Manuel Mundo-Ocampo

DESS: a versatile solution for preserving morphology and extractable DNA of nematodes

A solution containing dimethyl sulphoxide, disodium EDTA, and saturated NaCl (abbreviated here as DESS) was tested for various applications in the preservation of nematodes for combined morphological and molecular analyses. The solution can be used to preserve individual nematodes, nematode extracts, or entire soil/sediment samples. Preserved material can be easily stored for months at room temperature, shipped by mail, or carried in luggage. Morphological features are usually well preserved; specimen quality being comparable to formalin-based fixatives and much better than ethanol fixation. Specimens can be transferred to glycerin with little or no modification of traditional protocols. Unlike formalin-preserved material, routine PCR can be performed on individual specimens after any of these procedures with success rates and amplification sizes comparable to PCR of fresh specimens. At this point we have no data on long-term preservation quality. Nevertheless, DESS solution clearly enhances and simplifies a wide range of nematological studies due to its combined suitability for morphological and molecular analyses, as well as its less hazardous chemical properties.

Ditylenchus gallaeformans sp. n. (Tylenchida: Anguinidae) – a neotropical nematode with biocontrol potential against weedy Melastomataceae

Ditylenchus gallaeformans sp. n. was found on several hosts at numerous locations in Brazil and Costa Rica. In its native habitats it attacks several genera in the Melastomataceae, including two species ranked as among the worst invasive weeds of Pacific island forests, namely Miconia calvescens and Clidemia hirta. The new species causes a severe disease on infected plants involving the formation of gall-like structures on infected leaves, inflorescences and stems, and may cause significant impact on its hosts. Morphological study using light and scanning electron microscopy and analysis of the partial 18S rRNA, the D2-D3 expansion fragments of 28S rRNA and the ITS rRNA gene sequences showed little variations between populations from different hosts or geographical origins. The molecular study revealed that the new species is related to D. drepanocercus, which was recently found in association with M. calvescens but causing angular leaf spots on this host. Ditylenchus gallaeformans sp. n. is distinguished from D. drepanocercus by having a bursa reaching the tail tip (vs covering around 50% of tail in D. drepanocercus) and a conical tail, regularly tapering towards a variable tip (vs tail with a distinctive apical falciform appendage in both sexes in D. drepanocercus). PCR with species-specific primers was developed for diagnostics of both Ditylenchus species. Ditylenchus gallaeformans sp. n. deserves further investigation as a potential biocontrol agent against M. calvescens and C. hirta.

Redescription of Filenchus annulatus (Siddiqui & Khan, 1983) Siddiqi, 1986 based on specimens from Iran with contributions to the molecular phylogeny of the Tylenchidae

Filenchus annulatus is redescribed and males are characterised for the first time based on a population found in Northern Khorasan province, Iran. New morphological characterisation is based on light and scanning electron microscopy. In addition, molecular analyses based on 18S and 28S genes are included to test monophyly of the genus. Females from the Iranian population have a spermatheca typically filled with sperm. Generally males are similar to females, ranging from 306 to 426 μm long. Spicules are arcuate, cephalated and 11.5-14.0 μm long, the gubernaculum is minute and trough-shaped and the caudal alae are adanal. Phylogenetic analyses differed in results depending on the gene used: 28S gene strongly supports Filenchus as monophyletic whereas 18S shows Filenchus as polyphyletic. In both gene phylogenies, F. annulatus is placed as a sister taxon of F. quartus from Wyoming, USA. Although sequence divergence between these two species is only 3 base pairs and 1 base pair for 28S and 18S genes, respectively, strong morphological differences support their species status. Relationships between Filenchus and other Tylenchidae genera are also gene dependent. Such differences in tree topologies and branch support are related to the number of Filenchus species used in the analyses (greater for 18S gene) and gene resolution (greater for 28S gene). Molecular phylogenies also suggest that other Tylenchidae genera (i.e., Psilenchus, Cephalenchus and Eutylenchus) belong to separate clades, as is also suggested by some morphology-based classifications. The inclusion of more taxa and perhaps additional genes is needed further to clarify Filenchus relationships and further to test its monophyly.

Radopholus musicola n. sp., a new pathogenic species from Australia (Nematoda: Pratylenchidae).

