Given the morphological and molecular differences between this species and other Myxobolus species, we propose the name Myxobolus oralis sp. n. for this parasite from gibel carp.

Phylogeny, Ultrastructure, and Flagellar Apparatus of a New Marimonad Flagellate Abollifer globosa sp. nov. (Imbricatea, Cercozoa)

Abollifer is a little-known genus of marine heterotrophic flagellates with no ultrastructural and molecular information, and its taxonomic position remains uncertain. In this study, we report a new species of Abollifer, Abollifer globosa sp. nov., isolated from a seawater sample collected at Tokyo Bay. To reveal the taxonomic position and morphological characteristics of A. globosa, we performed light and electron microscopic observations and a phylogenetic analysis using small subunit ribosomal DNA sequences. A. globosa cells were 29.5 μm in length and 22.4 μm in width, oval or ovoid in shape with an apical projection. Two unequal flagella emerged from a deep subapical flagellar pit. The rim of the flagellar pit except for the ventral side was swollen. Electron microscopic observations showed that A. globosa possessed mitochondria with tubular cristae, a Golgi apparatus, microbodies, extrusomes, and many endosymbiotic bacteria. Basal bodies were arranged in parallel. The flagellar apparatus of A. globosa showed affinity with common gliding cercozoan flagellates. Our phylogenetic tree showed that A. globosa branched as the sister position of order Marimonadida (Imbricatea, Cercozoa). On the basis of the morphological and molecular phylogenetic analysis, we conclude that A. globosa is a new member of the order Marimonadida.

Quantitative PCR detection of Theileria equi using laboratory workflows to detect asymptomatic persistently infected horses

Equine piroplasmosis is the most important tick-borne disease of horses. Regulations on movement of horses into disease-free countries are in place to preserve international trade. Introduction of infectious disease, such as equine piroplasmosis, into non-endemic countries remains a substantial risk owing to the wide-spread distribution of vectors. Identification and restriction of movement of Theileria equi persistently infected horses is an integral part of control strategies, because persistently infected horses with low parasitaemia are an important reservoir. We used real-time PCR for diagnosis of T. equi DNA in clinically healthy horses in an equine piroplasmosis endemic area. The sensitivity was assessed using a synthetic plasmid DNA and a laboratory workflow was developed to maximise detection of persistently infected horses. The detection limit was 10 rDNA copies of the plasmid DNA. Assuming that each red blood cell contains a single T. equi genome the detection limit corresponded to 2.5T. equi/μl of total blood and parasitaemia as low as 2–3.8×10−5%. A laboratory workflow was developed and assessed on samples from Saudi Arabia. The laboratory workflow focused on samples returning no or single positive result in duplicate PCR. In total, we obtained 42% (59/141; 95% confidence interval: 33.85–50.15) T. equi positive samples, 26% (37/141) negative for T. equi samples. The remaining 45 samples were judged as suspect with no definitive diagnosis made. The Saudi Arabia's T. equi small subunit ribosomal DNA (SSU rDNA) sequencing (n=16) demonstrated A clade (n=15) as the dominant T. equi clade. Clade B was sequenced in a single case. We present an approach for diagnostic workflow to detect T. equi in clinically healthy but persistently infected horses. Results from Saudi Arabia confirm that T. equi is widespread in the Middle East region. High proportion of horses with low parasitaemia calls for caution with results based on a single blood sample. Understating of the fluctuation of the parasitaema in persistently infected horses in endemic areas is needed to establish the required sample numbers for reliable detection of T. equi.

