Paul De Ley

An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding

Molecular surveys of meiofaunal diversity face some interesting methodological challenges when it comes to interstitial nematodes from soils and sediments. Morphology-based surveys are greatly limited in processing speed, while barcoding approaches for nematodes are hampered by difficulties of matching sequence data with traditional taxonomy. Intermediate technology is needed to bridge the gap between both approaches. An example of such technology is video capture and editing microscopy, which consists of the recording of taxonomically informative multifocal series of microscopy images as digital video clips. The integration of multifocal imaging with sequence analysis of the D2D3 region of large subunit (LSU) rDNA is illustrated here in the context of a combined morphological and barcode sequencing survey of marine nematodes from Baja California and California. The resulting video clips and sequence data are made available online in the database NemATOL (http://nematol.unh.edu/). Analyses of 37 barcoded nematodes suggest that these represent at least 32 species, none of which matches available D2D3 sequences in public databases. The recorded multifocal vouchers allowed us to identify most specimens to genus, and will be used to match specimens with subsequent species identifications and descriptions of preserved specimens. Like molecular barcodes, multifocal voucher archives are part of a wider effort at structuring and changing the process of biodiversity discovery. We argue that data-rich surveys and phylogenetic tools for analysis of barcode sequences are an essential component of the exploration of phyla with a high fraction of undiscovered species. Our methods are also directly applicable to other meiofauna such as for example gastrotrichs and tardigrades.

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.

Metagenetic community analysis of microbial eukaryotes illuminates biogeographic patterns in deep-sea and shallow water sediments.

Microbial eukaryotes (nematodes, protists, fungi, etc., loosely referred to as meiofauna) are ubiquitous in marine sediments and probably play pivotal roles in maintaining ecosystem function. Although the deep-sea benthos represents one of the worlds largest habitats, we lack a firm understanding of the biodiversity and community interactions amongst meiobenthic organisms in this ecosystem. Within this vast environment, key questions concerning the historical genetic structure of species remain a mystery, yet have profound implications for our understanding of global biodiversity and how we perceive and mitigate the impact of environmental change and anthropogenic disturbance. Using a metagenetic approach, we present an assessment of microbial eukaryote communities across depth (shallow water to abyssal) and ocean basins (deep-sea Pacific and Atlantic). Within the 12 sites examined, our results suggest that some taxa can maintain eurybathic ranges and cosmopolitan deep-sea distributions, but the majority of species appear to be regionally restricted. For Operationally Clustered Taxonomic Units (OCTUs) reporting wide distributions, there appears to be a taxonomic bias towards a small subset of taxa in most phyla; such bias may be driven by specific life history traits amongst these organisms. In addition, low genetic divergence between geographically disparate deep-sea sites suggests either a shorter coalescence time between deep-sea regions or slower rates of evolution across this vast oceanic ecosystem. While high-throughput studies allow for broad assessment of genetic patterns across microbial eukaryote communities, intragenomic variation in rRNA gene copies and the patchy coverage of reference databases currently present substantial challenges for robust taxonomic interpretations of eukaryotic data sets.

Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae)

Derycke, S., De Ley, P., De Ley, I.T., Holovachov, O., Rigaux, A. & Moens, T. (2010). Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae).—Zoologica Scripta, 39, 276–289.

Culture-Independent Investigation of the Microbiome Associated with the Nematode Acrobeloides maximus

Background Symbioses between metazoans and microbes are widespread and vital to many ecosystems. Recent work with several nematode species has suggested that strong associations with microbial symbionts may also be common among members of this phylu. In this work we explore possible symbiosis between bacteria and the free living soil bacteriovorous nematode Acrobeloides maximus. Methodology We used a soil microcosm approach to expose A. maximus populations grown monoxenically on RFP labeled Escherichia coli in a soil slurry. Worms were recovered by density gradient separation and examined using both culture-independent and isolation methods. A 16S rRNA gene survey of the worm-associated bacteria was compared to the soil and to a similar analysis using Caenorhabditis elegans N2. Recovered A. maximus populations were maintained on cholesterol agar and sampled to examine the population dynamics of the microbiome. Results A consistent core microbiome was extracted from A. maximus that differed from those in the bulk soil or the C. elegans associated set. Three genera, Ochrobactrum, Pedobacter, and Chitinophaga, were identified at high levels only in the A. maximus populations, which were less diverse than the assemblage associated with C. elegans. Putative symbiont populations were maintained for at least 4 months post inoculation, although the levels decreased as the culture aged. Fluorescence in situ hybridization (FISH) using probes specific for Ochrobactrum and Pedobacter stained bacterial cells in formaldehyde fixed nematode guts. Conclusions Three microorganisms were repeatedly observed in association with Acrobeloides maximus when recovered from soil microcosms. We isolated several Ochrobactrum sp. and Pedobacter sp., and demonstrated that they inhabit the nematode gut by FISH. Although their role in A. maximus is not resolved, we propose possible mutualistic roles for these bacteria in protection of the host against pathogens and facilitating enzymatic digestion of other ingested bacteria.

