Accumulation and metabolism of aldicarb by the free-living nematodes Aphelenchus avenae and Panagrellus redivivus

The accumulation and metabolism of aldicarb has been compared in two species of free-living soil nematodes, Aphelenchus avenae and Panagrellus redivivus, which differ considerably in their sensitivity to this and other pesticides. Similar levels of aldicarb were found to accumulate in the two species. The results showed that although the rate of uptake of aldicarb was greater in P. redivivus, so also was the rate of metabolism and elimination. Levels of toxic metabolites of aldicarb were two to three times higher in the more susceptible species A. avenae after 24 h incubation than in the less susceptible P. redivivus. The toxicological significance of these findings is discussed in relation to previous work with the organophosphorus pesticide phorate and its effects on the same two nematode species.     

Two FMRFamide-like peptides from the free-living nematode Panagrellus redivivus.

Peptides of the FXRFamide family, where X = M, I or L, are broadly distributed among invertebrates. Two such peptides were purified and sequenced from the free-living nematode, Panagrellus redivivus. Immunohistochemical techniques localized FMRFamide-like material in several regions of these organisms, including the nerve cords and, most prominently, in paired groups of cells located caudally to the base of the pharynx. RIA determinations gave an estimate of 2.8 nmol immunoreactive peptide/g of an acetone extract of P. redivivus. Four sequential HPLC purification steps, followed by sequencing by automated Edman degradation and FAB-MS, led to the identification of Ser-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SDPNFLRFamide) and Ser-Ala-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SADPNFLRFamide) as members of the FXRFamide family in this nematode.     

Evolution of dnmt-2 and mbd-2-like genes in the free-living nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae

Whole genome sequencing of several metazoan model organisms provides a platform for studying genome evolution. How representative are the genomes of these model organisms for their respective phyla? Within nematodes, for example, the free-living soil nematode Caenorhabditis elegans is a highly derived species with unusual genomic characters, such as a reduced Hox cluster (Curr. Biol., 13, 37–40) and the absence of a Hedgehog signaling system. Here, we describe the recent loss of a DNA methyltransferase-2 gene (dnmt-2) in C.elegans. A dnmt-2-like gene is present in the satellite model organism Pristionchus pacificus, another free-living nematode that diverged from C.elegans 200–300 million years ago. In contrast, C.elegans, Caenorhabditis briggsae and P.pacificus all contain an mbd-2-like gene, which encodes another essential component of the methylation system of higher animals and fungi. Cel-mbd-2 is expressed throughout development and RNA interference (RNAi) experiments result in variable phenotypes. In contrast, Cbr-mbd-2 RNAi results in paralyzed larval or adult worms suggesting recent changes of gene function within the genus Caenorhabditis. We speculate that both genes were part of an ancestral DNA methylation system in nematodes and that gene loss and sequence divergence have abolished DNA methylation in C.elegans.     

Stimulatory effects of peptides on growth of the free-living nematode Caenorhabditis briggsae.

Evidence is presented that peptide products of hydrolysis of casein, including some di- and tripeptides, but not the constituent amino acids, can stimulate growth of C. briggsae in defined basal medium supplemented with cytochrome C and B-sitosterol. Peptide activity may raise from amino acid imbalances in the medium which causes competitive inhibition of uptake of essential amino acids. Such activity precludes facilitation of heme transport as the sole function of growth factor in C. briggsae. This is the first report in a nutritional role of peptides in invertebrate metazoa.     

Digestion of invertebrate neuropeptides by preparations from the free-living nematode Panagrellus redivivus

Proteases in the soluble fraction of homogenates prepared from the free-living nematode Panagrellus redivivus hydrolysed the amidated invertebrate neuropeptides FMRFa and FLRFa, and nematode FMRFa-like peptides (FLPs) KPNFLRFa (FLP-1-H), APKPKFIRFa (FLP-5-A), KNEFIRFa (FLP-8), KPSFVRFa (FLP-9), RNKFEFIRFa (FLP-12) and KHEYLRFa (FLP-14) in vitro. Results were assessed by analysing reaction components with RP-HPLC, UV detection at 210 nm and peak integration. Based upon substrate peak size, more than 90% of most of the peptide substrates was consumed after 1 h at 27 degrees C, but digestion was not complete even with a crude protease mixture. Two peptides, FLP-12 and FLP-14, were significantly less susceptible to digestion than the others. FLP-12 was the least susceptible of all sequences (71% loss; P < 0.0001), while FLP-14 was digested less (84% loss; P < 0.0004) than all but FLP-12. Product peak digestion patterns of FLP-12, a second nonapeptide (FLP-5-A), and FMRFa, incubated with aminopeptidase (amastatin) and serine endoprotease (AEBSF) inhibitors, demonstrated highly specific behaviours of each sequence to protease cleavage. Amastatin significantly (P < 0.03) reduced digestion of FLP-12 (54% loss) and FMRFa (61% loss; P < 0.0005), but had no effect on FLP-5-A. AEBSF had no protective effect on FMRFa but significantly decreased hydrolysis of FLP-5-A (77% loss; P < 0.0001) and FLP-12 (59% loss; P < 0.03). The combination of both inhibitors had additive effects only for FMRFa (34% loss; P < 0.0005). Further analysis of FMRFa digestion using peptides with D-amino acid substitutions demonstrated nearly complete protection of FdMRFa (2% loss; P < 0.0001) from all proteolytic digestion, whereas digestion of FMRdFa was complete. Results suggest that in addition to aminopeptidase and serine proteases, both deamidase and aminopeptidase P participate in neuropeptide metabolism in P. redivivus.     