Prevalence of Blastocystis in Shelter-Resident and Client-Owned Companion Animals in the US Pacific Northwest

Domestic dogs and cats are commonly infected with a variety of protozoan enteric parasites, including Blastocystis spp. In addition, there is growing interest in Blastocystis as a potential enteric pathogen, and the possible role of domestic and in-contact animals as reservoirs for human infection. Domestic animals in shelter environments are commonly recognized to be at higher risk for carriage of enteropathogens. The purpose of this study was to determine the frequency of infection of shelter-resident and client-owned domestic dogs and cats with Blastocystis spp in the Pacific Northwest region of the USA. Fecal samples were collected from 103 shelter-resident dogs, 105 shelter-resident cats, 51 client-owned dogs and 52 client-owned cats. Blastocystis were detected and subtypes assigned using a nested PCR based on small subunit ribosomal DNA sequences. Shelter-resident animals were significantly more likely to test positive for Blastocystis (P<0.05 for dogs, P = 0.009 for cats). Sequence analysis indicated that shelter-resident animals were carrying a variety of Blastocystis subtypes. No relationship was seen between Blastocystis carriage and the presence of gastrointestinal disease signs in either dogs or cats. These data suggest that, as previously reported for other enteric pathogens, shelter-resident companion animals are a higher risk for carriage of Blastocystis spp. The lack of relationship between Blastocystis carriage and intestinal disease in shelter-resident animals suggests that this organism is unlikely to be a major enteric pathogen in these species.

Toxisarcon taimyr sp. nov., a new large monothalamous foraminifer from the Kara Sea inner shelf

A large monothalamous foraminiferan, Toxisarcon taimyr sp. nov., has been isolated from the benthic samples from the Kara Sea inner shelf near the mouth of Yenisey river estuary, at a depth of 50–100 m. In its overall morphology, the new species closely resembles T. synsuicidica, one of the two species of Toxisarcon described to date. It possesses a large irregularly shaped cell body, covered by a thin layer of a fibrous organic coating. Numerous reticulopodia typically extend from all over the cell surface; the species is very motile and rapidly changes cell shape. Long and thick reticulopodial bundles form in the direction of movement. In the phylogenetic tree based on partial small-subunit ribosomal DNA (SSU rDNA) sequences, T. taimyr branches together with the two other known species of Toxisarcon within the clade C of monothalamous foraminifera.

The seagrass Zostera marina harbors growth-inhibiting bacteria against the toxic dinoflagellate Alexandrium tamarense

Seagrasses are known to have allelopathic activity to reduce growth of phytoplankton. We found growth-inhibiting bacteria (strains E8 and E9) from Zostera marina possessing strong activity against the toxic dinoflagellate Alexandrium tamarense. Strain E9 markedly inhibited growth of A. tamarense even with initial inoculum size as small as 2.9 cells ml−1. This bacterium also had growth-inhibiting effects on the red-tide raphidophytes Chattonella antiqua and Heterosigma akashiwo, the dinoflagellate Heterocapsa circularisquama, and the diatom Chaetoceros mitra. Small subunit (SSU) ribosomal DNA (rDNA) sequencing analysis demonstrated that the most probable affiliation of these strains was Flavobacteriaceae, and proved that another inhibitory bacterial strain (E8) was the same species as strain E9. Two other bacterial strains (E4-2 and E10), showing different colony color and isolated from the same seagrass sample, revealed no growth-inhibiting activity. Interestingly, strain E4-2 showed the same sequences as E8 and E9 (100 %), and strain E10 matched E8 and E9 with 99.80 % similarity. Growth-inhibiting bacteria against the toxic dinoflagellate Alexandrium tamarense associated with seagrass, such as Flavobacterium spp. E8 and E9, are able to repress shellfish poisoning besides the allelopathic activity of seagrass itself.

Updated morphology, histopathology and molecular phylogeny of Myxobolus hearti, cardiac myxosporea in gibel carp, Carassius gibelio (Bloch)

