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[
International Worm Meeting,
2009]
The ionome is defined as the complete elemental compositions of an organism, including essential mineral nutrients and trace elements. Ionomics, the study of the ionome, is utilized to uncover the genes and gene networks that regulate the ionome and the physiological stimuli that affect these networks. In ionomics, high throughput elemental analysis is integrated with genetics, genomics, and bioinformatics to gain a comprehensive picture of the ionome of an organism. Because of its invariant and precise somatic cell lineages, C. elegans is an excellent experimental model system to apply the concept of ionomics to gene networks within an intact animal. Wild-type N2 worms were grown axenically in modified CeHR liquid medium to mid L4 stage, collected on cellulose filters, and dried before analysis by inductively coupled plasma-mass spectrometry (ICP-MS) at the Purdue Ionomics Center. Using this approach, we have been able to precisely quantify the concentration of P, Ca, K, Mg, Cu, Fe, Zn, Mn, Co, Ni, B, Se, Mo, Na, As, and Cd in C. elegans. Our ultimate goals are to superimpose the ionome network with the regulatory circuit which defines developmental processes in an animal by combining functional ionomics with genome-wide RNAi screens.
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[
West Coast Worm Meeting,
2000]
In all animals, fertilization generates a pattern of intracellular calcium dynamics within the oocyte that constitutes an essential trigger for normal development. The spatiotemporal properties of the calcium dynamics differ among animals, e.g. cnidarians, nemerteans, fish and frogs have single calcium transients whereas annelids, ascidians, and mammals have multiple calcium oscillations. However, in all animals, fertilization-induced calcium dynamics are mediated by release of internal calcium stores by inositol 1,4,5-triphosphate (IP3). Although little is known about the signaling pathway intervening fertilization and the production of IP3, features of the pathway are likely to be widely shared among species [1]. Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism to study fertilization-induced calcium dynamics, we have characterized these dynamics in C. elegans. Owing to the transparency of the nematode, we have been able to study the calcium signal in C. elegans fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that originates at the point of sperm entry. This calcium elevation immediately spreads throughout the oocyte with an amplitude ~250 nM. The duration of this solitary calcium transient is ~6 min, after which the cytosolic calcium concentration returns to that preceding fertilization. Among other effects, this calcium signal may trigger the completion of meiosis and the formation of eggshell.
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[
International C. elegans Meeting,
2001]
We are analyzing the thermotactic response in C. elegans, both the behavior of the organism as it navigates thermal gradients and the activity of the neurons in response to thermal stimuli. Using custom-built equipment, we track worm motility on spatial and temporal gradients, and are establishing the rules of the thermotactic strategy. Using phluorins, we are attempting to measure vesicle recycling in the neurons underlying the thermotactic response. We are also attempting to detect calcium transients in these neurons using aequorin, a bioluminescent indicator of Ca2+.
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[
International Worm Meeting,
2013]
Like us, C. elegans lives in a microbial world. In its natural habitats of rotting fruits and vegetation, these nematodes proliferate as they dine on an array of microbes. Interactions with microbes span a spectrum from constant confrontation (pathogens) to relative indifference (food) to perhaps even mutual benefit (symbionts). This study identifies these natural microbes and addresses whether microbiome composition influences proliferation of C. elegans in the wild.
To examine this question, we sequenced bacterial 16S (SSU) rDNA amplicons from habitats with wild C. elegans populations collected in France and Spain. Our results show that C. elegans encounters a broad array of bacteria in the wild-especially the divisions (phyla) of Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. An abundance-weighted comparisons of phylogenetic differences (UniFrac) showed distinct clustering by habitat type, as rotting apples clustered separately from other habitats sequenced. Further, rotting apples clustered by large presence of proliferating or small non-proliferating (dauer) populations of worms. C. elegans appear to proliferate in apples with 'simpler' microbiomes (lower diversity, fewer species and Proteobacteria-rich). Specific alpha-proteobacteria were particularly enriched in apples with proliferating worms, while a number of genera were consistently found in apples with non-proliferating worms (e.g., Pseudomonas, several Bacteroidetes, etc.). Population size also correlated with apple rottenness, suggesting bacterial load is key to growth as well.
