C. elegans germline-deficient mutants respond to pathogen infection using shared and distinct mechanisms.

Reproduction extracts a cost in resources that organisms are then unable to utilize to deal with a multitude of environmental stressors. In the nematode C. elegans, development of the germline shortens the lifespan of the animal and increases its susceptibility to microbial pathogens. Prior studies have demonstrated germline-deficient nematodes to have increased resistance to gram negative bacteria. We show that germline-deficient strains display increased resistance across a broad range of pathogens including gram positive and gram negative bacteria, and the fungal pathogen Cryptococcus neoformans. Furthermore, we show that the FOXO transcription factor DAF-16, which regulates longevity and immunity in C. elegans, appears to be crucial for maintaining longevity in both wild-type and germline-deficient backgrounds. Our studies indicate that germline-deficient mutants glp-1 and glp-4 respond to pathogen infection using common and different mechanisms that involve the activation of DAF-16.

WormSizer: high-throughput analysis of nematode size and shape.

The fundamental phenotypes of growth rate, size and morphology are the result of complex interactions between genotype and environment. We developed a high-throughput software application, WormSizer, which computes size and shape of nematodes from brightfield images. Existing methods for estimating volume either coarsely model the nematode as a cylinder or assume the worm shape or opacity is invariant. Our estimate is more robust to changes in morphology or optical density as it only assumes radial symmetry. This open source software is written as a plugin for the well-known image-processing framework Fiji/ImageJ. It may therefore be extended easily. We evaluated the technical performance of this framework, and we used it to analyze growth and shape of several canonical Caenorhabditis elegans mutants in a developmental time series. We confirm quantitatively that a Dumpy (Dpy) mutant is short and fat and that a Long (Lon) mutant is long and thin. We show that daf-2 insulin-like receptor mutants are larger than wild-type upon hatching but grow slow, and WormSizer can distinguish dauer larvae from normal larvae. We also show that a Small (Sma) mutant is actually smaller than wild-type at all stages of larval development. WormSizer works with Uncoordinated (Unc) and Roller (Rol) mutants as well, indicating that it can be used with mutants despite behavioral phenotypes. We used our complete data set to perform a power analysis, giving users a sense of how many images are needed to detect different effect sizes. Our analysis confirms and extends on existing phenotypic characterization of well-characterized mutants, demonstrating the utility and robustness of WormSizer.

Metazoan operons accelerate recovery from growth-arrested states.

Existing theories explain why operons are advantageous in prokaryotes, but their occurrence in metazoans is an enigma. Nematode operon genes, typically consisting of growth genes, are significantly upregulated during recovery from growth-arrested states. This expression pattern is anticorrelated to nonoperon genes, consistent with a competition for transcriptional resources. We find that transcriptional resources are initially limiting during recovery and that recovering animals are highly sensitive to any additional decrease in transcriptional resources. We provide evidence that operons become advantageous because, by clustering growth genes into operons, fewer promoters compete for the limited transcriptional machinery, effectively increasing the concentration of transcriptional resources and accelerating recovery. Mathematical modeling reveals how a moderate increase in transcriptional resources can substantially enhance transcription rate and recovery. This design principle occurs in different nematodes and the chordate C. intestinalis. As transition from arrest to rapid growth is shared by many metazoans, operons could have evolved to facilitate these processes.

Later Life Consequences of Developmental Mitochondrial DNA Damage in C. elegans

Mitochondria are responsible for producing the vast majority of cellular ATP, and are therefore critical to organismal health [1]. They contain thir own genomes (mtDNA) which encode 13 proteins that are all subunits of the mitochondrial respiratory chain (MRC) and are essential for oxidative phosphorylation [2]. mtDNA is present in multiple copies per cell, usually between 103 and 104 , though this number is reduced during certain developmental stages [3, 4]. The health of the mitochondrial genome is also important to the health of the organism, as mutations in mtDNA lead to human diseases that collectively affect approximately 1 in 4000 people [5, 6]. mtDNA is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including the absence of Nucleotide Excision Repair (NER) in the mitochondria [7]. NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and also many environmental toxicants, including benzo[a]pyrene (BaP) [8]. While these lesions cannot be repaired, they are slowly removed through a process that involves mitochondrial dynamics and autophagy [9, 10]. However, when present during development in C. elegans, this damage reduces mtDNA copy number and ATP levels [11]. We hypothesize that this damage, when present during development, will result in mitochondrial dysfunction and increase the potential for adverse outcomes later in life.