The original description of Myxobolus hearti is supplemented with new data on spore morphology, histopathology and molecular phylogeny. Myxobolus hearti are found in the heart ventricle of the gibel carp, Carassius gibelio (Bloch), where they form whitish oval or irregularly shaped plasmodia. Mature spores are oval or shortly ellipsoidal in frontal view, lemon-shaped in sutural view and eye-shaped in apical view. The spores are 14.12 ± 0.35 (13.6-15) μm long (mean ± SD), 11.85 ± 0.34 ± 0.36 (11-12) μm wide and 7.32 ± 0.36 (7-8) μm thick. The two polar capsules are equal in size, 6.11 ± 0.29 (6-7) μm long and 3.89 ± 0.31(3-4) μm wide, and are long pyriform in shape. Polar filaments have six or seven coils situated perpendicular to the longitudinal axis of the polar capsules. Histopathology indicates that the plasmodia are encased by the host connective tissue, and no inflammatory responses are found in the heart ventricles. Phylogenetic analysis based on the 18S small-subunit ribosomal DNA sequences indicates that M. hearti is, genetically, most similar to Henneguya doneci, a gill-infecting species.

Kudoa thunnifrom Blackfin Tuna (Thunnus atlanticus) Harvested Off the Island of St. Kitts, West Indies

Numerous myxozoan cysts (∼ 1 mm) were found in the musculature of blackfin tuna (Thunnus atlanticus) harvested off the Caribbean island of St. Kitts. Myxospores were consistent with quadrate members of the Kudoidae, measuring 8.8 (8.2-9.4) μm wide, 7.3 (6.6-8.3) μm thick, and 6.2 (5.8-6.9) μm long with 4 uniform drop-like polar capsules measuring 2.7 (2.2-3.2) μm long and 2.0 (1.7-2.2) μm wide. The 18S small-subunit (SSU) and 28S large-subunit (LSU) ribosomal DNA sequences did not result in direct matches to any published sequences. However, the SSU sequences (1,786 base pairs [bp]) obtained from 6 individual cysts were identical and demonstrated high homology to Kudoa thunni (99.0%) from albacore (Thunnus alalunga). Alternatively, 33 unique sequences were obtained for the LSU (∼ 800 bp), demonstrating 0.1 to 5.0% variability between them, although a majority of these sequences (60%) demonstrated high homology (>99%) to K. thunni. Morphologically, the case isolate was smaller than published descriptions of K. thunni; however, rDNA sequence homology, and phylogenetic placement based on concatenated SSU and LSU rDNA sequences suggests this case isolate and K. thunni are conspecific. To our knowledge this is the first report of K. thunni infection in blackfin tuna from the Caribbean.

Multiple group I introns in the small-subunit rDNA of Botryosphaeria dothidea: implication for intraspecific genetic diversity.

Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea.

Multilamina teevani gen. et sp. nov., a microsporidian pathogen of the neotropical termite Uncitermes teevani

A new genus and species of microsporidia is described from adults of the termite Uncitermes teevani (Emerson) (n. comb., formerly Armitermes teevani), collected in Ecuador. Masses of elongate, ovoid, uninucleate spores were localized to the coelomic cavity of adult workers and measured 6.29×3.33μm (fresh) and 5.83×3.00μm (fixed). These spores were individually contained within a multi-layered sporophorous vesicle and contained an isofilar polar filament with 24–28 coils. Blast-n analysis revealed that the small subunit ribosomal DNA (ssrDNA) sequence of this new species exhibited 85% identity with that of a Varimorpha species from the fire ant, Solenopsis richteri, and slightly less (78–85% identity) to a large clade of microsporidian parasites from mosquitoes and microcrustacea. The morphological and sequence data support the conclusion that Multilamina teevani gen. et sp. nov. is a novel microsporidium and distinct from any previously described genera or species.

Phylogenetic position of Jaoa, a green algal genus endemic to China

SummaryJaoa prasina, a freshwater green alga endemic to China, was collected from a stream in Hubei province, China. Unialgal cultivation, morphological observation, and phylogenetic analyses of small subunit ribosomal DNA and RuBisCO large subunit sequences were performed. When cultured on agar medium, the alga was irregularly filamentous, similar to marine species of Acrochaete. Aplanospores were observed on solid medium. A vesicular‐like thallus without rhizoids developed in liquid medium, similar to specimen development in natural habitats. Molecular phylogenetic analyses revealed that Jaoa was closely related to the marine genera Acrochaete Pringsheim and Ulvella Crouan &amp; Crouan. The results suggested the genus Jaoa is a member of the family Ulvellaceae (Ulvophyceae), which contains mostly marine algae. The family name Jaoaceae should be abandoned. We speculate that Jaoa may have evolved from a marine Ulvellaceae ancestor.

