Genomes of parasitic nematodes (Meloidogyne hapla, Meloidogyne incognita, Ascaris suum and Brugia malayi) have a reduced complement of small RNA interference pathway genes: knockdown can reduce host infectivity of M. incognita

AbstractDicers and dicer-like enzymes play an essential role in small RNA processing in eukaryotes. Nematodes are thought to encode one dicer, DCR-1; only that for Caenorhabditis spp. is well-characterised. Using genomic sequences of eight root-knot nematodes (Meloidogyne spp.), we identified putative coding sequences typical of eukaryotic DICERS. We noted that the primary and secondary structures of DICERS they encode were different for different Meloidogyne species and even for isolates of the same species, suggesting paralogy for the gene. One of the genes for M. incognita (Midcr-1.1) expressed in eggs, juvenile stage 2 and adults, with the highest expression in the adult females. All the Meloidogyne DICERS had seven major domains typical of those for Caenorhabditis spp. and humans with very similar protein folding. RNAi of Midcr-1.1 in J2s using seven dsRNAs, each based on sequences encoding the domains, induced mild paralysis but measurable knockdown was detected in J2s treated with five of the dsRNAs. For four of the dsRNAs, the RNAi effect lasted and reduced the nematode’s infectivity. Also, host plant delivery of dsRNAs complementary to coding sequences of the Dicer Dimerisation domain impaired development, reducing nematode infection by 71%. These results confirm the importance of the gene to nematode health.

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RNA Interference of Dual Oxidase in the Plant Nematode Meloidogyne incognita

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-1099 ◽

2005 ◽

Vol 18 (10) ◽

pp. 1099-1106 ◽

Cited By ~ 75

Author(s):

Manjula Bakhetia ◽

Wayne Charlton ◽

Howard J. Atkinson ◽

Michael J. McPherson

Keyword(s):

Rna Interference ◽

Gene Function ◽

Meloidogyne Incognita ◽

Feeding Strategy ◽

Model Organisms ◽

Parasitic Nematodes ◽

In Planta ◽

Oxidase Gene ◽

Dual Oxidase

RNA interference (RNAi) is a powerful tool for the analysis of gene function in model organisms such as the nematode Caenorhabditis elegans. Recent demonstrations of RNAi in plant parasitic nematodes provide a stimulus to explore the potential of using RNAi to investigate disruption of gene function in Meloidogyne incognita, one of the most important nematode pests of global agriculture. We have used RNAi to examine the importance of dual oxidases (peroxidase and NADPH oxidase), a class of enzyme associated with extracellular matrix cross-linking in C. elegans. RNAi uptake by M. incognita juveniles is highly efficient. In planta infection data show that a single 4-h preinfection treatment with double-stranded RNA derived from the peroxidase region of a dual oxidase gene has effects on gene expression that are phenotypically observable 35 days postinfection. This RNAi effect results in a reduction in egg numbers at 35 days of up to 70%. The in vitro feeding strategy provides a powerful tool for identifying functionally important genes, including those that are potential targets for the development of new agrochemicals or transgenic resistance strategies.

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Allelopathy: a possible mechanism of suppression of plant-parasitic nematodes by entomopathogenic nematodes

Nematology ◽

10.1163/156854199508766 ◽

1999 ◽

Vol 1 (7) ◽

pp. 735-743 ◽

Cited By ~ 32

Author(s):

Parwinder S. Grewal ◽

Edwin E. Lewis ◽

Sudha Venkatachari

Keyword(s):