Similarly, Proteobacteria content does affect C. elegans (N2) growth rate in the lab, as worms grew faster on mixtures (and single isolates) with 80% Proteobacteria versus those with 40% Proteobacteria. Together, these studies define the microbial diet of C. elegans and implicate the natural microbiome as a key determinant of C. elegans' growth in the wild.
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[
International Worm Meeting,
2003]
The ability to tightly control solute and water balance is an essential prerequisite for cellular life. Osmotic homeostasis is maintained by the regulated accumulation and loss of inorganic ions and small organic solutes termed organic osmolytes. While cellular osmoregulation has been studied extensively in a variety of cells and tissues, major gaps exist in our molecular understanding of this essential process. Because of its many experimental advantages, C. elegans provides an ideal model system in which to define the genes and genetic pathways required for cellular osmoregulation in animals.To begin defining the genetic basis of cellular osmoregulation in C. elegans, we characterized the ability of worms to survive and adapt to extreme hypertonic stress by adding NaCl to the growth agar. Exposure to high salt agar causes rapid shrinkage, followed by slow recovery of body volume over several hours. Survival is normal on agar containing up to 200 mM NaCl. When grown on 200 mM NaCl for 2 weeks, worms are able to survive and reproduce on agar containing 400 mM NaCl. Worms adapted to hypertonic stress swell and then rapidly return to their initial body volume when returned to low salt agar. We are currently using forward and reverse genetic screens to identify genes required for adaptation to and recovery from hypertonic stress. The functions of these genes are being defined by molecular, biochemical, and physiological approaches.In response to hypertonicity, all organisms accumulate substances called organic osmolytes to counter the damaging effects of hypertonicity. Organic osmolytes are small uncharged or weakly charged compounds that are accumulated to concentrations of 10s to 100s of millimolar in the cytoplasm of hypertonically stressed cells. Importantly, the unique biophysical properties of organic osmolytes do not disturb cellular structure and function. Using HPLC analysis, we have detected a low molecular weight compound (~115 daltons) that increases 15-20 fold in nematodes grown on 200 mM NaCl plates. Accumulation of this compound begins after 6 h of high salt stress and peaks by 24 h. Further tests have shown that this compound is not an amino acid, one of the three major categories of known organic osmolytes. We are attempting to identify the molecular nature of this putative organic osmolyte using mass and NMR spectrometry.
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[
International Worm Meeting,
2015]
Like us, C. elegans lives in a microbial world. In its natural habitats of rotting fruits and vegetation, C. elegans encounters a diverse array of microbes, where they serve as diet, microbiota or pathogens. C. elegans is highly tuned and equipped to sense and response to the milieu of microbial products (xenobiotics) that they are bombarded with, as they navigate this microbial landscape. Indeed, several recent studies have demonstrated that animals monitor basic cellular subsystems for microbial targeting (presumably through decreased functionality/efficiency or the like), though the extent to which beneficial microbes may interface with these systems is unknown. Small RNA pathways play a central role in regulating many of the transcriptional and developmental programs that are responsive to microbes, in addition to directly mediating anti-viral immunity. Thus, this study takes a broad look at natural microbes that may specifically engage small RNA pathways to regulate C. elegans physiology.To examine this question, we screened a panel of 565 microbes ('BIGb collection') isolated from C. elegans' natural habitats for microbial-enhancers or -reducers of RNAi (mERI or mRDE, respectively) via co-feeding with several RNAi clones in E. coli (somatic and germline) or alone with a panel of transgenic reporters. In co-feeding experiments, nearly 20% of the microbes reduced RNAi effectiveness (113), while 7.5% enhanced RNAi (42). There was also a strong correlation between the impact of a microbe on C. elegans physiology-beneficial (faster growth, unstressed) or detrimental (slow growth/death, activation of stress reporters)-and its impact on RNAi pathways, with mRDE isolates being more likely to be detrimental and vice versa. Since co-feeding can affect uptake of dsRNA-producing E. coli, we also validated these microbes for impacts on RNAi using a panel of transgene-silencing reporter strains. Together, these data implicate a number of often pathogens (e.g., Pseudomonas, Stenotrophomonas and Enterobacter) as mRDE and beneficial microbes (e.g., Providencia and Bacillus) as mERI. In parallel, we also demonstrate that microbial RNA directly engages a subset of RNAi pathways in regulation of C. elegans growth. Together, these studies expand our understanding of the host systems that are under microbial influence and regulate host health. .