A parvicapsulid (Myxozoa) infecting Sprattus sprattus and Clupea harengus (Clupeidae) in the Northeast Atlantic uses Hydroides norvegicus (Serpulidae) as invertebrate host

A myxosporean producing actinospores of the tetractinomyxon type in Hydroides norvegicus Gunnerus (Serpulidae) in Denmark was identified as a member of the family Parvicapsulidae based on small-subunit ribosomal DNA (SSU rDNA) sequences. Myxosporean samples from various Danish and Norwegian marine fishes were examined with primers that detect the novel myxosporean. Sprattus sprattus (Linnaeus) and Clupea harengus Linnaeus (Teleostei, Clupeidae) were found to be infected. The sequences of this parvicapsulid from these hosts were consistently slightly different (0.8% divergence), but both these genotypes were found in H. norvegicus. Disporic trophozoites and minute spores of a novel myxosporean type were observed in the renal tubules of some of the hosts found infected through PCR. The spores appear most similar to those of species of Gadimyxa Køie, Karlsbakk et Nylund, 2007, but are much smaller. The actinospores of the tetractinomyxon type from H. norvegicus have been described previously. In GenBank, the SSU rDNA sequences of Parvicapsulidae gen. sp. show highest identity (82%) with Parvicapsula minibicornis Kent, Whitaker et Dawe, 1997 infecting salmonids (Oncorhynchus spp.) in fresh water in the western North America. A phylogenetic analysis places P. minibicornis and Parvicapsulidae gen. sp. in a sister clade to the other parvicapsulids (Parvicapsula spp. and Gadimyxa spp.).

Molecular phylogeny of rhynchonellide articulate brachiopods (Brachiopoda, Rhynchonellida)

We present here the first report based on phylogenetic analyses of small subunit (SSU/18S) and large subunit (LSU/28S) ribosomal DNA (rDNA) sequences from a wider-than-token sample of rhynchonellide articulate brachiopods, with data from 11 of ∼20 extant genera (12 species) belonging to all four extant superfamilies. Data exploration by network and saturation analyses shows that the molecular sequence data are free from major aberrations and are suitable for phylogenetic reconstruction despite the presence of large deletions in four SSU rDNA sequences. Although molecular sequence analyses cannot directly illuminate the systematics of fossils, the independent, genealogical evidence and phylogenetic inferences about extant forms that they make possible are highly relevant to paleontological systematics because they highlight the limitations of evolutionary inference from morphology. Parsimony, distance, maximum likelihood (no clock) and Bayesian (relaxed-clock) analyses all find a tree topology that disagrees strongly with the existing superfamily classification. All tested phylogenetic reconstructions agree that the taxa analyzed fall into three clades designated A1, A2, and B that reflect two major divergence events. The relaxed-clock analysis indicates that clades A and B diverged in the Paleozoic, while clades A1 and A2 reflect Permo-Triassic (and later) events. Morphological homoplasy and possible gene co-option are suggested as the main sources for the discord between the morpho-classification, the results of cladistic analyses of morphology, and the relationships reconstructed from molecular sequences. The origin, function and evolutionary implications of the deletion-bearing rhynchonellide SSU rDNA sequences are briefly discussed in relation to pseudogenes and concerted evolution in the rDNA genomic region.