Glutamine and asparagine synthesis in the nematodes Heligmosomoides polygyrus and Panagrellus redivivus

Low levels of glutamine synthetase were demonstrated in Panagrellus redivivus and Heligmosomoides polygyrus. Asparagine synthetase was detected in P. redivivus but activity was too low to be detected in H. polygyrus.     

Freeze-thaw survival of the free-living nematode Caenorhabditis briggsae.

Approximately 75% or more of the L2 and L3 juvenile stages of the free-living nematode Caenorhabditis briggsae survived freezing and thawing without loss of fertility. Optimum survival depended upon a combination of conditions: (1) pretreatment with 5% DMSO at 0 °C for 10 min, (2) 0.2 °C per minute cooling rate from 0 to ?100 °C prior to immersion into liquid nitrogen, and (3) a 27.6 °C per minute warming rate from ?196 °C to ?10 °C. Storage at ?196 °C for more than 100 days was without effect on viability or fertility. Some of the L4 (about 50%) and adult (about 3%) stages survive the routine freeze-thaw treatment. However, there was no recovery of either embryonic stages or embryonated eggs from ?196 °C under these standard conditions. Either very fast cooling (about 545 °C/min) or fast warming (about 858 °C/min) rates diminished survival of the L2 and L3 stages drastically.Scanning electron microscopy revealed that freeze-thaw survivors with aberrant swimming behavior had cuticular defects. In juvenile forms, the altered swimming motion was lost after a molt whereas as abnormal adults grew, sinusoidal movement resumed. In the L4 and adult forms the cuticular abnormalities lowered viability and fertility. It is concluded that survival of nematodes from a freeze-thaw cycle is contingent upon establishing specific cryobiological conditions by varying aspects of the procedure that gave high recoveries of L2 and L3 stages.     

In vitro studies on the functional maturation and locomotion of sperm in a free-living nematode, Panagrellus redivivus

In the seminal vesicle of male Panagrellus redivivus the sperm are normally rounded, non-motile and have cytoplasmic organelles randomly scattered throughout the whole cell body. Sperm become amoeboid in the uterus of the female with a clear anterior region capable of producing pseudopodia and an arch-shaped rigid posterior region containing numerous organelles. The sperm arrange themselves in the form of a chain in the uterus attaching themselves anterio-posteriorly, however sperm entering the post-vulvar uterine sac do not form a chain and remain scattered. Approximately eight hours after insemination the sperm in the uterus stop producing pseudopodia. Pseudopodial formation recommences in the seven to eight anteriormost sperm in the chain as they reach seminal receptacle.     

Panagrellus redivivus and Caenorhabditis elegans: evidence for the absence of sialic acids

Complementary experiments were performed to indicate the presence or absence of sialic acids in axenically cultured Panagrellus redivivus and Caenorhabditis elegans. Competitive displacement experiments with radiolabeled Limax flavus agglutinin demonstrated the presence of sialic acid in nematodes grown in medium which contained liver extract as a growth factor but the absence of sialic acid when heme was substituted for liver extract. This finding suggested that sialic acid present in the liver medium was responsible for conflicting results of other studies. Transmission electron microscopy of thin sections from nematodes labeled with an LFA-ferritin conjugate revealed no label to the surface area of the cephalic chemosensilla. Fluorometric analysis with a modification of the thiobarbituric acid assay was negative for sialic acid. Analyses by gas chromatography-mass spectrometry, sensitive to the high picomole range, were also negative for sialic acid. Taken together the results provide evidence for the absence of sialic acid in P. redivivus and C. elegans using the most sensitive and diagnostic technique currently available.     

The suitability of the free-living nematode Panagrellus redivivus as live food for first-feeding fish larvae

The free‐living nematode Panagrellus redivivus has been recommended as a suitable food source for first‐feeding fish. A new technology for mass production of P. redivivus enables fish hatchery operators to rely on an inexpensive, standardized and permanently available live food for first‐feeding fish larvae. The proximate composition, and the fatty acid and amino acid profiles of nematodes mass produced on oat‐based and purified ingredient media were determined. The quality of nematodes was significantly influenced by the culture medium used. The lipid content and fatty acid composition of nematodes could be modified by using lipid‐enriched media. Mass‐produced nematodes were tested on first‐feeding common carp (Cyprinus carpio L.) and whitefish (Coregonus lavaretus) larvae. Carp larvae, grown on nematodes cultured on oat medium enriched with sunflower oil, showed a higher survival rate (87.1%) than the control group fed frozen zooplankton (82.9%) at the end of the 1‐week feeding experiment. Differences in larval mass between the treatments disappeared after subsequent feeding of a dry diet for 2 weeks. Whitefish larvae can be reared exclusively on a dry diet; here, the initial feeding of nematodes had no effect on final biomass and survival of larvae.     