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[
International Worm Meeting,
2011]
Like all of us, C. elegans lives in a microbially dominated world. They naturally proliferate habitats rich in microbes, like rotting fruits and decaying vegetation. Interactions with these as yet unnamed microbes undoubtedly span a spectrum from constant confrontation (pathogens) to relative indifference (food) and perhaps even mutual benefit (symbionts). Interestingly, in contrast to N2, wild C. elegans harbor intestinal microbes. In addition to food, it is tantalizing to speculate that these worms might enlist microbes to improve its fitness (e.g., resistance to pathogens, harmful chemicals, etc.), just as we and many other animals have done. Within a habitat, C. elegans expansion is affected by a host of environmental and intrinsic (genetic) factors. Specifically, similar habitats in close proximity can harbor anywhere from no worms to dauers to actively proliferating populations; our hypothesis is that the mixture of microbes present is a key determinant of C. elegans' success. To examine this question, we performed culture-independent sequencing of microbial small subunit rDNA from habitats with wild C. elegans populations collected during several field seasons and different locations. In addition, we isolated worms to directly examine their more closely associated microbes. Using this dataset, we are able to address: (a) what are the commonly encountered microbes; (b) do groups of microbes correlate with population success; and (c) does C. elegans maintain a microbiota? Initial results from habitats indicate that bacteria belonging to four divisions (phyla), Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria are common in C. elegans' habitats, and occasionally seven more rare divisions are observed. However, there are also great variations in the bacterial diversity and richness among habitats (10-100+ species); we are testing bacterial species found in many habitats for their close association with worms. Notably, cultured microbial mimics of these habitats dramatically alter C. elegans' growth: e.g., proliferation on a Proteobacteria-rich mimic occurs faster than on a Bacteroidetes-rich mimic. Comparisons of responses of wild and lab-raised worms are also in progress. Determination of the microbes in C. elegans natural habitats is a first step in expanding our understanding of how microbes can influence host fitness and resistance to ecological pressures.