To test this hypothesis, 1st larval stage (L1) C. elegans are exposed to 3 doses of 7.5J/m2 ultraviolet C radiation 24 hours apart, leading to the accumulation of mtDNA damage [9, 11]. After exposure, many mitochondrial endpoints are assessed at multiple time points later in life. mtDNA and nucDNA damage levels and genome copy numbers are measured via QPCR and real-time PCR , respectively, every 2 day for 10 days. Steady state ATP levels are measured via luciferase expressing reporter strains and traditional ATP extraction methods. Oxygen consumption is measured using a Seahorse XFe24 extra cellular flux analyzer. Gene expression changes are measured via real time PCR and targeted metabolomics via LC-MS are used to investigate changes in organic acid, amino acid and acyl-carnitine levels. Lastly, nematode developmental delay is assessed as growth, and measured via imaging and COPAS biosort.

I have found that despite being removed, UVC induced mtDNA damage during development leads to persistent deficits in energy production later in life. mtDNA copy number is permanently reduced, as are ATP levels, though oxygen consumption is increased, indicating inefficient or uncoupled respiration. Metabolomic data and mutant sensitivity indicate a role for NADPH and oxidative stress in these results, and exposed nematodes are more sensitive to the mitochondrial poison rotenone later in life. These results fit with the developmental origin of health and disease hypothesis, and show the potential for environmental exposures to have lasting effects on mitochondrial function.

Lastly, we are currently working to investigate the potential for irreparable mtDNA lesions to drive mutagenesis in mtDNA. Mutations in mtDNA lead to a wide range of diseases, yet we currently do not understand the environmental component of what causes them. In vitro evidence suggests that UVC induced thymine dimers can be mutagenic [12]. We are using duplex sequencing of C. elegans mtDNA to determine mutation rates in nematodes exposed to our serial UVC protocol. Furthermore, by including mutant strains deficient in mitochondrial fission and mitophagy, we hope to determine if deficiencies in these processes will further increase mtDNA mutation rates, as they are implicated in human diseases.

To plasticity and back again.

Both the gain and the loss of flexibility in the development of phenotypes have led to an increased diversity of physical forms in nematode worms.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.

Efecto de una enmienda orgánica y nematicida sobre la dinámica poblacional de Paratylenchus sp. (Nematoda, Paratylenchinae) en apio

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Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).

Ecology of a North Sea pockmark with an active methane seep

ABSTRACT: At a large North Sea pockmark, with active methane seeps, surface sediments were found to have higher insoluble sulphide concentrations than sedlments from the surrounding area. The fauna of the pockmark was characterized by 2 species which have not pi-evlously been reported from the Fladen Ground in the northern North Sea. These species were a b~valve, Thyasira sarsi (which is known to contain endosymbiotic sulphur-oxidising bacteria) and a mouthless and gutless nematode, Astomonerna sp., which also contains endosymbiotic bacteria The nematode was the dominant meiofauna species in the pockmark sediments. Both macro-lnfauna and total nematodes were in low abundance in samples taken from the base of the pockmark. Sediment samples from the pockmark contained numerous otoliths, implying that substantial winnowing of the sediment had taken place. This was supported by studies on the sulphide concentrations in the sediment which showed multiple layering of the sediments on the sides of the pockmark, suggesting displacement. The carbon isotope compositions (6I3c) of the tissues of benthic animals from in and around the pockmark were generally in the range -16 to -2O%, indicating that little methane-derived carbon was contributing to their nutrition. T sarsi had the most 13c-depleted tissues, -31.4 to -35.1 L, confirming the nutritional dependence of this species on chemoautotrophic bacteria that utilize reduced sulphur.