Nematodes from vernal pools in the Santa Rosa Plateau Ecological Reserve, California I. Hirschmanniella santarosae sp. n. (Nematoda: Pratylenchidae), a cryptic sibling species of H. pomponiensis Abdel-Rahman & Maggenti, 1987.

Hirschmanniella santarosae sp. n. is described from the largest vernal pool in the Santa Rosa Plateau Ecological Reserve, Murrieta, California, USA. The cryptic new species is morphologically very close to H. pomponiensis and H. gracilis as it can be distinguished from the former only by a more anterior excretory pore position and by more flattened as well as laterally expanded stylet knobs, whilst it differs from the latter in the greater distance from phasmid to tail tip and in the bursal alae ending near the phasmids rather than near the tail tip. Analysis of the rDNA small subunit (SSU) and D2D3 expansion segments of the large subunit (LSU) shows that H. santarosae sp. n. is genetically distinct, having respective sequence homology of 98.89% and 95.9% with H. pomponiensis for these loci. Congruence in SSU and D2D3 gene trees as well as unambiguous character autapomorphies further support the new species status of H. santarosae sp. n. and sibling relationship with H. pomponiensis. Although many more isolates and species will need to be studied before informative biogeographic analyses can be performed, the presently available sequence data suggest that some Hirschmanniella lineages have diversified independently on either side of the Atlantic.

Description of Phasmarhabditis californica n. sp. and first report of P. papillosa (Nematoda: Rhabditidae) from invasive slugs in the USA

A new species of Phasmarhabditis and the known species P. papillosa were isolated from cadavers of invasive slugs in California. Phasmarhabditis papillosa is the type of the genus and has not previously been reported from the Americas. Both species are characterised based on morphology, morphometrics and molecular data. Molecular phylogenies were inferred from concatenated DNA sequence alignments of nearly complete SSU and the D2-D3 domains of the LSU rDNA. Phasmarhabditis californica n. sp. is characterised by a robust body, mature egg-laying specimens almost spindle-shaped when relaxed, 1.5 (1.3-1.8) mm long, tapering to a bluntly rounded anterior end with stoma about as long as lip region diam., six inner labial papillae and four outer cephalic papillae, pharynx with rounded to pyriform basal bulb, vulva located halfway along the body, hermaphroditic, didelphic, amphidelphic, and short, conoid tail constricted at one-third its length with prominent phasmids. Not a single male was found among five strains. Phasmarhabditis papillosa is gonochoristic and has a longer isthmus, pyriform basal bulb and longer, dome-shaped spicate female tail constricted halfway along its length. Sequence analysis revealed unambiguous autapomorphies for P. papillosa and P. californica n. sp. Phylogenetic analyses placed these two species in a monophyletic clade comprising Phasmarhabditis species and other gastropod-parasitic taxa. Morphology, genetic distance, reproductive strategy and nucleotide autapomorphies support the new taxon.

Morphological, molecular characterisation and phylogenetic position of Longidorus mindanaoensis n. sp. (Nematoda: Longidoridae) from a Philippine Avicennia mangrove habitat.

