Individual Worms Research Articles

A scanning electron microscope technique for studying the sclerites of Cichlidogyrus.

The genus Cichlidogyrus (Monogenea: Ancyrocephalidae) includes more than 90 species, most of which are gill parasites of African cichlid fishes. Cichlidogyrus has been studied extensively in recent years, but scanning electron microscope (SEM) investigations of the isolated hard parts have not yet been undertaken. In this paper, we describe a method for isolating and scanning the sclerites of individual Cichlidogyrus worms. Twenty-year-old, formol-fixed specimens of Cichlidogyrus casuarinus were subjected to proteinase K digestion in order to release the sclerites from the surrounding soft tissues. SEM micrographs of the haptoral sclerites and the male copulatory organ are presented. The ability to digest formol-fixed specimens makes this method a useful tool for the study of historical museum collections.

DNA barcoding of earthworms (Eisenia fetida/andrei complex) from 28 ecotoxicological test laboratories

Plenty of evidence indicates that the earthworms Eisenia fetida (Savigny, 1826) and Eisenia andreiBouché, 1972 (Lumbricidae) can be distinguished by morphological, physiological and molecular traits. However, the morphological differences alone do not allow to correctly identify these taxa. This may be a serious problem for the reliability of the standard ecotoxicological tests for which these worms are used. Recently, DNA barcoding (i.e., sequencing of a standard mtDNA COI gene fragment for identification purposes) has been proposed as a quick and simple method to identify species, including earthworms. In order to assess the practicability and reliability of this method, an international ring test was organized by the “Eisenia barcoding Initiative (EBI)”, a group of scientists from four public institutions and two contract laboratories. Coded samples of E. fetida,E. andrei, and Eisenia sp. were provided by 28 ecotoxicological laboratories from 15 countries on four continents. Five laboratories in Belgium, Canada, Germany, and Spain identified the specimen through DNA barcoding. All steps of the sample preparation were described by Standard Operating Procedures (SOP). The COI sequences (581bp) obtained were used to construct a neighbor-joining tree based on the uncorrected pairwise p-distance. This analysis revealed three distinct haplotype clusters: one including only E. andrei sequences (mean within-group p-distance 0.026±0.002) and two with only E. fetida sequences, referred to as E. fetida 1 and E. fetida 2. Each of the latter two in fact represented one single haplotype. The mean p-distance between E. fetida 1 and E. fetida 2 was 0.112, whereas the mean p-distances between these two taxa and E. andrei were 0.142 and 0.143, respectively. Such COI divergence levels are usually indicative of species level differentiation. Hence it is hypothesized that E. fetida 1 and E. fetida 2 refer to different cryptic species. Since the attribution of the individual worms to these three clusters was completely consistent among the five DNA barcoding laboratories the good applicability of DNA barcoding for the identification of these ecotoxicological test species is proven. Remarkably, specimens of the molecular E. fetida clusters were always identified morphologically as E. fetida. However, this was not true the other way round, i.e., some specimens of the molecular E. andrei cluster were identified morphologically as E. fetida. The results of this ring test are presented to standardization organizations (OECD, ISO) in order to improve the standardization and thus quality of ecotoxicological routine testing by using DNA barcoding.

Therapeutic efficacy and macrofilaricidal activity of doxycycline for the treatment of river blindness.

Onchocerca volvulus and lymphatic filariae, causing river blindness and elephantiasis, depend on endosymbiotic Wolbachia bacteria for growth, development, fertility, and survival. Clinical trials have shown that doxycycline treatment eliminates Wolbachia, causing long-term sterilization of adult female filariae and effecting potent macrofilaricidal activity. The continual reinfection by drug-naive worms that occurs in these trial settings dilutes observable anti-Wolbachia and antifilarial effects, making it difficult to estimate therapeutic efficacy and compare different doxycycline regimens, evaluated at different times after treatment. A meta-analytical modeling framework is developed to link all usable data collected from clinical trials measuring the Wolbachia status and viability of individual female adult worms collected at various times after treatment with 4, 5, or 6 weeks of daily 100 or 200 mg oral doxycycline. The framework is used to estimate efficacy parameters that are not directly measurable as trial outcomes. The estimated efficacy of doxycycline (the maximum proportional reduction in the percentage of adult female O. volvulus positive for Wolbachia) is 91%-94% on average, irrespective of the treatment regimen. Efficacy is >95% in the majority of trial participants. The life span of Wolbachia-depleted worms is reduced by 70%-80%, from approximately 10 years to 2-3 years. The efficacy parameters are pertinent to the prospects of using doxycycline on a "test and treat" basis for onchocerciasis control and confirm doxycycline as a potent macrofilaricidal therapy. The modeling approach is more generally relevant to the design and evaluation of clinical trials for antifilarial drugs conducted in endemic settings.

