Worm Physiology Research Articles

Uncovering the essential roles of glutamate carboxypeptidase 2 orthologs in Caenorhabditis elegans.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans to genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. The present study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to physiology of this protease group and in extension to human GCP2 involvement in pathophysiological processes.

Visualization of the biphasic calcium wave during fertilization in Caenorhabditis elegans using a genetically encoded calcium indicator.

Fertilization is a critical step in development, yet internal fertilization events are notoriously difficult to visualize. Taking advantage of the calcium response that is a hallmark of sperm-egg fusion, we adapted the genetically encoded calcium indicator jGCaMP7s to visualize the moment of fertilization in Caenorhabditis elegans using fluorescence. We termed this tool the 'CaFE' reporter, for 'calcium during fertilization in C. elegans'. The CaFE reporter produced a robust signal that recapitulated the previously reported, biphasic nature of the calcium wave and had no significant deleterious effects on worm physiology or fecundity. Calcium waves were not observed at the restrictive temperature in the spe-9(hc88) strain, in which sperm can still trigger meiotic maturation but can no longer fuse with the oocyte. Demonstrating the utility of the CaFE reporter, we analyzed polyspermy induced by inhibition of egg-3 via RNAi and observed late calcium waves in the uterus. This finding provides support to the idea that calcium release is not restricted to the first sperm fusion event during polyspermy. Establishment of the CaFE reporter in the genetically tractable and optically transparent worm provides a powerful tool to dissect the oocyte-to-embryo transition inside a living animal.

Effect of omega-6 linoleic acid on neurobehavioral development in Caenorhabditis elegans

Linoleic acid (LA, omega-6), an essential polyunsaturated fatty acid, is supplied by vegetable oils such as corn, sunflower and soybean. Supplementary LA in infants and children is required for normal growth and brain development, but has also been reported to induce brain inflammation and neurodegenerative diseases. This controversial role of LA development requires further investigation. Our study utilized Caenorhabditis elegans (C. elegans) as a model to clarify the role of LA in regulating neurobehavioral development. A mere supplementary quantity of LA in C. elegans larval stage affected the worm's locomotive ability, intracellular ROS accumulation and lifespan. We found that more serotonergic neurons were activated by supplementing LA above 10 μM thereby promoting locomotive ability with upregulation of serotonin-related genes. Supplementation with LA above 10 μM also inhibited the expression of mtl-1, mtl-2 and ctl-3 to accelerate oxidative stress and attenuate lifespan in nematodes; however, enhancement of stress-related genes such as sod-1, sod-3, mtl-1, mtl-2 and cyp-35A2 by supplementary LA under 1 μM decreased oxidative stress and increased the worm's lifespan. In conclusion, our study reveals that supplementary LA possesses both pros and cons in worm physiology and provides new suggestions for LA intake administration in childhood.

Exploring Effects of C. elegans Protective Natural Microbiota on Host Physiology.

The Caenorhabditis elegans natural microbiota was described only recently. Thus, our understanding of its effects on nematode physiology is still in its infancy. We previously showed that the C. elegans natural microbiota isolates Pseudomonas lurida MYb11 and P. fluorescens MYb115 protect the worm against pathogens such as Bacillus thuringiensis (Bt). However, the overall effects of the protective microbiota on worm physiology are incompletely understood. Here, we investigated how MYb11 and MYb115 affect C. elegans lifespan, fertility, and intestinal colonization. We further studied the capacity of MYb11 and MYb115 to protect the worm against purified Bt toxins. We show that while MYb115 and MYb11 affect reproductive timing and increase early reproduction only MYb11 reduces worm lifespan. Moreover, MYb11 aggravates killing upon toxin exposure. We conclude that MYb11 has a pathogenic potential in some contexts. This work thus highlights that certain C. elegans microbiota members can be beneficial and costly to the host in a context-dependent manner, blurring the line between good and bad.

Vitellogenins - Yolk Gene Function and Regulation in Caenorhabditis elegans.

Vitellogenins are a family of yolk proteins that are by far the most abundant among oviparous animals. In the model nematode Caenorhabditis elegans, the 6 vitellogenins are among the most highly expressed genes in the adult hermaphrodite intestine, which produces copious yolk to provision eggs. In this article we review what is known about the vitellogenin genes and proteins in C. elegans, in comparison with vitellogenins in other taxa. We argue that the primary purpose of abundant vitellogenesis in C. elegans is to support post-embryonic development and fertility, rather than embryogenesis, especially in harsh environments. Increasing vitellogenin provisioning underlies several post-embryonic phenotypic alterations associated with advancing maternal age, demonstrating that vitellogenins can act as an intergenerational signal mediating the influence of parental physiology on progeny. We also review what is known about vitellogenin regulation – how tissue-, sex- and stage-specificity of expression is achieved, how vitellogenins are regulated by major signaling pathways, how vitellogenin expression is affected by extra-intestinal tissues and how environmental experience affects vitellogenesis. Lastly, we speculate whether C. elegans vitellogenins may play other roles in worm physiology.