Kudoa azevedoi n. sp. (Myxozoa, Multivalvulida) from the oocytes of the Atlantic horse mackerel Trachurus trachurus (Perciformes, Carangidae) in Tunisian coasts

A new species Kudoa azevedoi sp. n. (Myxozoa, Multivalvulida) is described in Trachurus trachurus Linnaeus, 1758 (Carangidae) from fishing harbors in Tunisian coasts using spore morphology and SSU rDNA sequence data. The parasite occurs only in ovaries within oocytes of mature and immature specimens. Spores are quadrate in shape in apical view with rounded edges, having four shell valves and four symmetrical polar capsules. They are of small sizes and measure 3.5±0.41 (3-4.2)×4.5±0.44 (4-5.2) length by width. The polar capsules are pyriform in shape measuring 1.5±0.22 (1.5-2)×0.75±0.14 (0.5-1) μm. Infected oocytes are hypertrophied, whitish colored, and filled with mature spores. Plasmodia are tubular and ramified from the inner membrane toward the center of the oocyte. Phylogenetic analysis based on small subunit ribosomal DNA sequences shows the highest similarity (96%) with the ovary parasite Kudoa ovivora. Some morphological details and spore dimensions support the creation of a new species in the genus Kudoa. Mean prevalence among examined females is of about 55.5%. It varies between localities and length of fish. The present myxosporea is the second Kudoa species reported in fish ovaries.

Prevalence of Histomonas meleagridis in Broiler Chicken in Different Parts of Mizoram, India

A survey was carried out to detect the overall incidence of histomoniasis in broiler birds in different parts of Mizoram, India. Out of 4000 birds examined, 40 birds were found positive for Histomonas meleagridis revealing 1% infection rate. Heterakis gallinarum was recovered from the caeca of positive birds. Gross lesions were found in caeca and liver. Microscopic examination of stained section of liver tissue revealed numerous circular amoeboid Histomonas meleagridis together with cellular infiltration. The PCR selectively amplified 209 BP of the small subunit ribosomal DNA sequence of Histomonas meleagridis.

A new microsporidium, Triwangia caridinae gen. nov., sp. nov. parasitizing fresh water shrimp, Caridina formosae (Decapoda: Atyidae) in Taiwan

A new microsporidium was isolated from the endemic, Taiwanese shrimp, Caridina formosae (Decapoda, Atyidae) from northern Taiwan. A conspicuous symptom of infection was presence of opaque white xenomas located in the proximity of the alimentary tract, the surface of the hepatopancreas, and the gills. A fully developed xenoma consisted of a hard, thick capsule filled with sporophorous vesicles containing multiple spores. Microsporidia developed synchronously within the same sporophorous vesicle, although the stage of parasite development differed among the vesicles. Fresh spores were pyriform, mononucleated and measured 6.53×4.38μm. The polar filament was anisofilar with 9–11 coils. Phylogenetic analysis based on the small subunit ribosomal DNA sequence showed that the isolate is most similar to the fish microsporidian clade containing the genera Kabatana, Microgemma, Potaspora, Spraguea, and Teramicra. The highest sequence identity, 80%, was with Spraguea spp. Based on pathogenesis, life cycle and phylogenetic analysis, we erect a new genus and species, Triwangia caridinae for the novel microsporidium.

Genetic Diversity and Environmental Preferences of Monothalamous Foraminifers Revealed through Clone Analysis of Environmental Small-Subunit Ribosomal DNA Sequences

Foraminifers form an important link between bacteria and macrobenthos in natural food webs and thereby play a major role in biogeochemical cycling. There have been few studies conducted to clarify these relationships, although the biomass of foraminifera, particularly the soft-shelled monothalamous taxa, is high in the deep seas. Molecular tools are now being used to study evolution and genetic diversity in widely separated oceanic environments. However, the relationship between genetic diversity and the ambient environment is not well understood. To test the idea that environmental conditions can affect genetic diversity, we conducted molecular phylogenetic analyses based on clone analysis of environmental DNA for benthic foraminifers and environmental measurements in sediments from various oceanic environments. Monothalamous foraminifers were found in dysoxic ( 120 ?M) environments. The number of phylotypes was higher in the oxic environments of central Sagami Bay, Japan, and the east equatorial Pacific Ocean, than in the lower-oxygen areas of Sagami Bay. Phylotypes closely related to the genus Ovammina and some other saccamminids, as well as the calcareous genus Stainforthia, were preferentially found in lower dissolved-oxygen (DO) environments, while phylotypes of other saccamminids, Hippocrepinella, and an unidentified foraminiferal species, occurred at higher DO concentrations. Our analysis suggests that novel monothalamous phylotypes exist in habitats ranging from the sulfide-enriched environments of chemosynthetic ecosystems to those with high concentrations of DO as at the abyssal depths and in well-mixed bioturbated sediments.

