Roy Neilson

Greater coverage of the phylum Nematoda in SSU rDNA studies

The small subunit ribosomal gene (SSU rDNA) is used in both environmental and phylogenetic studies of nematodes. However, currently described SSU rDNA primers near to the 5′ end of the gene show mismatches with a number of different nematode sequences. Here a new SSU rDNA 5′ forward primer, Nem_SSU_F74, is designed from existing database sequences and its performance compared with a previously described forward primer, SSU_F04, by sequencing from nematode assemblage DNA, in combination with a previously described reverse primer. DNA was extracted from three nematode assemblages isolated from arable soil and, in total, six clone libraries were created: three amplified with established forward primer SSU_F04 and three with the novel primer Nem_SSU_F74. Ninety six clones were sequenced from each library. Nem_SSU_F74 libraries yielded a higher number of nematode sequences than SSU_F04 libraries, and a greater number of nematode taxa were found using the novel forward primer. The most abundant sequences were common to libraries created with either forward primer. Data from a morphological survey of the same samples revealed that biomass was more closely related to molecular analysis than simple counts of nematodes. For all but one nematode order (Aphelenchida), percentage assemblage composition was not significantly different between biomass and sequences obtained with the novel forward primer Nem_SSU_F74.

DNA Barcoding and Morphological Identification of Benthic Nematodes Assemblages of Estuarine Intertidal Sediments: Advances in Molecular Tools for Biodiversity Assessment

Concerns regarding the status of marine ecosystems have increased in part due to traditional and emerging human activities in marine waters, driving a demand for approaches with high sample throughput capability to improve ecosystem monitoring. Nematodes are already used as indicator species in biodiversity assessments and biomonitoring of terrestrial and marine systems, with molecular approaches offering the opportunity to utilize these organisms further in large scale ecological surveys and environmental assessments. Based on an available nematode dataset for estuarine sediments of the Mira estuary (SW coast, Portugal), we evaluated the diversity of the nematode community of this system, using the molecular markers 18S rRNA and COI genes. These approaches were compared to voucher specimens from a morphological characterization of the same samples allowing validation and comparison between nematode communities. The spatial and temporal variability of the density and diversity of the nematode assemblages was analyzed based on morphological characterization to allow the validation and efficiency of the genetic characterization. A PCO ordination plot showed a distinct separation of the assemblages between sampling occasions confirmed by PERMANOVA analysis, which showed significant differences, although no significant differences were detected between sampling sites. The morphological characterization identified 50 genera of which only 26 and 25 distinct 18S rRNA and COI DNA barcodes, respectively, were obtained. 90.2% of the morphologically identified specimens representing eleven different genera, successfully generated DNA barcodes for both 18S rRNA and COI genes. This study confirmed that the success of the 18S rRNA gene PCR amplification is higher than of COI gene with 43 species amplified against 34. The study highlights a limitation of available sequences for both targets in databases when compared to the known diversity of marine nematodes. The gene sequences of this study enriched the databases, contributing gene sequences from 7 and 16 new genera for the 18S rRNA and COI genes, respectively. A robust database of gene sequences is a prerequisite for the development of robust high sample throughput techniques to be applied in marine assessing and monitoring programs.

Morphological and molecular characterisation of Aphelenchoides besseyi and A. fujianensis (Nematoda: Aphelenchoididae) from rice and forage grass seeds in Brazil

Morphologically similar Aphelenchoides spp. populations extracted from rice and forage grass seeds from different geographical regions in Brazil were morphologically and molecularly characterised. Overall, the populations studied separated into two groups based on morphological and phylogenetic analyses, referred to herein as ‘Group-rice’ and ‘Group-forage’. Bayesian phylogenetic analyses of SSU, LSU and mtCOI regions strongly supported the presence of two dichotomous groups with Group-rice and Group-forage populations genetically similar to A. besseyi and A. fujianensis , respectively. This study reports the presence of a morphologically similar species to A. besseyi associated with seeds of grasses, but genetically distinct based on three genomic regions, which our results strongly suggest to be A. fujianensis , this being a new geographical record for Brazil. Additional information regarding spicule morphology of male A. besseyi is also reported.