The Nematode-Copepod Ratio And Its Use In Pollution Ecology

The nematode/copepod ratio is critically examined with a view to adding some precision to its proposed use in pollution ecology. At two unpolluted intertidal sites, differing markedly in sediment grade, the metabolic requirements of copepods are shown to be equivalent to the requirements of that fraction of the nematode population which feeds in the same way. The partitioning of this total energy requirement among individuals depends on the distribution of individual metabolic body sizes and the relative rates of metabolism. The distribution of body sizes is constrained by the sediment granulometry, which affects nematodes and copepods differently. These considerations enable precise predictions of the nematode/copepod ratios expected in unpolluted situations, against which observed ratios can be compared.

The Influence Of Temperature And Salinity On Energy Partitioning In The Marine Nematode Diplolaimelloides-Bruciei

Measurements of population growth, generation time, fecundity and respiration in laboratory culture have been made, in relation to temperature and salinity, for the nematode Diplolaimelloides bruciei Hopper, a species normally associated with decayed material of the marsh grass Spartina. The intrinsic rate of increase (r) is high: it is related to temperature between 5° and 25°C by a sigmoid function which is steepest between 10° and 15°C, and is maximum at 26‰ salinity. Generation time is related to temperature by a power function and is shortest at 26‰ salinity. The effect of temperature on generation time is consistent with other data for marine nematodes, and the steep slope of r against temperature is largely due to the marked effect of temperature on fecundity. A sex ratio of 2:1 in favour of males is maintained regardless of culture conditions or population density. Respiration increases exponentially with temperature between 5° and 25°C, with a very high Q10 (3.94), but is not affected by salinity. At 30°C respiration is no higher than at 25°C. A high and relatively stable production efficiency (P/A) is maintained between 10 and 30°C with a maximum of 87% at 15°C; there is a stable reproductive effort (Pr/A) of about 10%. At 5°C both these ratios are zero. Data for the harpacticoid copepod Tachidius discipes, derived from the literature, show that this too has a high and stable production efficiency, which may be a characteristic of meiofaunal species in general, but in this species efficiency is relatively high at 5°C. Many features of the energy balance in D. bruciei can be related to an opportunistic mode of life.

Pattern of benthic biomass size spectra from shallow waters in the East China Seas

Benthic biomass size spectra (BSS) and normalized biomass size spectra were constructed, and benthic secondary production was estimated by a size spectrum equation in the shallow waters in the East China Sea, ranging latitudinally from 40A degrees N to 29A degrees N. The BSS patterns were bimodal, two biomass peaks corresponding to meiofauna and macrofauna, respectively, separated by a trough of low biomass at 8-256 mu g individual dry weight which varied in position with median sediment particle size. The BSS also displayed bimodality within meiofauna size ranges, which in most stations was due to the relative proportions of nematodes and other meiofauna taxa. Re-analysis of data from sites in the UK, South Africa, and Antarctic showed a similar bimodality in the adult species body size distribution within the meiofauna size range. Macrofaunal production estimated by the size spectrum equation was very similar to the results of Brey90 empirical equation. However, these production values were much lower than those calculated by Brey01. Different individual dry-to-wet conversion ratios, temperature deviation, and macrofauna taxonomic composition might be responsible for the between-model differences. The macrofaunal P/B ratios calculated by this equation ranged from 0.3 to 3.4 which were in accordance with values from Northern Hemisphere mid-latitudes. Meiofaunal production estimates will need further empirical support.

Interactions between multiple large macrofauna species and nematode communities — Mechanisms for indirect impacts of trawling disturbance