Radopholus musicola n. sp. is described from Musa acuminata cv. Williams at Berrimah Research Station, Darwin, Northern Territory, Australia. The new species is distinguished from other species by female lateral lines with deep folds on the outer edge and by males with a uniquely long thin tail (73.6-92.8 μm) having a pointed terminus. Radopholus musicola n. sp. is most similar to R. similis and R. bridgei but males further differ from R. similis by a smaller stylet (8.8-12 vs 12-17 μm) and from R. bridgei by well-developed separated stylet knobs vs tiny or obscure knobs. The rate of reproduction of R. musicola n. sp. is much greater at higher temperatures (>31.5°C) than that of R. similis, and some banana cultivars resistant to R. similis are less resistant to R. musicola n. sp. The relevance of previous sequence-based (rDNA) analysis for distinguishing R. musicola n. sp. from R. similis and R. bridgei is discussed.

Morphology, molecular characterisation and systematic position of the genus Cynura Cobb, 1920 (Nematoda: Plectida)

Descriptions of three known species of Cynura, i.e., C. cerambus, C. klunderi and C. papillata, are given, including SEM micrographs of C. cerambus and a tabular compendium for all species of the genus. The phylogenetic relationships of C. klunderi are inferred from molecular data. Bayesian analyses of small subunit (SSU) of rRNA sequences support a position nested among the Plectidae suggesting the secondary simplification in the morphology of pharyngeal valvular apparatus in Cynura and the ‘return’ from a terrestrial to a marine environment in this genus.

Phylogeny and biogeography of the genus Cephalenchus (Tylenchomorpha, Nematoda)

The phylogenetic position of Cephalenchus is enigmatic with respect to other tylench nematodes. In this study, Cephalenchus populations representing 11 nominal species were sampled worldwide for molecular and morphological characterization. Species identification was based on light microscopy (LM) and scanning electron microscopy (SEM). Molecular analyses were based on the genes (i.e. 18S, 28S, 5.8S) and internal transcribed spacers (ITS‐1 and ITS‐2) of the ribosomal RNA (rRNA). Phylogenetic analyses (i.e. full and reduced alignments) of either concatenated or single genes always supported the monophyly of Cephalenchus. A sister relationship between Cephalenchus and Eutylenchus excretorius was recovered by most analyses, although branch support varies depending on the dataset used. The position of Cephalenchus + E. excretorius within Tylenchomorpha nevertheless remains ambiguous, thus highlighting the importance of sampling additional genes as well as taxa. Placement of Cephalenchus + E. excretorius as sister of Tylenchinae or Boleodorinae could not be rejected on the basis of 18S and 28S rRNA genes. Within Cephalenchus, amphidial opening morphology shows congruence with molecular‐based phylogenetic relationships, whereas the number of lines in the lateral field is likely to be a convergent trait. Morphometric analyses clearly distinguished short tail from medium–long tail species, and SEM observations seem to suggest a relation between tail length and amphidial opening. In addition, molecular phylogenies support the non‐monophyly of Cephalenchus cephalodiscus, Cephalenchus cylindricus, Cephalenchus daisuce and Cephalenchus leptus. The known extent of Cephalenchus diversity is increased with the inclusion of two new species, and the biogeography of the genus is discussed.

Metacrobeles amblyurus n. sp. (Nematoda: Cephaloboidea) from Death Valley, California.

Metacrobeles amblyurus n. sp. (Cephalobina, Cephaloboidea) is described from sand dunes of Death Valley, California, USA. It shares with other Metacrobeles species a far posterior vulva as well as five lateral lines but is distinguished from previously known species in having a blunt (vs more tapering) conical tail. Probolae of M. amblyurus n. sp. are low conical-rounded, adjacent lips are fused into three pairs, and guard processes are lacking. The very narrow vulva in M. amblyurus n. sp. appears to be plugged in adult females, and juveniles apparently hatch exclusively within the body of the dead mother. Type localities of the two previously known species of Metacrobeles are in western Africa and this is the first confirmed report of Metacrobeles from another continent.