New live screening of plant-nematode interactions in the rhizosphere

Free living nematodes (FLN) are microscopic worms found in all soils. While many FLN species are beneficial to crops, some species cause significant damage by feeding on roots and vectoring viruses. With the planned legislative removal of traditionally used chemical treatments, identification of new ways to manage FLN populations has become a high priority. For this, more powerful screening systems are required to rapidly assess threats to crops and identify treatments efficiently. Here, we have developed new live assays for testing nematode responses to treatment by combining transparent soil microcosms, a new light sheet imaging technique termed Biospeckle Selective Plane Illumination Microscopy (BSPIM) for fast nematode detection, and Confocal Laser Scanning Microscopy for high resolution imaging. We show that BSPIM increased signal to noise ratios by up to 60 fold and allowed the automatic detection of FLN in transparent soil samples of 1.5u2009mL. Growing plant root systems were rapidly scanned for nematode abundance and activity, and FLN feeding behaviour and responses to chemical compounds observed in soil-like conditions. This approach could be used for direct monitoring of FLN activity either to develop new compounds that target economically damaging herbivorous nematodes or ensuring that beneficial species are not negatively impacted.

Hydrolysis probe-based PCR for detection of Pratylenchus crenatus , P. neglectus and P. penetrans

Molecular detection of pest and pathogens relies on rapid and dependable methods for their identification as well as an assessment of their abundance. This study describes the development and evaluation of a diagnostic method for detection of Pratylenchus crenatus , P. penetrans and P. neglectus , based on a hydrolysis probe qPCR assay. Primer/probe sets were designed targeting the ITS-1 rDNA. In order to assess the specificity, primer/probe sets were tested with samples of non-target Pratylenchus species and Radopholus similis . Experiments using dilutions of purified plasmid standards tested the sensitivity of the hydrolysis assay against detection of DNA extracted from individual nematodes. Target DNA was detected in soil samples collected from potato fields and this indicated that P. crenatus , P. neglectus and P. penetrans are widely distributed in Scotland, frequently co-existing in mixed populations, with P. crenatus more prevalent than either P. neglectus or P. penetrans .

Diversity of endosymbiont bacteria associated with a non-filarial nematode group

There is a significant knowledge gap with regard to non-filarial nematodes and their relationships, if any, with intracellular bacteria, with only sporadic reports in the literature. An intracellular bacteria Xiphinematobacter , belonging to subdivision 2 of the Verrucomicrobia, was previously reported in the ovaries of three species of the non-filarial Xiphinema americanum -group of nematodes. We explored the diversity of Xiphinematobacter in 22 populations of X. americanum sourced from six continents and conservatively have identified nine phylotypes, six of which have not previously been reported. A geographic basis to the phylotypes was noted with phylotypes A and B only found in Europe, whereas phylotypes F, G, H and I were mainly found in North America. Phylotypes C, D and E showed greater geographical variation. Sequences of Xiphinematobacter from this study help to inform the taxonomy of Verrucomicrobia such that the status and composition of Verrucomicrobia subdivision 2 potentially requires reflection.

Plant parasitic nematode assemblages associated with sweet potato in Kenya and their relationship with environmental variables

Sweet potato is one of the most important staple food crops consumed in Kenya and throughout Africa but yields are greatly reduced by plant parasitic nematodes (PPN). The aim of this study was to determine the prevalence of PPN in Kenyan sweet potato fields and their relationship with soil and climatic variables. Soil samples were collected from sweet potato fields in Busia, Teso, Kisii, Embu and Makueni counties. Thirteen nematode genera were identified across the five counties with Meloidogyne, Pratylenchus and Rotylenchus being the most prevalent. There was a significant (P <0.05) relationship between PPN abundance and sodium, calcium and iron. Canonical correspondence analysis of climatic variables revealed that the relationship between rainfall and nematode genera was significant (P <0.05) while maximum and minimum temperatures were not significant. This description of PPN assemblages associated with sweet potato in Kenya and their relationship with environmental variables provides a starting point from which appropriate nematode management strategies can be implemented.