Highlights •We exposed meiofauna to 7 different large macrofauna species at high and low densities. •Macrofauna presence altered nematode community structure and reduced their abundance. •Macrofauna species had similar effects by reducing the few dominant nematode species. •Meio–macrofauna resource competition and spatial segregation are the main drivers. •Trawling effects on macrofauna affect nematode communities indirectly. Diverse assemblages of infauna in sediments provide important physical and biogeochemical services, but are under increasing pressure by anthropogenic activities, such as benthic trawling. It is known that trawling disturbance has a substantial effect on the larger benthic fauna, with reductions in density and diversity, and changes in community structure, benthic biomass, production, and bioturbation and biogeochemical processes. Largely unknown, however, are the mechanisms by which the trawling impacts on the large benthic macro- and megafauna may influence the smaller meiofauna. To investigate this, a mesocosm experiment was conducted whereby benthic nematode communities from a non-trawled area were exposed to three different densities (absent, low, normal) of 7 large (> 10 mm) naturally co-occurring, bioturbating species which are potentially vulnerable to trawling disturbance. The results showed that total abundances of nematodes were lower if these large macrofauna species were present, but no clear nematode abundance effects could be assigned to the macrofauna density differences. Nematode community structure changed in response to macrofauna presence and density, mainly as a result of the reduced abundance of a few dominant nematode species. Any detectable effects seemed similar for nearly all macrofauna species treatments, supporting the idea that there may be a general indirect, macrofauna-mediated trawling impact on nematode communities. Explanations for these results may be, firstly, competition for food resources, resulting in spatial segregation of the meio- and macrobenthic components. Secondly, different densities of large macrofauna organisms may affect the nematode community structure through different intensities of bioturbatory disturbance or resource competition. These results suggest that removal or reduced densities of larger macrofauna species as a result of trawling disturbance may lead to increased nematode abundance and hints at the validity of interference competition between large macrofauna organisms and the smaller meiofauna, and the energy equivalence hypothesis, where a trade-off is observed between groups of organisms that are dependent on a common source of energy.

Microsporidia-nematode associations in methane seeps reveal basal fungal parasitism in the deep sea

The deep sea is Earth’s largest habitat but little is known about the nature of deep-sea parasitism. In contrast to a few characterized cases of bacterial and protistan parasites, the existence and biological significance of deep-sea parasitic fungi is yet to be understood. Here we report the discovery of a fungus-related parasitic microsporidium, Nematocenator marisprofundi n. gen. n. sp. that infects benthic nematodes at Pacific Ocean methane seeps on the Pacific Ocean floor. This infection is species-specific and has been temporally and spatially stable over two years of sampling, indicating an ecologically consistent host-parasite interaction. A high distribution of spores in the reproductive tracts of infected males and females and their absence from host nematodes’ intestines suggests a sexual transmission strategy in contrast to the fecal-oral transmission of most microsporidia. N. marisprofundi targets the host’s body wall muscles causing cell lysis, and in severe infection even muscle filament degradation. Phylogenetic analyses placed N. marisprofundi in a novel and basal clade not closely related to any described microsporidia clade, suggesting either that microsporidia-nematode parasitism occurred early in microsporidia evolution or that host specialization occurred late in an ancient deep-sea microsporidian lineage. Our findings reveal that methane seeps support complex ecosystems involving interkingdom interactions between bacteria, nematodes, and parasitic fungi and that microsporidia parasitism exists also in the deep sea biosphere.

Effects of elevated CO2 and temperature on an intertidal meiobenthic community

In the near future, the marine environment is likely to be subjected to simultaneous increases in temperature and decreased pH. The potential effects of these changes on intertidal, meiofaunal assemblages were investigated using a mesocosm experiment. Artificial Substrate Units containing meiofauna from the extreme low intertidal zone were exposed for 60 days to eight experimental treatments (four replicates for each treatment) comprising four pH levels: 8.0 (ambient control), 7.7 & 7.3 (predicted changes associated with ocean acidification), and 6.7 (CO2 point-source leakage from geological storage), crossed with two temperatures: 12 °C (ambient control) and 16 °C (predicted). Community structure, measured using major meiofauna taxa was significantly affected by pH and temperature. Copepods and copepodites showed the greatest decline in abundance in response to low pH and elevated temperature. Nematodes increased in abundance in response to low pH and temperature rise, possibly caused by decreased predation and competition for food owing to the declining macrofauna density. Nematode species composition changed significantly between the different treatments, and was affected by both seawater acidification and warming. Estimated nematode species diversity, species evenness, and the maturity index, were substantially lower at 16 °C, whereas trophic diversity was slightly higher at 16 °C except at pH 6.7. This study has demonstrated that the combination of elevated levels of CO2 and ocean warming may have substantial effects on structural and functional characteristics of meiofaunal and nematode communities, and that single stressor experiments are unlikely to encompass the complexity of abiotic and biotic interactions. At the same time, ecological interactions may lead to complex community responses to pH and temperature changes in the interstitial environment