FMRFamide-like Immunoactivity in Heterodera glycines (Nemata: Tylenchida)

Material antigenically related to the neuromodulatory peptide FMRFamide was detected and examined in preparations of the soybean cyst nematode, Heterodera glycines, and in the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. FMRFamide-related peptides were quantified by an enzyme-linked immunosorbent assay. Specific activities were remarkably similar among all of the vermiform members of the three species. FMRFamide-related peptide immunoactivity was present in both sexes and all stages of H. glycines examined. The highest specific activity was present in second-stage juveniles and in males, and the lowest in white and yellow females. Total FMRFamide-related peptide level per individual was highest in brown females, with 90% of the activity associated with the eggs. Peptide levels in these eggs and in second-stage juveniles were comparable and increased in adults, especially in females. Chromatographic analysis of FMRFamide-related peptide preparations from H. glycines juveniles, C. elegans, and P. redivivus revealed distinct qualitative differences between the infective plant parasite and the free-living nematodes.     

Regulation of nematode behavior by physical means

Application of microwave radiation, laser microbeam irradiation, and short pulses of electric current stimulates changes in normal swimming behavior of the free-living nematode Panagrellus silusiae. The altered behavior, termed activated behavior, involves increased movement of the ends of the nematode, rapid changes in orientation, and an increase in the proportion of the swimming time that the nematode is curved such that the ends are pointing in the same direction. Laser microbeam studies indicate that the receptor for mating attraction is situated on the spicule. Panagrellvs silusiae shows mating attraction with two strains of P. redivivus, but the P. redivivus strains only attract P. silusiae or the other P. redivivus strain. Experiments with pulses of electric current indicate that the nematodes can be conditioned and that this conditioning requires RNA and protein synthesis.     

Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome

Expression profiling of the 5′ ends of uncapped mRNAs (“degradome” sequencing) can be used to empirically catalog microRNA (miRNA) targets, to probe patterns of miRNA hairpin processing, to examine mRNA decay, and to analyze accumulation of endogenous short interfering RNA (siRNA) precursors. We sequenced and analyzed the degradome of the moss Physcomitrella patens, an important model system for functional genomic analyses in plant evolution. A total of 52 target mRNAs of 27 different Physcomitrella miRNA families were identified. Many targets of both more conserved and less conserved miRNA families encoded putative regulatory proteins. Remnants of MIRNA hairpin processing also populated the degradome data and indicated an unusual “loop-first” mode of precise processing for the MIR319 gene family. Precise loop-first processing was confirmed for native Physcomitrella, rice, and Arabidopsis MIR319 hairpins, as well as an Arabidopsis artificial MIRNA (aMIRNA) based upon a MIR319 backbone. MIR319 is thus a conserved exception to the general rule of loop-last processing of MIRNA hairpins. Loop-first MIR319 processing may contribute to the high efficacy of a widely used MIR319-based strategy for aMIRNA production in plants.     

Identification,expression, and molecular evolution of microRNAs in the “living fossil” Triops cancriformis (tadpole shrimp)

MicroRNAs have been identified and analyzed in various model species, but an investigation of miRNAs in nonmodel species is required for a more complete understanding of miRNA evolution. In this study, we investigated the miRNAs of the nonmodel species Triops cancriformis (tadpole shrimp), a “living fossil,” whose morphological form has not changed in almost 200 million years. Dramatic ontogenetic changes occur during its development. To clarify the evolution of miRNAs, we comparatively analyzed its miRNAs and the components of its RNAi machinery. We used deep sequencing to analyze small RNA libraries from the six different developmental stages of T. cancriformis (egg, first–fourth instars, and adult), and also analyzed its genomic DNA with deep sequencing. We identified 180 miRNAs (87 conserved miRNAs and 93 novel candidate miRNAs), and deduced the components of its RNAi machinery: the DICER1, AGO1–3, PIWI, and AUB proteins. A comparative miRNA analysis of T. cancriformis and Drosophila melanogaster showed inconsistencies in the expression patterns of four conserved miRNAs. This suggests that although the miRNA sequences of the two species are very similar, their roles differ across the species. An miRNA conservation analysis revealed that most of the conserved T. cancriformis miRNAs share sequence similarities with those of arthropods, although T. cancriformis is called a “living fossil.” However, we found that let-7 and DICER1 of T. cancriformis are more similar to those of the vertebrates than to those of the arthropods. These results suggest that miRNA systems of T. cancriformis have evolved in a unique fashion.     

Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.     

A Nematode Growth Factor from Baker's Yeast

An extract prepared from commercially available yeast supported maturation of the free-living nematode Caenorhabditis briggsae. The extract can be used to supplement a chemically defined medium or, after a limited dialysis, as a complete medium. Several biologically active fractions were prepared; those containing larger amounts of ribonucleic acid (RNA) had greater biological activity, the most active being a pellet resuspended after centrifugation at 30,000 × g for 30 min. This fraction could be substituted for serum in a medium which supports the maturation of the animal parasites Trichinella spiralis and Hymenolepis nana. Addition of protamine sulfate decreased the RNA content, leaving inactive protein fractions which could be reactivated by specific treatments that caused protein precipitation. It is postulated that biological activity is associated with protein sedimented with ribosomes.     

Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants

Background and Aims

The OVATE gene encodes a nuclear-localized regulatory protein belonging to a distinct family of plant-specific proteins known as the OVATE family proteins (OFPs). OVATE was first identified as a key regulator of fruit shape in tomato, with nonsense mutants displaying pear-shaped fruits. However, the role of OFPs in plant development has been poorly characterized.

Methods

Public databases were searched and a total of 265 putative OVATE protein sequences were identified from 13 sequenced plant genomes that represent the major evolutionary lineages of land plants. A phylogenetic analysis was conducted based on the alignment of the conserved OVATE domain from these 13 selected plant genomes. The expression patterns of tomato SlOFP genes were analysed via quantitative real-time PCR. The pattern of OVATE gene duplication resulting in the expansion of the gene family was determined in arabidopsis, rice and tomato.

Key Results

Genes for OFPs were found to be present in all the sampled land plant genomes, including the early-diverged lineages, mosses and lycophytes. Phylogenetic analysis based on the amino acid sequences of the conserved OVATE domain defined 11 sub-groups of OFPs in angiosperms. Different evolutionary mechanisms are proposed for OVATE family evolution, namely conserved evolution and divergent expansion. Characterization of the AtOFP family in arabidopsis, the OsOFP family in rice and the SlOFP family in tomato provided further details regarding the evolutionary framework and revealed a major contribution of tandem and segmental duplications towards expansion of the OVATE gene family.

Conclusions

This first genome-wide survey on OFPs provides new insights into the evolution of the OVATE protein family and establishes a solid base for future functional genomics studies on this important but poorly characterized regulatory protein family in plants.     

Operons Are a Conserved Feature of Nematode Genomes

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and “spliced leader” (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.     

Characterization of Aminopeptidase in the Free-living Nematode Panagrellus redivivus: Subcellular Distribution and Possible Role in Neuropeptide Metabolism

Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide. Subcellular distribution of activity was 80% soluble and 20% membrane-associated. Aminopeptidases in the two fractions differed in affinity for Ala-4-NA, with Km''s of 0.65 mM (soluble) and 2.90 mM (membrane). Specific activities (units/mg) at pH 7.8, 27°C were 9.10 (soluble) and 14.30 (membrane). Each enzyme was competitively inhibited by amastatin (90% at 100 μM inhibitor, IC₅₀ = 3.7 μM) and inhibited by puromycin (30% at 500 μM) and 1,10-phenanthroline (IC_50''s:; 148 μM, soluble; 89 μM, membrane). Activity was restored by Zn⁺⁺, with maximum recoveries of 50% (soluble) and 90% (membrane), each at 23 μM ZnCl₂. Estimated molecular masses for each were ∼150 kDa. FMRFamide-like neuropeptides behaved as competitive inhibitors. Modification of the N-terminal F of FMRFamide weakened inhibition by 95%, suggesting that the N-terminus is essential for binding to the enzyme. Two nematode FMRFamides, APKPFIRFa and RNKFEFIRFa, were the most potent tested. This is the first biochemical characterization of aminopeptidase in a free-living nematode other than Caenorhabditis elegans and demonstrates the high selectivity of the P. redivivus enzymes for neuropeptide substrates.     