Biomass measurement of living Lumbriculus variegatus with impedance spectroscopy

Abstract Impedance spectroscopy is a useful tool for non-invasive and real time measurements of cell suspensions and a variety of biological tissues. The objective of this study was the investigation of the dielectric properties of living aquatic worms (Lumbriculus variegatus) using impedance spectroscopy in a frequency range between 100 Hz and 10 MHz. We demonstrate a linear relation between the worm biomass and the phase response of the signal thereby providing a quick and precise method to determine the biomass of aquatic worms in situ. Possible applications for non-destructive online biomass monitoring of aquatic worms and other aqueous organisms are discussed. Furthermore, we show that groups of worms fed different diets can be distinguished by the method presented. These results reveal a close relationship between the nutritional composition of the worms and the measured phase response. We also demonstrate that the phase response at 90 kHz does not depend on the worm size. In contrast, the response function for the signal at 440 Hz reveals a linear correlation of average individual worm size and phase. Therefore, we conclude that the measured phase response at 90 kHz qualifies as a measure of the total amount of worm biomass present in the measuring cell, whereas the phase measurement at 440 Hz can be used to estimate the average individual worm size.

A fully automated microfluidic femtosecond laser axotomy platform for nerve regeneration studies in C. elegans.

Femtosecond laser nanosurgery has been widely accepted as an axonal injury model, enabling nerve regeneration studies in the small model organism, Caenorhabditis elegans. To overcome the time limitations of manual worm handling techniques, automation and new immobilization technologies must be adopted to improve throughput in these studies. While new microfluidic immobilization techniques have been developed that promise to reduce the time required for axotomies, there is a need for automated procedures to minimize the required amount of human intervention and accelerate the axotomy processes crucial for high-throughput. Here, we report a fully automated microfluidic platform for performing laser axotomies of fluorescently tagged neurons in living Caenorhabditis elegans. The presented automation process reduces the time required to perform axotomies within individual worms to ∼17 s/worm, at least one order of magnitude faster than manual approaches. The full automation is achieved with a unique chip design and an operation sequence that is fully computer controlled and synchronized with efficient and accurate image processing algorithms. The microfluidic device includes a T-shaped architecture and three-dimensional microfluidic interconnects to serially transport, position, and immobilize worms. The image processing algorithms can identify and precisely position axons targeted for ablation. There were no statistically significant differences observed in reconnection probabilities between axotomies carried out with the automated system and those performed manually with anesthetics. The overall success rate of automated axotomies was 67.4±3.2% of the cases (236/350) at an average processing rate of 17.0±2.4 s. This fully automated platform establishes a promising methodology for prospective genome-wide screening of nerve regeneration in C. elegans in a truly high-throughput manner.

First identification of the F200Y SNP in the β-tubulin gene linked to benzimidazole resistance in Ancylostoma caninum

Single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene in codons 167, 198 and 200 have been associated with benzimidazole resistance in some nematodes, although no polymorphisms in this gene have been described in the hookworm Ancylostoma caninum to date. This study aimed to screen for SNPs in the β-tubulin isotype 1 gene at codons 198 and 200 associated with benzimidazole resistance in A. caninum worms recovered from naturally infected dogs from two Brazilian states (Minas Gerais and Piauí). To perform this analysis, a molecular technique based on the amplification refractory mutation system (ARMS-PCR) was standardized. A total of 110 individual worms recovered from 9 dogs in Piauí and 124 individual worms recovered from 10 dogs in Minas Gerais were analyzed using this methodology. The presence of a SNP at position 200 was observed at a low frequency (0.8%), which was only detected in the worm population from Minas Gerais State. A total of 75 randomly selected samples were sequenced to validate this technique, and the presence of the SNP was confirmed. Furthermore, SNP at position 198 were evaluated by sequencing, and none were detected in this region. This is the first report of the presence of a SNP in the β-tubulin isotype 1 gene associated with benzimidazole resistance in an A. caninum population.