Testudinema gilchristi n. gen., n. sp. (Nematoda: Monhysteridae) from the perianal folds of stinkpot turtles, Sternotherus odoratus (Testudines), USA

Summary – Examination of perianal folds of stinkpot turtles, Sternotherus odoratus, from Virginia, USA, revealed several monhysterid nematodes. The general morphological characteristics of this population, such as circular amphids, unstriated cuticle under light microscope, single outstretched ovary, single testis, caudal glands, terminal spinneret and the absence of an apophysis on the gubernaculum, with the exception of the position of the gonad in relation to the intestine, shows the affinity of this population to the Monhysteridae. Microscopic observations of the inner stoma show three (one dorsal and two ventro-sublateral) buccal wall extensions towards the lumen and numerous (ca 50) distinct denticles on its wall. Because this stoma structure is a morphological novelty within the Monhysterida and this is the first monhysterid group associated with perianal folds of turtles, we propose to establish Testudinema n. gen. within the Monhysteridae to accommodate this morphologically distinct population. We provide a detailed comparison with known monhysterid genera including those that possess a cup-shaped stoma, stoma with denticles or those genera reported to be commensals (Gammarinema, Monhystrium, Tripylium, Odontobius). Stinkpot turtles feed on benthic fauna and may acquire these commensals when the sediment is disturbed. The denticles and structure of the buccal cavity could be morphological adaptations for transition to ecto-parasitism.

Revision and phylogeny of Tarvaia Allgén, 1934 (Nematoda: Tarvaiidae)

Descriptions of eight new and three known species of Tarvaia are given. Tarvaia pacifica sp. n. has cephalic capsule reaching posterior part of short amphids, amphidial shield without ornamentation, straight vagina with pars refringens vaginae, slightly asymmetrical spicules, gubernaculum with two dorsal apophyses, precloacal ridge present; T. algicola sp. n. has cephalic capsule with unevenly thickened walls reaching posterior part of short amphids, amphidial shield without ornamentation, straight vagina, apophyses absent, precloacal ridge present; T. carolinensis sp. n. has cephalic capsule reaching posterior part of short amphids, amphidial shield with ornamentation, sigmoid vagina, gubernaculum with a dorsal apophysis; T. longispiculata sp. n. has elongated cephalic capsule reaching posterior part of elongate amphids, amphidial shield without ornamentation, sigmoid vagina, unusually long spicules, gubernaculum with dorsal apophysis, precloacal ridge present; T. inaequalis sp. n. has short cephalic capsule reaching anterior third of elongate amphids, amphidial shield with ornamentation, straight vagina, strongly anisometric spicules, gubernaculum with caudal apophysis, precloacal ridge present; T. sakhalinensis sp. n. has short cephalic capsule reaching middle of short amphids, amphidial shield with distinct pores, symmetrical spicules, apophyses absent; T. tchesunovi sp. n. has cephalic capsule with unevenly thickened walls reaching posterior part of short amphids, amphidial shield without ornamentation, symmetrical spicules, apophyses absent; and T. ricordensis sp. n. has cephalic capsule reaching posterior part of short amphids, amphidial shield without ornamentation, straight vagina, symmetrical spicules, gubernaculum with dorsal apophyses. Updated descriptions of T. donsi, T. cladara and T. gerlachi are provided. Phylogenetic analysis based on 18S rDNA suggests a close relationship between Tarvaia and Desmoscolecida.

Nothacrobeles borregi n. sp. (Nematoda: Cephalobidae) from Anza Borrego, California and N. laticollaris (De Ley & Vandebroek, 1992) n. comb.

Nothacrobeles borregi n. sp. is described from blow-sand in the vicinity of the roots of creosote bush (Larrea tridentata) from Anza Borrego State Park, San Diego, California. The new species is distinctive from all others of the genus by the specific morphology of the labial probolae and lips, as viewed with both interference light and scanning electron microscopy. The probolae include a basal ridge unique by its large size, smooth rim (lacking tines), and unusual large triangular platelet projections on each side; the probolae are also distinctive in having a knob-like ledge separate and posterior to the basal ridge. Nothacrobeles borregi n. sp. provides a new insight into homology of characters important for addressing problems with generic level classification in Cephalobinae. These hypotheses of homology suggest a basis for transferring N. laticollaris n. comb. (syn. Cervidellus laticollaris). Characteristics of Nothacrobeles in relation to Cervidellus and Stegelletina are discussed.