A study on Maruca vitrata infestation of Yard-long beans (Vigna unguiculata subspecies sesquipedalis).

Globally, Maruca vitrata (Geyer) is a serious yield constraint on food legumes including Yard-long bean (Vigna unguiculata subspecies sesquipedalis). However, there is a dearth of information on its damage potential, distribution and population dynamics in Yard-long beans. In the present study, the level of M. vitrata larval infestation on flowers and pods of Yard-long beans in Sri Lanka was determined with respect to three consecutive cropping seasons, Yala, Off and Maha. Results indicated that larval infestation and abundance varied with developmental stage of flowers and pods, cropping season and their combined interactive effects. Flowers of Yard-long beans were more prone to M. vitrata larval attack compared to pods. Abundance and level of infestation of M. vitrata varied with plant parts, having a ranking of flower buds (highest) > open flowers > mature pods > immature pods (lowest). Peak infestation was observed six and eight weeks after planting on flowers and pods, respectively. Among the three cropping seasons, M. vitrata infestation was found to be higher during Maha and Off seasons compared to Yala. The findings of this study contribute to the identified knowledge gap regarding the field biology of an acknowledged important pest, M. vitrata, in a previously understudied crop in Sri Lanka.

Temperate grass allergy season defined by spatio-temporal shifts in airborne pollen communities

Grass pollen is the world’s most harmful outdoor aeroallergen and sensitivity varies between species. Different species of grass flower at different times, but it is not known how airborne communities of grass pollen change in time and space. Persistence and high mobility of grass pollen could result in increasingly diverse seasonal pollen communities. Conversely, if grass pollen does not persist for an extended time in the air, shifting pollen communities would be predicted throughout the summer months. Here, using targeted high throughput sequencing, we tracked the seasonal progression of airborne Poaceae pollen biodiversity across Britain, throughout the grass allergy season. All grass genera displayed discrete, temporally restricted peaks of pollen incidence which varied with latitude, revealing that the taxonomic composition of grass pollen exposure changes substantially across the allergy season. By developing more refined aeroallergen profiling, we predict that our findings will facilitate the exploration of links between taxon-specific exposure of harmful grass pollen and disease, with concomitant socio-economic benefits.

Variable changes in nematode infection prevalence and intensity after Rabbit Haemorrhagic Disease Virus emerged in wild rabbits in Scotland and New Zealand

The myxoma virus (a microparasite) reduced wild rabbit numbers worldwide when introduced in the 1950s, and is known to interact with co-infecting helminths (macroparasites) causing both increases and decreases in macroparasite population size. In the 1990s Rabbit Haemorrhagic Disease Virus (RHDV) infected rabbits and also significantly reduced rabbit numbers in several countries. However, not much is known about RHDV interactions with macroparasites. In this study, we compare prevalence and intensity of infection for three gastrointestinal nematode species (Trichostrongylus retortaeformis, Graphidium strigosum and Passalurus ambiguus) before and after RHDV spread across host populations in Scotland and New Zealand. During one common season, autumn, prevalence of T. retortaeformis was higher after RHDV spread in both locations, whereas it was lower for G. strigosum and P. ambiguus after RHDV arrived in New Zealand, but higher in Scotland. Meanwhile, intensity of infection for all species decreased after RHDV arrived in New Zealand, but increased in Scotland. The impact of RHDV on worm infections was generally similar across seasons in Scotland, and also similarities in seasonality between locations suggested effects on infection patterns in one season are likely similar year-round. The variable response by macroparasites to the arrival of a microparasite into Scottish and New Zealand rabbits may be due to differences in the environment they inhabit, in existing parasite community structure, and to some extent, in the relative magnitude of indirect effects. Specifically, our data suggest that bottom-up processes after the introduction of a more virulent strain of RHDV to New Zealand may affect macroparasite co-infections by reducing the availability of their shared common resource, the rabbits. Clearly, interactions between co-infecting micro- and macroparasites vary in host populations with different ecologies, and significantly impact parasite community structure in wildlife.