Meloidogyne Incognita ◽

Entomopathogenic Nematodes ◽

Galleria Mellonella ◽

Steinernema Feltiae ◽

Symbiotic Bacteria ◽

Parasitic Nematodes ◽

Plant Parasitic Nematodes ◽

Tomato Roots ◽

Xenorhabdus Nematophilus ◽

Plant Parasitic

Abstract A possible mechanism of suppression of a plant-parasitic nematode Meloidogyne incognita by entomopathogenic nematodes is described. Heat-killed entomopathogenic nematodes Steinernema feltiae and S. riobrave temporarily suppressed penetration of the root-knot nematode M. incognita into tomato roots, but live nematodes had no effect. Infective juvenile M. incognita were repelled from all entomopathogenic nematode treatments that included their symbiotic bacteria. They were repelled by Galleria mellonella cadavers infected with S. carpocapsae, S. feltiae, and S. riobrave and from cell-free culture filtrates of the symbiotic bacteria Xenorhabdus nematophilus, X. bovienii, and Xenorhabdus sp. "R" from the three nematode species, respectively. Cell-free filtrates from all three Xenorhabdus spp. were toxic to M. incognita infective juveniles causing 98-100% mortality at 15% concentration. Cell-free filtrate of Xenorhabdus sp. "R" also reduced the hatch of M. incognita eggs. Application of formulated bacterial cell-free filtrates temporarily suppressed M. incognita penetration into tomato roots in a greenhouse trial. The short-term effects of cell-free bacterial filtrates, namely toxicity and repellency, were almost entirely due to ammonium. These results demonstrate allelopathic interactions between plant-parasitic nematodes, entomopathogenic nematodes and their symbiotic bacteria. The likely role of allelopathy in the suppression of plant-parasitic nematodes by innundative applications of entomopathogenic nematodes is discussed. Allelopathie: Ein moglicher Mechanismus zur Unterdruckung pflanzenparasitarer Nematoden durch insektenpathogene Nematoden - Es wird ein moglicher Mechanismus zur Unterdruckung des pflanzenparasitaren Nematoden Meloidogyne incognita durch insektenpathogene Nematoden beschrieben. Durch Hitze abgetotete insektenpathogene Nematoden Steinernema feltiae und S. riobrave underdruckten das Eindringen des Wurzelgallenalchens M. incognita in Tomatenwurzeln, lebende Nematoden hatten keine Wirkung. Infektionsjuvenile von M. incognita wurden von allen Behandlungen mit insektenpathogenen Nematoden abgestossen, die auch die symbiontischen Bakterien einschlossen. Sie wurden durch die Kadaver von Galleria mellonella abgestossen, die mit S. carpocapsae, S. feltiae und S. riobrave infiziert waren sowie durch zellfreie Kultursubstrate der symbiontischen Bakterien Xenorhabdus nematophilus, X. bovienii und Xenorhabdus sp. "R" aus den drei genannten Nematodenarten. Zellfreie Kultursubstrate von allen drei Xenorhabdus spp. waren giftig fur die Infektionsjuvenilen von M. incognita und verursachten in einer Konzentration von 15% Abtotungsraten von 98-100%. Zellfreie Kultursubstrate von Xenorhabdus sp. "R" vermiderten ausserdem das Schlupfen von M. incognita-Eiern. In einem Gewachshausversuch unterdruckten formulierte zellfreie Bakterienfiltrate vorubergehend das Eindringen von M. incognita in Tomatenwurzeln. Die Kurzzeitwirkungen von zellfreien Bakterien filtraten, namentlich Giftigkeit und Abstossung, waren nahezu ganz bedingt durch Ammoniak. Diese Ergebnisse zeigen das Vorhandensein von allelopathischen Wechselwirkungen zwischen pflanzenparasitaren Nematoden, insektenpathogenen Nematoden und deren symbiontischen Bakterien. Die wahrscheinliche Rolle von Allelopathie bei der Unterdruckung pflanzenparasitarer Nematoden durch eine Massenanwendung insektenpathogener Nematoden wird diskutiert.

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First Report of the Root-Knot Nematode Meloidogyne hapla Parasitizing Roses in Ethiopia

Plant Disease ◽

10.1094/pdis-04-14-0383-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1286-1286 ◽

Cited By ~ 5

Author(s):

Beira H. Meressa ◽

H. Heuer ◽

H.-W. Dehne ◽

J. Hallmann

Keyword(s):