Description of Meloidoderita salina sp. n. (Nematoda, Sphaeronematidae) from a micro-tidal salt marsh at Mont-Saint-Michel Bay in France

Meloidoderita salina sp. n. is described and illustrated from the halophytic plant Atriplex portulacoides L. (sea purslane) growing in a micro-tidal salt marsh in the Mont-Saint-Michel Bay in France. This new species is the first member of Meloidoderita Poghossian, 1966 collected from a saline environment, and is characterized by the following features: sedentary mature females having a small swollen body with a clear posterior protuberance; slightly dorsally curved stylet, 19.9 µm long, with posteriorly sloping knobs; neck region irregular in shape and twisted; well developed secretory-excretory (S–E) pore, with markedly sclerotized S-E duct running posteriorly; prominent uterus bordered by a thick hyaline wall and filled with eggs. The adult female transforms into a cystoid. Eggs are deposited in both egg-mass and cystoid. Cystoids of Meloidoderita salina sp. n. display a unique sub-cuticular hexagonal beaded pattern. Male without stylet, pharyngeal region degenerated, S-E duct prominent, deirids small, developed testis 97.5 µm long, spicules 18.4 µm long, cloacal opening ventrally protruded, small phasmids posterior to cloaca opening and situated at 5.9 (3.2–7.7) µm from tail end, and conical tail ending in a rounded terminus marked with one (rarely two) ventrally positioned mucro. Additionally, some young malesof the new species were observed enveloped in the last J2 cuticle. Second-stage juvenile body 470 µm long, with a 16.4 µm long stylet, prominent rounded knobs set off from the shaft, hemizonid anterior and adjacent to S-E pore, small deirids located just above S-E pore level, genital primordium located at 68–77% of body length, phasmids small and located at about 19 µm from tail tip, and tail 38.7 µm long, tapering to finely pointed terminus with a finger-like projection. Phylogenetic analyses based on the nearly full length small subunit ribosomal DNA sequences of Meloidoderita salina sp. n. revealed a close relationship of the new species with Sphaeronema alni Turkina & Chizhov, 1986 and placed these two species sister to the rest of Criconematina.

Small Subunit Ribosomal DNA-Based Phylogenetic Analysis of Foliar Nematodes (Aphelenchoidesspp.) and Their Quantitative Detection in Complex DNA Backgrounds

Foliar nematodes, plant-parasitic representatives of the genus Aphelenchoides, constitute a minority in a group dominated by fungivorous species. Distinction between (mostly harmless) fungal feeding Aphelenchoides species and high impact plant parasites such as A. besseyi, A. fragariae, A. ritzemabosi, and A. subtenuis is severely hampered by the scarcity of informative morphological characters, some of which are only observable in specific developmental stages. Poor description of a number of non-plant-parasitic Aphelenchoides species further complicates identification. Based on (nearly) full-length small subunit ribosomal DNA (SSU rDNA) sequences (≈1,700 bp), a phylogenetic tree was generated, and the four target species appeared as distinct, well-supported groups. Notably, this genus does not constitute a monophyletic group: A. besseyi and A. ritzemabosi cluster together and they are phylogenetically isolated from A. fragariae, A. subtenuis, and most other fungivorous species. A phylum-wide SSU rDNA framework was used to identify species-specific DNA motifs. For the molecular detection of four plant-parasitic Aphelenchoides species, polymerase chain reaction primers were developed with high, identical annealing temperatures (63°C). Within the molecular framework presented here, these primers can be used for the rapid screening of plant material and soil for the presence of one or multiple foliar nematode species.