Colin C. Fleming

A molecular identification key for economically important thrips species (Thysanoptera: Thripidae) using direct sequencing and a PCR‐RFLP‐based approach

Abstract 1 Treatments against pathogens or pests are often very specific and, as a fundamental first step, require the ability to identify taxa correctly and unambiguously. We used PCR amplification techniques to successfully establish a molecular identification key for economically important thrips species. 2 A PCR amplified 433 bp long fragment of the mitochondrial COI coding gene was analysed by automated direct sequencing and RFLP. Sequencing of 264 individual thrips representing 10 named species detected 17 haplotypes. Variation within species was low, whereas among species variation was high resulting in an average sequence divergence of 18.6% and an average pairwise species differentiation (calculated as FST‐value) of 0.9896. 3 Two restriction enzymes (AluI, Sau3AI) produced patterns that allowed unambiguous identification of all thrips species. 4 Statistical support for the quality of the key was given by (i) a highly significant permutation approach, assigning individual haplotypes to the correct species groups and (ii) a hierarchical NJ cluster analysis in which all conspecific individual sequences clustered together with maximal (100%) bootstrap support. 5 This study has shown that the use of genetic markers represents a valuable alternative for situations, such as epidemiological research, in which correct identification with classical morphological methods is either very difficult and time consuming or virtually impossible.

flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference

The potato cyst nematode Globodera pallida is a serious pest of potato crops. Nematode FMRFamide‐like peptides (FLPs) are one of the most diverse neuropeptide families known, and modulate sensory and motor functions. As neuromuscular function is a well‐established target for parasite control, parasitic nematode FLP signaling has significant potential in novel control strategies. In the absence of transgenic parasitic nematodes and the reported ineffectiveness of neuronal gene RNAi in Caenorhabditis elegans, nothing is known about flp function in nematode parasites. In attempts to evaluate flp function in G. pallida, we have discovered that, unlike in C. elegans, these genes are readily susceptible to RNAi. Silencing any of the five characterized G. pallida flp genes (Gp‐flp‐1, ‐6, ‐12, ‐14, or ‐18) incurred distinct aberrant behavioral phenotypes consistent with key roles in motor function. Further delineation of these effects revealed that double‐stranded RNA exposure time (≥18 h) and concentration (≥0.1 μg/ml) were critical to the observed effects, which were reversible. G. pallida flp genes are essential to coordinated locomo‐tory activities, do not display redundancy, and are susceptible to RNAi, paving the way for the investigation of RNAi‐mediated flp gene silencing as a novel plant parasite control strategy.—Kimber, M. J., McKinney, S., McMaster, S., Day, T. A., Fleming, C. C., Maule, A. G. flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference. FASEB J. 21, 1233–1243 (2007)

Plant biostimulants: a review on the processing of macroalgae and use of extracts for crop management to reduce abiotic and biotic stresses

A biostimulant is an organic material that, when applied in small quantities, enhances plant growth and development such that the response cannot be attributed to the application of traditional plant nutrients. This review is aimed at highlighting developments in the processing of macroalgae for agricultural biostimulants (AB), summarising the biologically active components of brown macroalgae and examining the factors supporting the use of macroalgal AB for managing abiotic and biotic stresses in crop plants. The policy drivers supporting the use of macroalgal-derived ABs in agriculture are also emphasised. We examine the use of macroalgal ABs in crop production and evaluated the benefits of seed priming, foliar application, soil drenches and hydroponic treatments. The use of macroalgal ABs on crop plants can generate multiple benefits with reported effects including enhanced rooting, higher crop and fruit yields, freezing, drought and salt tolerance, enhanced photosynthetic activity and resistance to fungi, bacteria and virus. ABs can be applied as an alternative, or used in conjunction with synthetic crop protection products and plant growth regulators, and may have a role in maintaining crop production levels, health and quality in the future when many active ingredients will be lost to the industry due to changes in European Union regulations. Worldwide, macroalgae remain largely unexploited, we highlight some of the future research and development priorities.

RNAi effector diversity in nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.

Taxonomy: Species status of hybridizing oaks

The two widespread species of oak tree in Europe, Quercus robur L. and Q. petraea (Matt.) Liebl., hybridize extensively, calling their taxonomic status into question. Here we use microsatellite DNA, a highly informative genetic marker, to show that Q. robur and Q. petraea are discrete taxonomic units despite this intensive hybridization. Furthermore, individual oaks can be assigned to separate species.

DETECTION OF RHOPALOSIPHUM INSERTUM (APPLE-GRASS APHID) PREDATION BY THE PREDATORY MITE ANYSTIS BACCARUM USING MOLECULAR GUT ANALYSIS

Abstract  1 A simple, yet sensitive polymerase chain reaction based technique was developed for the detection of the apple‐grass aphid Rhopalosiphum insertum in the gut of Anystis baccarum, a predatory mite.