Biophysical and biological meanings of healthspan from C. elegans cohort

Lifespan among individuals ranges widely in organisms from yeast to mammals, even in an isogenic cohort born in a nearly uniform environment. Needless to say, genetic and environmental factors are essential for aging and lifespan, but in addition, a third factor or the existence of a stochastic element must be reflected in aging and lifespan. An essential point is that lifespan or aging is an unpredictable phenomenon. The present study focuses on elucidating the biophysical and biological meanings of healthspan that latently indwells a stochastic nature. To perform this purpose, the nematode Caenorhabditis elegans served as a model animal. C. elegans fed a healthy food had an extended healthspan as compared to those fed a conventional diet. Then, utilizing this phenomenon, we clarified a mechanism of healthspan extension by measuring the single-worm ATP and estimating the ATP noise (or the variability of the ATP content) among individual worms and by quantitatively analyzing biodemographic data with the lifespan equation that was derived from a fluctuation theory.

Genetically distinct isolates of Spirocerca sp. from a naturally infected red fox (Vulpes vulpes) from Denmark

Spirocerca lupi causes formation of nodules that may transform into sarcoma in the walls of aorta, esophagus and stomach of infected canids. In February 2013, post mortem examination of a red fox (Vulpes vulpes) hunted in Denmark revealed the presence of several nodules containing adult worms of Spirocerca sp. in the stomach and the omentum. The nodules largely consisted of fibrous tissue with infiltration of mononuclear cells, neutrophilic granulocytes and macrophages with hemosiderin deposition. Parasitological examination by three copromicroscopic methods, sedimentation, flotation with saturated sugar-salt solution, and sieving failed to detect eggs of Spirocerca sp. in feces collected from the colon. This is the first report of spirocercosis in Denmark, and may have been caused by a recent introduction by migrating paratenic or definitive host. Analysis of two overlapping partial sequences of the cox1 gene, from individual worms, revealed distinct genetic variation (7–9%) between the Danish worms and isolates of S. lupi from Europe, Asia and Africa. This was confirmed by phylogenetic analysis that clearly separated the Danish worms from other isolates of S. lupi. The distinct genetic differences of the current worms compared to other isolates of S. lupi may suggest the presence of a cryptic species within Spirocerca.

Molecular study of bacterial diversity within the trophosome of the vestimentiferan tubeworm Ridgeia piscesae

AbstractA large proportion of the faunal biomass in hydrothermal vent ecosystems relies on symbiotic relationships, with bacteria as a source of nutrition. Whereas multiple symbioses have been observed in diverse vent hosts, siboglinid tubeworms have been thought to harbour a single endosymbiont phylotype affiliated to the Gammaproteobacteria. In the case of the Northeast Pacific vestimentiferan Ridgeia piscesae, two previous studies suggested the presence of more than one symbiont. The possibility of multiple, and possibly habitat‐specific, symbionts in R. piscesae provided a potential explanation for the tubeworm's broad ecological niche, compared with other hydrothermal vent siboglinids. This study further explored the diversity of trophosome bacteria in R. piscesae using two methodological approaches not yet applied to this symbiosis. We carried out 454‐pyrosequencing on trophosome samples from 46 individual worms and used catalyzed reporter deposition‐fluorescence in situ hybridization (CARD‐FISH) to verify the presence of the major groups detected in the pyrotag data. Both methods yielded inconsistent and sometimes contradictory results between sampling sites, and neither provided irrefutable evidence for the presence of symbionts other than the expected Gammaproteobacteria. We therefore conclude that the other adaptive mechanisms must be considered to explain the broad physico‐chemical niche occupied by the different growth forms of R. piscesae.

Helminth parasites of freshwater fishes from Cuatro Ciénegas, Coahuila, in the Chihuahuan Desert of Mexico: inventory and biogeographical implications.

As part of an ongoing inventory of the helminth parasites of freshwater fishes in Mexico, 570 individual fish were collected between Apr 2008 and Oct 2011 in 26 localities along the Cuatro Ciénegas region in Coahuila State, northern Mexico. Seventeen species of hosts, mostly corresponding to Nearctic freshwater elements, were studied. A total of 8324 individual worms were collected during this survey, representing 25 species of helminths, of which 9 were digeneans, 3 monogeneans, 3 acanthocephalans, 9 nematodes and 1 cestode. Most of the records in this checklist represent new host or locality records. The information provided in this checklist may be helpful for our understanding of the biodiversity and historical biogeography of this host-parasite system, because in the Cuatro Ciénegas region occur a Nearctic freshwater fish fauna, along with Neotropical and endemic elements, and from a biogeographical point of view, this may represent a transitional area.