Nematicidal Activity of α -Chaconine: Effect of Hydrogen-ion Concentration

α-Chaconine, a steroid-glycoalkaloid from Solanum tuberosum L., was increasingly more toxic to a free-living nematode, Panagrellus redivivus, with decreasing acidity from about pH 5 to 7. A study of the toxicity to adult nematodes at three concentrations of α-chaconine in buffer from pH 4 to 7.5 indicated that the free base is the nematicidal form of the compound. The median effective doses (ED₅₀) of α-chaconine to inhibit the motility of P. redivivus were estimated as 85 μg/ml at pH 6.7, 170 μg/ml at pH 6.5, and 340 μg/ml at pH 6.2.     

Complete genome sequence of Halorhabdus utahensis type strain (AX-2)

Halorhabdus utahensis Wainø et al. 2000 is the type species of the genus, which is of phylogenetic interest because of its location on one of the deepest branches within the very extensive euryarchaeal family Halobacteriaceae. H. utahensis is a free-living, motile, rod shaped to pleomorphic, Gram-negative archaeon, which was originally isolated from a sediment sample collected from the southern arm of Great Salt Lake, Utah, USA. When grown on appropriate media, H. utahensis can form polyhydroxybutyrate (PHB). Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the a member of halobacterial genus Halorhabdus, and the 3,116,795 bp long single replicon genome with its 3027 protein-coding and 48 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Molecular Characterization of Aldolase from Heterodera glycines and Globodera rostochiensis

Fructose-bisphosphate aldolase (EC 4.1.2.13) is a key enzyme in glycolysis. We have characterized full-length coding sequences for aldolase genes from the cyst nematodes Heterodera glycines and Globodera rostochiensis, the first for any plant-parasitic nematode. Nucleotide homology is high (83% identity), and the respective sequences encode 40 kDa proteins with 89% amino acid identity. Genomic sequences contain six introns located at identical positions in both genes. Intron 4 in the H. glycines gene is >500 bp. Partial genomic sequences determined for seven other cyst nematode species reveal that the large fourth intron is characteristic of Heterodera but not Globodera aldolase genes. Total aldolase-like specific activity in homogenates from H. glycines was 2-fold lower than in either Caenorhabditis elegans or Panagrellus redivivus (P = 0.001). Activity in H. glycines samples was higher in juvenile stages than in adults (P = 0.003). Heterodera glycines aldolase has Km = 41 µM and is inhibited by treatment with carboxypeptidase A or sodium borohydride.     

Acanthocytes of Stropharia rugosoannulata Function as a Nematode-Attacking Device

Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.     

Endogenous siRNAs and piRNAs derived from transposable elements and genes in the malaria vector mosquito Anopheles gambiae

Background

The siRNA and piRNA pathways have been shown in insects to be essential for regulation of gene expression and defence against exogenous and endogenous genetic elements (viruses and transposable elements). The vast majority of endogenous small RNAs produced by the siRNA and piRNA pathways originate from repetitive or transposable elements (TE). In D. melanogaster, TE-derived endogenous siRNAs and piRNAs are involved in genome surveillance and maintenance of genome integrity. In the medically relevant malaria mosquito Anopheles gambiae TEs constitute 12-16% of the genome size. Genetic variations induced by TE activities are known to shape the genome landscape and to alter the fitness in An. gambiae.