Addis Ababa ◽

Sequence Similarity ◽

Infected Plant ◽

Export Market ◽

Economic Losses ◽

Parasitic Nematodes ◽

Morphological Identification ◽

Meloidogyne Hapla ◽

Root Knot Nematode ◽

Damage Potential

Meloidogyne hapla is one of the most damaging plant-parasitic nematodes in temperate regions. This nematode has a wide host range with more than 500 plant taxa including roses. In Ethiopia, rose production has developed over the past 10 years to the second most important export market after coffee. Considering the high damage potential of M. hapla, infestation of roses in Ethiopia with this nematode could result in major economic losses. Therefore, awareness of this nematode species is extremely important. During two surveys conducted in August 2011 and April 2012, M. hapla was detected in soil samples from six out of nine rose producing farms located in the districts of Ziway, Holleta, Sebeta, and Menagesha. At infested farms, rose plants appeared stunted and less productive and often showed symptoms of chlorosis and wilting. Identification was based on morphological and morphometrical characters of females, males, and second-stage juveniles, which were all within the range of variability known for this species (4). Shape of juvenile stylet knobs, shape of male head, and perennial pattern of the females with characteristic punctuations between the anus and tail terminus were also typical for M. hapla. The morphological identification was confirmed by sequence analysis of the D2-D3 expansion segment of the 28S rDNA gene following amplification with the primers D2A (5′-ACAAGTACCGTGAGGGAAAGTT-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (1). PCR products were purified and sequenced at the Macrogene sequencing facility service (Amsterdam, The Netherlands). Sequences were deposited in GenBank (KJ645427 to 33). The sequences were compared with previously published sequences in NCBI database and showed 96 to 100% sequence similarity with M. hapla accession nos. GQ130139, DQ328685, KF430798, and DQ145641. Unfortunately, comparison of sequences did not provide further information about the origin of this Ethiopian population, if it is native to Ethiopia or was imported with infected plant material. To the best of our knowledge, this is the first record of M. hapla occurring in Ethiopia. M. hapla is known as a serious pest of roses in colder climate regions. In Africa, it was previously reported from Tanzania (3) and South Africa (2). Thus, it appears that this species has now become also established in Ethiopia at higher altitudes (1,400 to 2,100 m above sea level) within the urban hinterland of Addis Ababa. References: (1) Baldwin et al. Mol. Phy. Evol. 8:248, 1887. (2) J. H. O'Bannon. Institute Agri. Res. 29, 1975. (3) E. Onkendi and L. N. Moleleki. Eur. J. Pl. Pathol. 136:1, 2013. (4) A. G. Whitehead. Trans. Zool. Soc. Lon.31:263, 1968.

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The impact of certain flavourings and preservatives on the survivability of eggs of Ascaris suum and Trichuris suis

Regulatory Mechanisms in Biosystems ◽

10.15421/022053 ◽

2020 ◽

Vol 11 (2) ◽

pp. 344-348

Author(s):

O. O. Boyko ◽

V. V. Brygadyrenko

Keyword(s):

Benzoic Acid ◽

Formic Acid ◽

Negative Impact ◽

Ascaris Suum ◽

Food Additives ◽

Antibacterial Properties ◽

Parasitic Nematodes ◽

Trichuris Suis ◽

The Impact ◽

Eggs And Larvae

The article describes a laboratory study of nematocidal properties of flavourings with antibacterial effect against Ascaris suum (Goeze, 1782) and Trichuris suis Schrank, 1788. In the experiments, eight concentrations of food additives with antibacterial properties were used: cinnamaldehyde, benzoic acid, formic acid, linalool, citral, β-ionone. Minimum LC50 value for eggs of A. suum was observed while using cinnamaldehyde and benzoic acid – 1.62 ± 0.37% and 1.69 ± 0.14%, and for eggs of T. suis – 0.57 ± 0.03% and 1.80 ± 0.11% respectively. The lowest influence on the development of eggs of nematodes of pigs’ A. suum and T. suis was exerted by formic acid, linalool, citral and β-ionone. In eggs of A. suum and T. suis, larvae formed in 21 and 50 days even during exposure to 3% emulsions of these substances. The strongest negative impact on the eggs of parasitic nematodes was displayed by cinnamaldehyde flavouring. Further study on nematocidal properties of flavourings, as well as their mixtures, would contribute to the development of preparations which would have a strong effect on eggs and larvae of nematodes of animals and humans.

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Single pass cDNA sequencing - a powerful tool to analyse gene expression in preparasitic juveniles of the southern root-knot nematode Meloidogyne incognita

Nematology ◽

10.1163/156854101750236259 ◽

2001 ◽

Vol 3 (2) ◽

pp. 129-139 ◽

Cited By ~ 38

Author(s):

Jaap Bakker ◽

Fred Gommers ◽

Geert Smant ◽

Pierre Abad ◽

Marie-Noëlle Rosso ◽

...