24-Methyl-23-dehydrocholesterol: a new sterol intermediate in C-24 demethylation from the nematodes Panagrellus redivivus and Caenorhabditis elegans?

Panagrellus redivivus produced 24-methyl-23-dehydrocholesterol as 4.0% of the 4-desmethylsterols when propagated in a medium containing campesterol as the dietary sterol. The re-examination of previous data revealed that Caenorhabditis elegans produced 1.8% 24-methyl-23-dehydrocholesterol when propagated in medium containing campesterol. 24-Methyl-23-dehydrocholesterol was not detected when the nematodes were propagated in medium containing 22-dihydrobrassicasterol or 24-methylenecholesterol. This may be a result of the greater efficiency of dealkylation of the latter two sterols. This is the first report of the natural occurrence of this sterol in a non-photosynthetic organism, and the first report in organisms that dealkylate 24-alkylsterols.     

Sex and tissue specific patterns of protein synthesis and turnover in the free-living nematode Panagrellus redivivus

• 1.1. A pulse-chase autoradiographic study of incorporation of ³H-leucine into body-wall, digestive and reproductive tissue of virgin adult Panagrellus redivivus demonstrates both tissue and sex-specific patterns of protein biosynthesis and turnover.
• 2.2. Female body wall has a higher rate of protein synthesis and turnover than male body wall.
• 3.3. Uptake of leucine into macromolecule in the digestive tissue is more rapid in males than females, as is the rate of turnover.
• 4.4. The ovary is more rapidly labelled than the testis, with a much more rapid turnover rate.

     

Growth Inhibition of Caenorhabditis elegans and Panagrellus redivivus by Selected Mammalian and Insect Hormones

Caenorhabditis elegans and Panagrellus redivivus were cultured in axenic medium in microwells. The addition of selected steroids and terpenoids to the medium caused quantitative inhibition of numbers of offspring produced per well. Three out of 14 vertebrate sex hormones and analogs, and seven out of 10 insect juvenile hormones and analogs inhibited growth at 25 or 50 micrograms per ml. In addition, two insecticide synergists which mimic juvenile hormones, propyl 2-propynyl phenyl phosphonate and piperonyl butoxide, inhibited growth at 7 μg/ml. Total lipids from Panagrellus and from Nematospiroides dubius were inhibitory. Separation by silicic acid column chromatography yielded active and inactive portions. We concluded that the inhibition observed was non-specific.     

Aminopeptidases in Caenorhabditis elegans and Panagrellus redivivus: detection using peptide and non-peptide substrates

Aminopeptidase activities were detected in extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus using the aminoacyl substrate L-alanine-4-nitroanilide. The activities exhibited similarities in Km (C. elegans = 2.22 mM; P. redivivus = 2.09 mM) and specific activity (C. elegans = 1.38 +/- 0.43 mAU min(-1) x g(-1); P. redivivus, 1.23 +/- 0.18m AU min(-1) microg(-1). Each is inhibited competitively by amastatin (C. elegans IC50 = 0.46 microM; P. redivivus IC50 = 15.90 microM) and non-competitively by leuhistin (C. elegans IC50 = 3.00 microM; P. redivivus IC50 = 37.35 microM). The bioactive peptides adipokinetic hormone and substance P decrease the apparent aminopeptidase activities of each extract suggesting that the peptides compete with the Ala-pNA as substrates. With each extract, adipokinetic hormone appeared to be the more effective substrate. Digestion of adipokinetic hormone by C. elegans and P. redivivus extracts in the presence and absence of 1 mM amastatin produced distinct chromatographic profiles that suggest different digestion patterns for the two species. However, amastatin had clear effects on chromatographic profiles from each species indicating that an aminopeptidase is involved in the digestion of the peptide substrates. The data presented indicate that extracts of free-living nematodes are capable of metabolizing peptide hormones, and that this metabolism involves substrate-selective aminopeptidases.     

Prediction of structured non-coding RNAs in the genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae

We present a survey for non-coding RNAs and other structured RNA motifs in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae using the RNAz program. This approach explicitly evaluates comparative sequence information to detect stabilizing selection acting on RNA secondary structure. We detect 3,672 structured RNA motifs, of which only 678 are known non-translated RNAs (ncRNAs) or clear homologs of known C. elegans ncRNAs. Most of these signals are located in introns or at a distance from known protein-coding genes. With an estimated false positive rate of about 50% and a sensitivity on the order of 50%, we estimate that the nematode genomes contain between 3,000 and 4,000 RNAs with evolutionary conserved secondary structures. Only a small fraction of these belongs to the known RNA classes, including tRNAs, snoRNAs, snRNAs, or microRNAs. A relatively small class of ncRNA candidates is associated with previously observed RNA-specific upstream elements.     

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.