The presence of benzimidazole resistance mutations in Haemonchus placei from US cattle

Haemonchus populations were collected from cattle from mid-western and eastern Southern US (four and six populations, respectively) to determine the relative prevalence of Haemonchus contortus and Haemonchus placei and the frequency of the three isotype-1 β-tubulin polymorphisms associated with benzimidazole resistance. A minimum of 32 individual adult worms were genotyped at position 24 of the rDNA ITS-2 for each population to determine species identity (296 worms in total). One population from Georgia was identified as 100% H. contortus with the remaining nine populations identified as 100% H. placei. For the H. contortus population, 29 out of 32 worms carried the P200Y (TAC) isotype-1 β-tubulin and 2 out of 32 worms carried the P167Y (TAC) benzimidazole resistance associated polymorphisms respectively. For H. placei, six out of the nine populations contained the P200Y (TAC) isotype-1 β-tubulin benzimidazole resistance associated polymorphism at low frequency (between 1.6% and 9.4%) with no resistance associated polymorphisms being identified at the P198 and P167 codons. This is the first report of the P200Y (TAC) isotype-1 β-tubulin benzimidazole resistance associated polymorphism in H. placei. The presence of this mutation in multiple independent H. placei populations indicates the risk of resistance emerging in this parasite should benzimidazoles be intensively used for parasite control in US cattle.

Making the in vitro breeding of Schistocephalus solidus more flexible

Schistocephalus solidus is one of the few cestodes that can be bred in vitro. Worms have typically been bred in pairs, so the parents of each offspring can clearly be assigned. From a genetic perspective, it would be useful to be able to mate an individual worm to multiple partners while still being able to distinguish among different parents. As each adult S. solidus possesses numerous reproductive complexes, cutting worms and breeding the pieces separately would facilitate such breeding designs. We halved worms before in vitro breeding and evaluated whether this affected outcrossing rates and reproductive output. Cutting did not influence clutch mass, i.e. egg number and size, or outcrossing rates, but eggs from cut worms had a lower hatching rate than eggs from uncut worms. We found that when two anterior worm halves were bred together, they produced fewer, smaller eggs with higher hatching rates, compared to two posterior halves. Moreover, once we controlled for this effect of ‘worm half’, the two halves of an individual worm tended to reproduce similarly under comparable circumstances. We conclude that cutting plerocercoids increases the flexibility with which this tapeworm can be experimentally bred without dramatically affecting the production of viable, outcrossed eggs.

Zoonotic Trichuris trichiura Infections in Non-Human Primates at Samsun Zoo, Turkey: First Molecular Characterization

Summary Trichuris trichiura were collected from rhesus macaque (Macaca mulatta) and Hamadryas baboon (Papio hamadryas) in Samsun Zoo, Turkey. DNA was isolated from individual worm in baboon for molecular characterization. The ITS region was amplified and sequencing in both directions using NC5-NC2 primers. Pairwise comparison between ITS region of the T. trichiura isolates from Turkey (KC877992) and other isolates China (AM992981, AM992985, AM992998), Czech Republic (JF690940, JF690941, JF690950), Netherlands (JF690948) and South Africa (GQ301551, GQ301553) showed differences ranging from 0.1% to 1.2%. With the present study, T. trichiura from Turkey were characterized for the first time by sequencing of the ITS. Keywords: Trichuris trichiura, Macaca mulatta, Papio hamadryas, Molecular Characterization, Samsun, Turkey Samsun Hayvanat Bahcesi’ndeki Primatlarda Zoonotik Trichuris trichiura Enfeksiyonu: Ilk Molekuler Karakterizasyon Ozet Samsun Hayvanat Bahcesi’ndeki rehesus maymunu (Macaca mulatta)

Conserved behavioral and genetic mechanisms in the pre-hatching molt of the nematode Pristionchus pacificus.