Keyword(s):

Gene Expression ◽

Cdna Library ◽

Meloidogyne Incognita ◽

Sequence Data ◽

Homo Sapiens ◽

Large Fraction ◽

Nematode Species ◽

Cdna Libraries ◽

Parasitic Nematodes ◽

Root Knot Nematode

AbstractExpressed sequence tags (EST) have been widely used to assist in gene discovery in various organisms (e.g., Arabidopsis thaliana, Caenorhabditis elegans, Mus musculus, and Homo sapiens). In this paper we describe an EST project, which aims to investigate gene expression in Meloidogyne incognita at the onset of parasitism. Approximately 1000 5′-end sequence tags were produced from a cDNA library made of freshly hatched preparasitic second stage juveniles (J2). The EST were identified in the primary transformants of the cDNA library, and assigned to nine different functional groups, including (candidate) parasitism genes. A large fraction of the EST (45%) did not have a putative homologue in public databases. Sixty five percent of the EST that could be clustered into a functional group had putative homologues in other nematode species. EST were found for virtually all parasitism related genes that have been cloned from M. incognita to date. In addition, several novel genes were tagged, including a xylanase and a chitinase gene. The efficiency of EST projects, which produce sequence data for thousands of genes in months time without any difficult pre-selections of mRNA pools, makes random sequencing cDNA libraries a superior method to identify candidates for parasitism related genes in plant-parasitic nematodes. The sequences in this paper are retrievable from Genbank with the accession numbers BE191640 to BE191741, BE217592 to BE217720, BE225324 to BE225598, BE238852 to BE239221, and BE240829 to BE240865.

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FOXO regulates RNA interference in Drosophila and protects from RNA virus infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1517124112 ◽

2015 ◽

Vol 112 (47) ◽

pp. 14587-14592 ◽

Cited By ~ 33

Author(s):

Michael J. Spellberg ◽

Michael T. Marr

Keyword(s):

Rna Interference ◽

Virus Infection ◽

Gene Silencing ◽

Small Rna ◽

Stress Responses ◽

Posttranscriptional Regulation ◽

Rna Virus ◽

Regulation Of Gene Expression ◽

Cellular Physiology ◽

Metabolic Conditions

Small RNA pathways are important players in posttranscriptional regulation of gene expression. These pathways play important roles in all aspects of cellular physiology from development to fertility to innate immunity. However, almost nothing is known about the regulation of the central genes in these pathways. The forkhead box O (FOXO) family of transcription factors is a conserved family of DNA-binding proteins that responds to a diverse set of cellular signals. FOXOs are crucial regulators of cellular homeostasis that have a conserved role in modulating organismal aging and fitness. Here, we show that Drosophila FOXO (dFOXO) regulates the expression of core small RNA pathway genes. In addition, we find increased dFOXO activity results in an increase in RNA interference (RNAi) efficacy, establishing a direct link between cellular physiology and RNAi. Consistent with these findings, dFOXO activity is stimulated by viral infection and is required for effective innate immune response to RNA virus infection. Our study reveals an unanticipated connection among dFOXO, stress responses, and the efficacy of small RNA-mediated gene silencing and suggests that organisms can tune their gene silencing in response to environmental and metabolic conditions.

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Combinatorial in vitro RNAi of two neuropeptide genes and a pharyngeal gland gene on Meloidogyne incognita

Nematology ◽

10.1163/15685411-00002859 ◽

2015 ◽

Vol 17 (2) ◽

pp. 155-167 ◽

Cited By ~ 6

Author(s):

Prakash Banakar ◽

Amita Sharma ◽

Catherine J. Lilley ◽

Nagavara Prasad Gantasala ◽

Mukesh Kumar ◽

...