Longidorus mindanaoensis n. sp. is described from samples associated with riverine mangroves in the Philippines and is the first Longidorus species collected from such a habitat. Its relationship within the genus is inferred from molecular and morphological data. Phylogenetic inferences were performed for D2-D3 and SSU rDNA using appropriate phylogenetic algorithms. The new species is characterised by a thickened basal layer of the body cuticle with prominent spiral fibres, a dome-shaped continuous lip region, an oval to elliptical fovea with pore-like opening, the guiding ring at 2.2-2.5 lip region diam. from the anterior end, very weakly developed odontophore, all three pharyngeal gland nuclei at about the mid-point of the basal bulb, vulva at 30-39%, tail convex-conoid to hemispherical, shorter than anal body diam., males with 19-24 ventromedian supplements, and four juvenile stages, all with the replacement odontostyle at a short distance from the functional one. On the basis of amphid shape and structure, shape of anterior body region and general body shape, L. mindanaoensis n. sp. comes close to L. caespiticola, L. eridanicus, L. helveticus, L. macrosoma, L. poessneckensis, L. orongorongensis, L. waikouaitii and L. belondiroides. D2-D3 analysis placed the new species as sister to L. pisi and the two combined are sister clade to a monophyletic group that includes (L. poessneckensis (L. caespiticola (L. helveticus, L. macrosoma))). However, on the basis of SSU phylogeny, L. mindanaoensis n. sp. is positioned within a clade that includes (Longidorus sp. (L. mindanaoensis n. sp. (L. poessneckensis (L. helveticus, L. macrosoma)))). The ‘close’ relationship of the new species with L. pisi in the D2-D3 tree is incongruent with the widely different morphology of the two species. The other species that are close to the new species in the cladogram are in agreement with the morphology.

Acromoldavicus mojavicus n. sp. (Nematoda: Cephaloboidea) from the Mojave Desert, California

Acromoldavicus (Cephalobina, Cephaloboidea) with its highly distinctive lip region has only a single species, Acromoldavicus skrjabini Nesterov & Lisetskaya, originally described from Moldova and subsequently also detected at sites in the Middle East and near the Mediterranean. Herein, Acromoldavicus mojavicus n. sp. is described from sandy soil surrounding a Joshua tree (Yucca brevifolia) in a remote area of the Mojave Desert, California, USA. The lip region of A. mojavicus n. sp. is bilaterally symmetrical with three triangular probolae surrounded by three pairs of plate-like lips. The lip region is organised along similar lines as that of A. skrjabini, but differs in several respects, such as its larger size, presence of elongate posterior processes on each lip and division of the lateral lips into two lobes (excluding the dorso-sublateral guard processes). In addition, phylogenetic interpretation of sequence data from the large-subunit of ribosomal DNA provides further evidence for autapomorphies and separate species status for A. skrjabini and A. mojavicus n. sp. Characteristics shared with Cephaloboidea include the offset spermatheca and males with eight pairs of genital papillae. Both species of Acromoldavicus have a buccal capsule with a reduced gymnostom, a character that seems to be shared with the cephalobid Elaphonema and in part is a basis for placement of both genera in Elaphonematidae. The species A. mojavicus n. sp. exhibits additional similarities with Elaphonema spp. that further support this placement.

Morphological, molecular and biological characterization of Mehdinema alii (Nematoda: Diplogasterida) from the decorated cricket (Gryllodes sigillatus).

The nematode Mehdinema alii was recovered from the decorated cricket Gryllodes sigillatus (Walker). Morphometric comparisons are presented from 3 populations. The nematode is characterized by dense arrays of spines on the cuticle of the anterior half of the body and a highly elongate, tubular stoma with a dorsal denticle in the glottoid region. Females have a protruding vulva. Young females are amphidelphic, but the anterior ovary disappears in older females bearing multiple developing juveniles. The male is monorchic with asymmetrically placed genital papillae, distally fused spicules, and a highly complex gubernaculum bearing 2 cuticularized thorns that protrude through a separate, postcloacal opening. Adult nematodes are located primarily in the hindgut, whereas juveniles or dauers occur mainly in the genital chamber of both male and female crickets. Male crickets are significantly more likely to be infected than females. This male-biased infection may be linked to the venereal transmission mechanism of the dauers. Although morphologically unusual in many respects, placement of M. alii in Diplogasterida is supported by both the morphology of the anterior digestive tract as well as analysis of its 18S rDNA sequence. These sequence data suggest that M. alii groups most closely with members of the Cylindrocorporidae.