BackgroundDuring development, juvenile nematodes undergo four molts. Although the number of molts appears to be constant within the Nematoda, the timing of the first molt can occur either before or after hatching. A previous study indicates that, as in some parasitic nematode lineages, a pre-hatching juvenile stage also exists in Diplogastrid nematodes. A detailed description of these sequence of events has yet to be shown for any single species.FindingsTo delineate the timing of the pre-hatching molt in the beetle-associated Pristionchus pacificus, we tracked individual mid-J1 stage worms inside the eggshell through the J1-J2 transition and hatching. We found that active movement ended 21 hours after egg-laying, followed by lethargus and hatching. We inferred that lethargus behavior represents the onset of the first molt, which precedes each post-hatching molt in C. elegans and P. pacificus. The onset of the J1-J2 molt was also marked by the upregulation of the P. pacificus molting marker Ppa-pnhr-1. We further corroborated the pre-hatching molt with the isolation of two genetic mutants that exhibited aberrant molting both inside the egg and after hatching, as characterized by protracted and often-aborted shedding of the old cuticle.ConclusionOur results describe in detail the pre-hatching juvenile molt in P. pacificus, provide strong visual evidence of a pre-hatching molt, and show support for common genetic mechanisms regulating molting in the pre-hatching and post-hatching developmental stages. Our findings support the hypothesis that the evolution of pre-hatching development in Diplogastrid nematodes is likely due to a heterochronic shift between the timing of the first molt and hatching.

Probing the anti-aging role of polydatin in Caenorhabditis elegans on a chip

C. elegans is widely used as a model organism in the study of aging and evaluation of anti-aging drugs due to its unique characteristics. In this work, we set out to investigate polydatin, a natural resveratrol glycoside, and its role in extending lifespan, improving oxidative stress resistance, and the possible regulation mechanism involved in the Insulin/IGF-1 signaling (IIS) pathway for the first time by using a flexible microfluidic device. The effects of polydatin on the lifespan, oxidative stress resistance, mobility and the expression of aging-related proteins and genes were explored. Polydatin was found to significantly extend the mean lifespan of worms by up to 30.7% and 62.1% under normal and acute stress conditions respectively. It improved the expression of the inducible oxidative stress protein (GST-4) and corresponding stroke frequencies in the transgenic CL2166 strain. Moreover, it also increased SOD-3::GFP expression in CF1553 worms and promoted DAF-16 nucleus translocation in TJ356 worms. The longevity-extending role of polydatin is partly attributed to its anti-oxidative activity and increased oxidative stress resistance by regulating the stress-resistance related proteins SOD-3, and daf-16 expression at protein and mRNA levels involved in the IIS pathway. The established microfluidic platform is capable of flexible operation with multiple functions, which not only supports the individual worm's long-term culture with sufficient nutrient exchange, but also facilitates mobility monitoring of the worm, immobilizing and imaging in a controllable and parallel manner. These interesting findings reported here highlight the significance of the natural compound polydatin in the study of aging-related diseases, and the utility of the microfluidic platform for applications in aging studies.

Pharmacological knowledge and sustainable anthelmintic therapy in ruminants

Considering the increasing concern for the development of anthelmintic resistance, the use of pharmacology-based information is critical to design successful strategies for the future of parasite control in livestock. Integrated evaluation of the available knowledge on pharmacological features is required to optimize the activity and to achieve sustainable use of the existing anthelmintic drugs. The assessment of the drug disposition in the host and the comprehension of the mechanisms of drug influx/efflux/detoxification in different target helminths, has signified a relevant progress on the understanding of the pharmacology of anthelmintic drugs in ruminant species. However, additional scientific knowledge on how to improve the use of available and novel molecules is required to avoid/delay resistance development. Different pharmacokinetic-based approaches to enhance parasite exposure and the use of mixtures of drugs from different chemical families have been proposed as valid strategies to delay the development of anthelmintic resistance. The rationale behind using drug combinations is based on the fact that individual worms may have a lower degree of resistance to a multiple component formulation (each chemical with different mode of action/resistance) compared to that observed when a single anthelmintic is used. However, the limited available information is unclear on the potential additive or synergistic effects occurring after co-administration of two (or more) drugs with different mode of action. This review article contributes to the topic with some pharmacology-based data emerging from the assessment of combined anthelmintic preparations. The activity against multi-drug-resistant isolates based on novel modes of action is a highly favorable element to judge the future of some of the recently developed anthelmintic compounds. More specific knowledge on the basic host–parasite kinetic behavior as well as a highly responsible use of those novel compounds will be necessary to secure their maximum lifespans. Overall, the outcome from integrated pharmaco-parasitological research approaches has greatly contributed to optimize drug activity, which seems relevant to preserve existing and particularly novel active ingredients as useful tools for parasite control in livestock animals.