Keyword(s):

Meloidogyne Incognita ◽

Parasitic Nematodes ◽

Specific Gene ◽

Plant Parasitic Nematodes ◽

Rnai Silencing ◽

Transcript Quantification ◽

The Central Nervous System ◽

Pharyngeal Gland

Root-knot nematodes are the most economically important group of plant-parasitic nematodes. In the present study, functional validation using in vitro RNAi was carried out on Meloidogyne incognita with two FMRFamide-like peptide genes, flp-14 and flp-18, and a subventral pharyngeal gland specific gene, 16D10. It was found that RNAi silencing of each gene reduced the attraction of M. incognita at different time intervals both in combination and individually. Silencing of the genes reduced nematode infection by 23-30% and development as indicated by a reduction in the number of females by 26-62%. Reproduction was decreased by 27-73% and fecundity was decreased by 19-51%. In situ hybridisation revealed the expression of flp-18 in cells associated with the ventral and retro vesicular ganglia of the central nervous system. qRT-PCR supported the correlation between phenotypic effects of silencing with that of transcript quantification.

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Short-Term Effects of Sublethal Doses of Nematicides on Meloidogyne incognita

Phytopathology ◽

10.1094/phyto-11-18-0420-r ◽

2019 ◽

Vol 109 (9) ◽

pp. 1605-1613 ◽

Cited By ~ 10

Author(s):

Catherine L. Wram ◽

Inga A. Zasada

Keyword(s):

Population Density ◽

Meloidogyne Incognita ◽

Cultivated Plants ◽

Parasitic Nematodes ◽

Initial Population ◽

Plant Parasitic Nematodes ◽

Water Control ◽

Effective Doses ◽

Sublethal Doses ◽

Plant Parasitic

Meloidogyne species are one of the most important groups of plant-parasitic nematodes globally because of their ability to damage most cultivated plants. Although they are widespread and economically important, there are limited control measures to combat these nematodes. New nonfumigant nematicides have been discovered that have the potential to be widely utilized for the management of plant-parasitic nematodes. Because of the longer half-lives in soil and lower toxicity of new nematicides compared with traditional fumigant and nonfumigant nematicides, understanding how nematodes respond to sublethal doses of nematicides is imperative to understanding whether nematicide resistance has the potential to develop. Characterizing responses of nematodes to sublethal doses will provide the foundation for future work, such as gene expression studies. In this study, the nematicides oxamyl (Vydate), fluazaindolizine (Salibro), fluensulfone (Nimitz), and fluopyram (Velum), were evaluated to understand how sublethal doses affect the fecundity and mobility of Meloidogyne incognita second-stage juveniles (J2). Using a microwell assay system, dose-response curves for each nematicide were established for M. incognita J2. Fluopyram was the most toxic nematicide, with effective doses up to 230 times lower than that of other nematicides. The other nematicides had predicted ED50 values (effective doses that resulted in 50% of the population becoming inactive) of 89.4, 131.7, and 180.6 ppm for oxamyl, fluensulfone, and fluazaindolizine, respectively. The 24-h ED50 of each nematicide was then used in both motility and infectivity assays. The motility and activity of M. incognita J2 exposed to ED50 doses of fluazaindolizine and fluensulfone was significantly reduced, with nematodes initially being motile but eventually becoming inactive. However, the motility of M. incognita J2 exposed to ED50 doses of fluopyram and oxamyl was not different from a water control. In a pot assay, M. incognita J2 exposed to ED50 doses of fluazaindolizine, oxamyl, and fluensulfone were unable to reproduce on tomato, with reproduction factors (RF = final population density/initial population density) of 0 to 0.03. Fluopyram did not reduce reproduction of M. incognita, with a mean RF of 38.7 ± 4.5, which was similar to the RF of 46.3 ± 4.6 for the water control. This study is the first comprehensive evaluation of M. incognita activity, motility, and fecundity after exposure to the traditional nematicide, oxamyl, as well as three new nematicides, fluazaindolizine, fluopyram, and fluensulfone.

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Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽

10.1017/s0031182019001689 ◽

2019 ◽

Vol 147 (8) ◽

pp. 855-864

Author(s):

Collette Britton ◽

Roz Laing ◽

Eileen Devaney

Keyword(s):

Gene Expression ◽

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Parasitic Nematodes ◽

Small Rna Sequencing ◽

Small Interfering Rnas ◽

Rna Sequences ◽

C Elegans

AbstractSmall RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

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