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[
International Worm Meeting,
2003]
Osmotic stress alters the expression of genes required for cellular osmoregulation and for the detection and repair of osmotically-induced cellular and molecular damage. The identity and function of many of these genes are unknown. C. elegans provides numerous experimental advantages for identifying osmotically regulated genes and for defining their molecular functions.We have demonstrated recently C. elegans survives and adapts readily to extreme hypertonic stress. Using microarray analysis, we have begun to identify the complement of genes that are transcriptionally regulated during exposure to high NaCl growth agar. When nematodes are adapted to 200 mM NaCl agar for 24 h, approximately 150 genes showed average changes in transcription of >1.7 fold. These included genes that encode proteases, cytoskeletal proteins, extracellular matrix proteins and components of stress response and signaling pathways. Putative metabolic genes accounted for 41% of the genes that were transcriptionally upregulated. Transport of inorganic ions and small organic solutes termed organic osmolytes plays an important role in the adaptation of cells to hypertonic stress. However, upregulation of relatively few channel- or transporter-encoding genes was observed. One putative channel gene that was upregulated (mean change in expression level
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2010]
Like all of us, C. elegans lives in a microbially dominated world. It naturally colonizes habitats rich in microbes, such as rotting fruits and vegetation. Interactions with these as yet unnamed microbes undoubtedly span a spectrum from constant confrontation (pathogens) to relative indifference (food) and perhaps even mutual benefit (symbionts). Many other animals, including some nematodes, acquire fitness benefits by relying on microbes for nutrients, protection from pathogen colonization, and detoxification of harmful xenobiotics. Indeed, C. elegans adults have been observed to harbor undigested microbes in their intestines under some circumstances. In any case, C. elegans has certainly adapted to be highly tuned to microbial cues in order to evaluate their food quality or potential to support growth. However, little is known about how this complex microbial calculus occurs with natural microbes. To more broadly examine the microbes encountered by wild C. elegans populations, we performed both culture-based and comprehensive culture-independent sequencing of microbial SSU rDNA from habitats with proliferating and non-proliferating (mostly dauer) wild C. elegans populations. In addition, we isolated animals away from their habitats to directly sequence their more closely associated microbes and potential natural microbiota. Preliminary analyses of all habitats indicate that these animals most commonly encounter bacteria belonging to four phylogenetic divisions (phyla), Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, along with seven more rare divisions. However, there are also great variations in the diversity and richness of bacteria found within the habitats: some rotting apples have more simple microbial communities (<10 phylotypes) while other rotting vegetation harbors nearly 100 phylotypes. Additionally, several phylotypes were identified from quite disparate habitats (e.g., a snail, a rotting apple and a rotting orange), which could indicate their close association with C. elegans animals themselves. Further analyses of enrichment of specific microbes in isolated worms compared to their habitats (natural or culture-based mimics) will inform these correlations. Determination of what microbes C. elegans associate with naturally is a first step in elaborating the ecological pressures that they may both instigate and ameliorate. Further, by comparing responses of lab-raised strains that have been domesticated on E. coli and wild strains with their naturally associated microbes, we hope to identify conserved host-microbial response pathways that influence C. elegans physiology and metabolism.
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[
International Worm Meeting,
2015]
The temperature dependence of muscle function has been well documented 1. Whether this dependence is due to the intrinsic properties of muscles, their control by motor neurons, regulatory input from thermoreceptor neurons, or some combination of these factors is unclear. We are investigating this question using the nematode C. elegans and egg-laying behavior. We first measured steady-state egg-laying rate as a function of temperature in several wild strains isolated from a range of climates and used these data to estimate the peak egg-laying rate (Rmax) and the temperature at which it occurs (Tmax). We find that Tmax, but not Rmax correlates with the distance from the equator for each strain's origin. In a companion presentation (Lasse, McPherson, et al), we share initial work directed towards identifying genetic polymorphisms that might account for the variation in Tmax, focusing on the two most divergent strains: Bristol (N2) and Hawaiian (CB4856). To learn more about the contribution of thermoreceptor neurons, we leveraged existing and novel mutations in the
tax-4 gene, which is required for the function of all thermoreceptor neurons operating in the innocuous thermal zone2. In both Bristol (N2) and Hawaiian (CB4856) strains, loss of
tax-4 function decreases Tmax. This manipulation reduces Rmax by a factor of three in the Bristol (N2) background but does not alter Rmax in the Hawaiian (CB4856) background. We propose that the motor program has similar intrinsic temperature dependence across worm strains and that thermoreceptor neuron input is necessary to shift this dependence to a range that is best matched to a particular environment.We thank Mario de Bono and Changchun Chen for assistance with the CRISPR technique and the NIH/IRACDA for fellowship funding to S. Lasse.1. Bennett, A. F. Thermal dependence of muscle function. Am. J. Physiol. 247, R217-29 (1984).2. Mori, I. & Ohshima, Y. Neural regulation of thermotaxis in Caenorhabditis elegans. Nature 376, 344-348 (1995).