Dose dependence of Phasmarhabditis isolates (P. hermaphrodita, P. californica, P. papillosa) on the mortality of adult invasive white garden snails (Theba pisana).

Theba pisana is an invasive snail pest which has established itself in San Diego County and some areas of Los Angeles County, California. The snail has grown to large populations in some areas and mitigation is becoming necessary to stop the spread of the species. In a previous study, three US strains of Phasmarhabditis species (P. californica, P. papillosa, and P. hermaphrodita) effectively killed juvenile (0.25 gram each, 4-6 mm wide) T. pisana in laboratory conditions at 5 times (150 IJs/cm2) the recommended dose. Based on laboratory assays, we demonstrated that the same three US strains of Phasmarhabditis can effectively kill larger adult T. pisana (0.4-1.2 gram, 11.5-15mm wide) in two weeks at the same dose. The strains were more efficient at killing T. pisana than the compared molluscicide Sluggo Plus®. Results further showed that the most virulent P. californica did not effectively kill T. pisana at lower doses of 30 IJs/cm2 and 90 IJs/cm2. Additional research is needed to develop the most efficient means of application of Phasmarhabditis to mitigate T. pisana in the field.

TARANTOBELUS JEFFDANIELSI N. SP. (PANAGROLAIMOMORPHA; PANAGROLAIMIDAE), A NEMATODE PARASITE OF TARANTULAS.

Multiple tarantula deaths for a wholesale breeder were reported in 2018. The breeder noticed white discharge in the oral cavities of the tarantulas. Upon inspection, it was discovered that the white discharge was a large group of nematodes intertwined inside the tarantula's oral cavity. We examined the nematodes and propose a new species, Tarantobelus jeffdanielsi n. sp., in the currently monotypic genus Tarantobelus based on a combination of morphological and morphometrical data and unique nuclear rDNA 28S and 18S sequences. Based on phylogenetic analyses, the previously described Tarantobelus arachnicida was relocated, along with T. jeffdanielsi, into the family Panagrolaimidae. We also provide evidence of the ability of T. jeffdanielsi to parasitize Galleria mellonella larvae and the tarantula Grammostola pulchra. The life span and fecundity of the new species were also assessed, resulting in an 11.2-d average life span, and a total fertility rate of 158 nematodes/adult.

Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand.

Biological control (biocontrol) as a component of pest management strategies reduces reliance on synthetic chemicals, and seemingly offers a natural approach that minimizes environmental impact. However, introducing a new organism to new environments as a classical biocontrol agent can have broad and unanticipated biodiversity effects and conservation consequences. Nematodes are currently used in a variety of commercial biocontrol applications, including the use of Phasmarhabditis hermaphrodita as an agent targeting pest slug and snail species. This species was originally discovered in Germany, and is generally thought to have European origins. P. hermaphrodita is sold under the trade name Nemaslug®, and is available only in European markets. However, this nematode species was discovered in New Zealand and the western United States, though its specific origins remained unclear. In this study, we analyzed 45 nematode strains representing eight different Phasmarhabditis species, collected from nine countries around the world. A segment of nematode mitochondrial DNA (mtDNA) was sequenced and subjected to phylogenetic analyses. Our mtDNA phylogenies were overall consistent with previous analyses based on nuclear ribosomal RNA (rRNA) loci. The recently discovered P. hermaphrodita strains in New Zealand and the United States had mtDNA haplotypes nearly identical to that of Nemaslug®, and these were placed together in an intraspecific monophyletic clade with high support in maximum likelihood and Bayesian analyses. We also examined bacteria that co-cultured with the nematode strains isolated in Oregon, USA, by analyzing 16S rRNA sequences. Eight different bacterial genera were found to associate with these nematodes, though Moraxella osloensis, the bacteria species used in the Nemaslug® formulation, was not detected. This study provided evidence that nematodes deriving from the Nemaslug® biocontrol product have invaded countries where its use is prohibited by regulatory agencies and not commercially available.

An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa.

Phylogenetic reconstructions of relations within the phylum Nematoda are inherently difficult but have been advanced with the introduction of large-scale molecular-based techniques. However, the most recent revisions were heavily biased towards terrestrial and parasitic species and greater representation of clades containing marine species (e.g. Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, Enoplida, and Monhysterida) is needed for accurate coverage of known taxonomic diversity. We now add small subunit ribosomal DNA (SSU rDNA) sequences for 100 previously un-sequenced species of nematodes, including 46 marine taxa. SSU rDNA sequences for >200 taxa have been analysed based on Bayesian inference and LogDet-transformed distances. The resulting phylogenies provide support for (i) the re-classification of the Secernentea as the order Rhabditida that derived from a common ancestor of chromadorean orders Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, and Monhysterida and (ii) the position of Bunonema close to the Diplogasteroidea in the Rhabditina. Other, previously controversial relationships can now be resolved more clearly: (a) Alaimus, Campydora, and Trischistoma belong in the Enoplida, (b) Isolaimium is placed basally to a big clade containing the Axonolaimidae, Plectidae, and Rhabditida, (c) Xyzzors belongs in the Desmodoridae, (d) Comesomatidae and Cyartonema belongs in the Monhysterida, (e) Globodera belongs in the Hoplolaimidae and (f) Paratylenchus dianeae belongs in the Criconematoidea. However, the SSU gene did not provide significant support for the class Chromadoria or clear evidence for the relationship between the three classes, Enoplia, Dorylaimia, and Chromadoria. Furthermore, across the whole phylum, the phylogenetically informative characters of the SSU gene are not informative in a parsimony analysis, highlighting the short-comings of the parsimony method for large-scale phylogenetic modelling.

Phylogeny of Cephalobina (Nematoda): molecular evidence for recurrent evolution of probolae and incongruence with traditional classifications.

Nematodes of the suborder Cephalobina include an ecologically and morphologically diverse array of species that range from soil-dwelling microbivores to parasites of vertebrates and invertebrates. Despite a long history of study, certain of these microbivores (Cephaloboidea) present some of the most intractable problems in nematode systematics; the lack of an evolutionary framework for these taxa has prevented the identification of natural groups and inhibited understanding of soil biodiversity and nematode ecology. Phylogenetic analyses of ribosomal (LSU) sequence data from 53 taxa revealed strong support for monophyly of taxa representing the Cephaloboidea, but do not support the monophyly of most genera within this superfamily. Historically these genera have primarily been recognized based on variation in labial morphology, but molecular phylogenies show the same general labial (probolae) morphotype often results from recurrent similarity, a result consistent with the phenotypic plasticity of probolae previously observed for some species in ecological time. Phylogenetic analyses of LSU rDNA also recovered strong support for some other groups of cephalobs, including taxa representing most (but not all) Panagrolaimoidea. In addition to revealing homoplasy of probolae, molecular trees also imply other unexpected patterns of character evolution or polarity, including recurrent similarity of offset spermatheca presence, and representation of complex probolae as the ancestral condition within Cephaloboidea. For Cephalobidae, molecular trees do not support traditional genera as natural groups, but it remains untested if deconstructing probolae morphotypes or other structural features into finer component characters may reveal homologies that help delimit evolutionary lineages.

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.

Phylogenetic Analyses of Meloidogyne Small Subunit rDNA.

Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species.