Unexpected occurrence of Haemonchus placei in cattle in southern Western Australia

Haemonchus placei is an abomasal parasite of cattle, primarily in tropical and subtropical areas of the world. In Australia, this nematode can be extremely pathogenic in summer rainfall areas, particularly in the hot, sub-tropical Kimberley region, in the far north of the state of Western Australia (WA). Although cattle are occasionally transferred to southern parts of WA, it was believed that H. placei did not occur in southern regions of WA, as it is less cold-adapted than Haemonchus contortus, and the free-living stages would not develop during the cold winter and dry summer periods. Here, we show that, although H. contortus is found in cattle in the temperate southern region of WA, it appears that H. placei also occurs in southern WA. While investigating the prevalence of anthelmintic resistance in nematodes of cattle in WA, the existence of H. placei was suspected on a range of participating farms, following the morphological examination of third-stage larvae cultured from faeces, and of adult worms recovered from sheep experimentally infected with these larvae. Genomic DNAs from individual worms as well as eggs from pooled faecal samples from seven farms in southern WA were subjected to PCR-based mutation scanning and sequence analyses of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA. The results showed that both H. contortus and H. placei were harboured by cattle. This first record of H. placei in cattle in southern WA raises questions as to the prevalence and distribution of this parasite in other temperate and cool climatic regions of Australia. Although clinical disease due to H. placei has not yet been seen in southern WA, global, climatic trends might suggest an increased importance of this parasite in the longer term.

Genetic variability within and among Haemonchus contortus isolates from goats and sheep in China

BackgroundHaemonchus contortus (order Strongylida) is a common parasitic nematode infecting small ruminants and causing significant economic losses worldwide. Knowledge of genetic variation within and among H. contortus populations can provide a foundation for understanding transmission patterns, the spread of drug resistance alleles and might assist in the control of haemonchosis.Methods152 H. contortus individual adult worms were collected from seven different geographical regions in China. The second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA and mitochondrial nicotinamide dehydrogenase subunit 4 gene (nad4) were amplified by polymerase chain reaction (PCR) and sequenced directly. The sequence variations and population genetic diversities were determined.ResultsNucleotide sequence analyses revealed 18 genotypes (ITS-2) and 142 haplotypes (nad4) among the 152 worms, with nucleotide diversities of 2.6% and 0.027, respectively, consistent with previous reports from other countries, including Australia, Brazil, Germany, Italy, Malaysia, Sweden, the USA and Yemen. Population genetic analyses revealed that 92.4% of nucleotide variation was partitioned within populations; there was no genetic differentiation but a high gene flow among Chinese populations; some degree of genetic differentiation was inferred between some specimens from China and those from other countries.ConclusionsThis is the first study of genetic variation within H. contortus in China. The results revealed high within-population variations, low genetic differentiation and high gene flow among different populations of H. contortus in China. The present results could have implications for studying the epidemiology and ecology of H. contortus in China.

Dampening of expression oscillations by synchronous regulation of a microRNA and its target

The complexity of metazoan organisms requires precise spatiotemporal regulation of gene expression during development. We find that in the nematode Caenorhabditis elegans approximately 2,000 transcripts undergo expression oscillations synchronized with larval transitions while thousands of genes are expressed in temporal gradients, similar to known timing regulators. By counting transcripts in individual animals, we show that the pulsatile expression of the microRNA (miRNA) lin-4 maintains the temporal gradient of its target lin-14 by dampening its expression oscillations. Our results demonstrate that this insulation is optimal when pulsatile expression of the miRNA and its target is synchronous. We propose that such a miRNA-mediated incoherent feed-forward loop is a potent filter that prevents propagation of potentially deleterious gene expression fluctuations during the development of an organism.

Age-related micro-RNA abundance in individual C. elegans

Non-coding small RNAs of the micro-RNA class (miRNA) are conserved regulators of gene function with a broad impact on biological processes. We screened miRNA levels for age-related changes in individual worms and investigated their influence on the lifespan of the nematode C. elegans. We measured the abundance of 69 miRNAs expressed in individual animals at different ages with over thirty five thousand discrete quantitative nano-fluidic polymerase chain reactions. We found that miRNA abundance was highly variable between individual worms raised under identical conditions and that expression variability generally increased with age. To identify expression differences associated with either reproductive or somatic tissues, we analyzed wild type and mutants that lacked germlines. miRNAs from the mir-35-41 cluster increased in abundance with age in wild type animals, but were nearly absent from mutants lacking a germline, suggesting their age-related increase originates from the germline. Most miRNAs with age-dependent levels did not have a major effect on lifespan, as corresponding deletion mutants exhibited wild-type lifespans. The major exception to this was mir-71, which increased in abundance with age and was required for normal longevity. Our genetic characterization indicates that mir-71 acts at least partly in parallel to insulin/IGF like signals to influence lifespan.