Results

Here, using bioinformatics approaches we analyzed the small RNA data sets from 6 libraries formally reported in a previous study and examined the expression of the mixed germline/somatic siRNAs and piRNAs produced in adult An. gambiae females. We characterized a large population of TE-derived endogenous siRNAs and piRNAs, which constitutes 56-60% of the total siRNA and piRNA reads in the analysed libraries. Moreover, we identified a number of protein coding genes producing gene-specific siRNAs and piRNAs that were generally expressed at much lower levels than the TE-associated small RNAs. Detailed sequence analysis revealed that An. gambiae piRNAs were produced by both “ping-pong” dependent (TE-associated piRNAs) and independent mechanisms (genic piRNAs). Similarly to D. melanogaster, more than 90% of the detected piRNAs were produced from TE-associated clusters in An. gambiae. We also found that biotic stress as blood feeding and infection with Plasmodium parasite, the etiological agent of malaria, modulated the expression levels of the endogenous siRNAs and piRNAs in An. gambiae.

Conclusions

We identified a large and diverse set of the endogenously derived siRNAs and piRNAs that share common and distinct aspects of small RNA expression across insect species, and inferred their impact on TE and gene activity in An. gambiae. The TE-specific small RNAs produced by both the siRNA and piRNA pathways represent an important aspect of genome stability and genetic variation, which might have a strong impact on the evolution of the genome and vector competence in the malaria mosquitoes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1436-1) contains supplementary material, which is available to authorized users.     

Coprinus comatus Damages Nematode Cuticles Mechanically with Spiny Balls and Produces Potent Toxins To Immobilize Nematodes

We reported recently a unique fungal structure, called the spiny ball, on the vegetative hyphae of Coprinus comatus (O. F. Müll.:Fr.) Pers. Although some observations regarding the role of this structure were presented, its function remained largely unknown. In this study, we showed that purified (isolated and washed) spiny balls could immobilize and kill the free-living nematode Panagrellus redivivus Goodey highly efficiently. Scanning electron microscopy studies illustrated that the spiny structure damaged the nematode cuticle, suggesting the presence of a mechanical force during the process of nematode immobilization. Severe injuries on nematode cuticles caused the leakage of inner materials of the nematodes. When these structures were ground in liquid nitrogen, their killing efficacy against nematodes was lost, indicating that the shape and the complete structure of the spiny balls are indispensable for their function. However, extraction with organic solvents never lowered their activity against P. redivivus, and the extracts showed no obvious effect on the nematode. We also investigated whether C. comatus was able to produce toxins which would aid in the immobilization of nematodes. In total, we identified seven toxins from C. comatus that showed activity to immobilize the nematodes P. redivivus and Meloidogyne incognita (Kofoid et White) Chitwood. The chemical structures of these toxins were identified with nuclear magnetic resonance, mass spectrometry, infrared, and UV spectrum analysis. Two compounds were found to be novel. The toxins found in C. comatus are O-containing heterocyclic compounds.     

Caenorhabditis elegans: A Genetic Guide to Parasitic Nematode Biology

The advent of parasite genome sequencing projects, as well as an increase in biology-directed gene discovery, promises to reveal genes encoding many of the key molecules required for nematode-host interactions. However, distinguishing parasitism genes from those merely required for nematode viability remains a substantial challenge. Although this will ultimately require a functional test in the host or parasite, the free-living nematode Caenorhabditis elegans can be exploited as a heterologous system to determine function of candidate parasitism genes. Studies of C. elegans also have revealed genetic networks, such as the dauer pathway, that may also be important adaptations for parasitism. As a more directed means of identifying parasitism traits, we developed classical genetics for Heterodera glycines and have used this approach to map genes conferring host resistance-breaking phenotypes. It is likely that the C. elegans and H. glycines genomes will be at least partially syntenic, thus permitting predictive physical mapping of H. glycines genes of interest.