Localisation of Globodera pallida FMRFamide-related peptide encoding genes using in situ hybridisation

The present study employed an in situ hybridisation technique to detect the expression of a number of FMRFamide-like peptide encoding (flp) genes, previously identified from Globodera pallida, in whole-mount preparations of the J(2) stage of this worm. gpflp-1, encoding the FMRFamide-related peptide (FaRP) KSAYMRFamide, was expressed in neurones associated with the circumpharyngeal nerve ring and specifically in a number of cell bodies in the lumbar ganglia of the perianal nerve ring. The lumbar ganglia and pre-anal ganglia along with the BDU neurones and a number of cells in the retrovesicular ganglion were observed to express gpflp-2, encoding KNKFEFIRFamide. gpflp-3 (encoding KHEYLRFamide) expression was localised to the anterior ganglion and a number of paired cells posterior to the circumpharyngeal nerve ring whilst expression of gpflp-4, encoding a number of -P(G/Q)VLRFamides, was localised to the retrovesicular ganglion. No expression of gpflp-5 was observed. Identification of the reactive cells has implicated distinct roles for the FaRPs encoded on these genes in regulation of both dorsal and ventral body wall muscles, the musculature of the vulva and in the function of a number of sensory structures in both the head and tail of G. pallida. Comparison with the expression patterns of analogous genes in Caenorhabditis elegans suggests that, whilst some of the encoded peptides are conserved between nematode species, their functions therein are distinct. Furthermore, the expression of some of these genes in a number of interneurones supports the idea that FaRPs fulfil neuromodulatory as well as neurotransmitter roles.

Non-nematode-derived double-stranded RNAs induce profound phenotypic changes in Meloidogyne incognita and Globodera pallida infective juveniles

Nine non-nematode-derived double-stranded RNAs (dsRNAs), designed for use as controls in RNA interference (RNAi) screens of neuropeptide targets, were found to induce aberrant phenotypes and an unexpected inhibitory effect on motility of root knot nematode Meloidogyne incognita J2s following 24h soaks in 0.1 mg/ml dsRNA; a simple soaking procedure which we have found to elicit profound knockdown of neuronal targets in Globodera pallida J2s. We have established that this inhibitory phenomenon is both time- and concentration-dependent, as shorter 4h soaks in 0.1 mg/ml dsRNA had no negative impact on M. incognita J2 stage worms, yet a 10-fold increase in concentration to 1 mg/ml for the same 4h time period had an even greater qualitative and quantitative impact on worm phenotype and motility. Further, a 10-fold increase of J2s soaked in 0.1 mg/ml dsRNA did not significantly alter the observed phenotypic aberration, which suggests that dsRNA uptake of the soaked J2s is not saturated under these conditions. This phenomenon was not initially observed in potato cyst nematode G. pallida J2s, which displayed no aberrant phenotype, or diminution of migratory activity in response to the same 0.1 mg/ml dsRNA 24h soaks. However, a 10-fold increase in dsRNA to 1mg/ml was found to elicit comparable irregularity of phenotype and inhibition of motility in G. pallida, to that initially observed in M. incognita following a 24h soak in 0.1 mg/ml dsRNA. Again, a 10-fold increase in the number of G. pallida J2s soaked in the same volume of 1 mg/ml dsRNA preparation did not significantly affect the observed phenotypic deviation. We do not observe any global impact on transcript abundance in either M. incognita or G. pallida J2s following 0.1 mg/ml dsRNA soaks, as revealed by reverse transcriptase-PCR and quantitative PCR data. This study aims to raise awareness of a phenomenon which we observe consistently and which we believe signifies a more expansive deficiency in our knowledge and understanding of the variables inherent to RNAi-based investigation.

FMRFamide-related peptides in potato cyst nematodes

This study presents data demonstrating the presence of FMRFamide-related peptides (FaRPs) in potato cyst nematodes (PCN). Five transcripts of FaRP encoding genes, designated gpflp-1 to gpflp-5, were characterised using RACE. In terms of ORFs, gpflp-1 was 444 base pairs (bp) long and coded for four copies of the FaRP, PF3 (KSAYMRFamide) whilst gpflp-2 was 309 bp long and encoded one copy of the peptide, KNKFEFIRFamide. gpflp-3 (420 bp) Encoded two copies of KHEYLRFamide (AF2) and the genes gpflp-4 and gpflp-5 encoded a total of 11 FaRPs, most of which are novel to PCN. FMRFamide-related peptide (FaRP)-like immunoreactivity was observed in both PCN species, Globodera pallida and Globodera rostochiensis, using an antiserum raised against the invertebrate peptide, FMRFamide. Immunopositive neurones were found throughout the central nervous system in the ventral and dorsal nerve cords and the circumpharyngeal and perianal nerve rings. Reactive neurones were also present peripherally, innervating the highly muscular pharynx with a nerve net and ring-like structures. Positive immunostaining was also observed in neurones running toward the stylet protractor muscles and/or the anterior sensory apparatus. This study implicates a role for FaRPs in feeding, host penetration and sensory function of PCN. This is the first study to characterise FaRP encoding genes from a plant-parasitic nematode using a targeted PCR based RACE approach and further underlines the importance and diversity of this neuropeptide group in the phylum Nematoda.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality.

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control. On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (si)RNA-soaked M. incognita eggs. Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH(2))-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal. Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality. To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s, which recovered within 24h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M. incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-eri-1 transcript which encodes an RNAi-inhibiting siRNA exonuclease. We observe a marked up-regulation of Mi-eri-1 transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RNAi-based reverse genetic screens in plant parasitic nematodes.