Role of CYP eicosanoids in the regulation of pharyngeal pumping and food uptake in Caenorhabditis elegans

Cytochrome P450 (CYP)-dependent eicosanoids comprise epoxy- and hydroxy-metabolites of long-chain PUFAs (LC-PUFAs). In mammals, CYP eicosanoids contribute to the regulation of cardiovascular and renal function. Caenorhabditis elegans produces a large set of CYP eicosanoids; however, their role in worm's physiology is widely unknown. Mutant strains deficient in LC-PUFA/eicosanoid biosynthesis displayed reduced pharyngeal pumping frequencies. This impairment was rescued by long-term eicosapentaenoic and/or arachidonic acid supplementation, but not with a nonmetabolizable LC-PUFA analog. Short-term treatment with 17,18-epoxyeicosatetraenoic acid (17,18-EEQ), the most abundant CYP eicosanoid in C. elegans, was as effective as long-term LC-PUFA supplementation in the mutant strains. In contrast, 20-HETE caused decreased pumping frequencies. The opposite effects of 17,18-EEQ and 20-HETE were mirrored by the actions of neurohormones. 17,18-EEQ mimicked the stimulating effect of serotonin when added to starved worms, whereas 20-HETE shared the inhibitory effect of octopamine in the presence of abundant food. In wild-type worms, serotonin increased free 17,18-EEQ levels, whereas octopamine selectively induced the synthesis of hydroxy-metabolites. These results suggest that CYP eicosanoids may serve as second messengers in the regulation of pharyngeal pumping and food uptake in C. elegans.

The soft genome

Caenorhabditis elegans (C. elegans) nematodes transmit small RNAs across generations, a process that enables transgenerational regulation of genes. In contrast to changes to the DNA sequence, transgenerational transmission of small RNA-mediated responses is reversible, and thus enables "soft" or "flexible" inheritance of acquired characteristics. Until very recently only introduction of foreign genetic material (viruses, transposons, transgenes) was shown to directly lead to inheritance of small RNAs. New discoveries however, demonstrate that starvation also triggers inheritance of endogenous small RNAs in C.elegans. Multiple generations of worms inherit starvation-responsive endogenous small RNAs, and starvation also results in heritable extension of the progeny's lifespan. In this Commentary paper we explore the intriguing possibility that large parts of the genome and many additional traits are similarly subjected to heritable small RNA-mediated regulation, and focus on the potential influence of transgenerational RNAi on the worm's physiology. While the universal relevance of this mechanism remains to be discovered, we will examine how the discoveries made in worms already challenge long held dogmas in genetics and evolution.

Imatinib treatment causes substantial transcriptional changes in adult Schistosoma mansoni in vitro exhibiting pleiotropic effects.

BackgroundSchistosome parasites cause schistosomiasis, one of the most important infectious diseases worldwide. For decades Praziquantel (PZQ) is the only drug widely used for controlling schistosomiasis. The absence of a vaccine and fear of PZQ resistance have motivated the search for alternatives. Studies on protein kinases (PKs) demonstrated their importance for diverse physiological processes in schistosomes. Among others two Abl tyrosine kinases, SmAbl1 and SmAbl2, were identified in Schistosoma mansoni and shown to be transcribed in the gonads and the gastrodermis. SmAbl1 activity was blocked by Imatinib, a known Abl-TK inhibitor used in human cancer therapy (Gleevec/Glivec). Imatinib exhibited dramatic effects on the morphology and physiology of adult schistosomes in vitro causing the death of the parasites.Methodology/Principal FindingsHere we show modeling data supporting the targeting of SmAbl1/2 by Imatinib. A biochemical assay confirmed that SmAbl2 activity is also inhibited by Imatinib. Microarray analyses and qRT-PCR experiments were done to unravel transcriptional processes influenced by Imatinib in adult schistosomes in vitro demonstrating a wide influence on worm physiology. Surface-, muscle-, gut and gonad-associated processes were affected as evidenced by the differential transcription of e.g. the gynecophoral canal protein gene GCP, paramyosin, titin, hemoglobinase, and cathepsins. Furthermore, transcript levels of VAL-7 and egg formation-associated genes such as tyrosinase 1, p14, and fs800-like were affected as well as those of signaling genes including a ribosomal protein S6 kinase and a glutamate receptor. Finally, a comparative in silico analysis of the obtained microarray data sets and previous data analyzing the effect of a TGFβR1 inhibitor on transcription provided first evidence for an association of TGFβ and Abl kinase signaling. Among others GCP and egg formation-associated genes were identified as common targets.Conclusions/SignificanceThe data affirm broad negative effects of Imatinib on worm physiology substantiating the role of PKs as interesting targets.

Bacterial ncRNAs alter gene expression in worms

This study shows that endogenous bacterial non-coding RNAs (ncRNAs) can affect gene expression in animals that eat the bacteria. Liu and colleagues showed that the Escherichia coli ncRNAs OxyS and DsrA suppress the expression of two Caenorhabditis elegans genes — che-2 and F42G9.6, respectively — when bacteria are ingested by worms that express these ncRNAs. These effects on gene expression have resulting effects on worm physiology, resulting in impaired chemosensation in the case of che-2 and decreased lifespan in the case of F42G9.6.

Vaccination of goats with glyceraldehyde-3-phosphate dehydrogenase DNA vaccine induced partial protection against Haemonchus contortus

Owing to its critical functions in worm physiology, glyceraldehyde-3-phosphate dehydrogenase in Haemonchus contortus (HcGAPDH) is potential candidates for vaccine to control haemonchosis. In this study, DNA vaccine expressing HcGAPDH antigen was tested for protection against experimental H. contortus infections in goats. Fifteen goats (9–10months of age) were allocated into three trial groups. Group 1 was vaccinated with HcGAPDH DNA vaccine twice on days 0 and 14, and then challenged with 5000 infective H. contortus L3 (third stage larvae) on days 28. Group 2 was an unvaccinated positive control group challenged with H. contortus L3 on days 28. Group 3 was an unvaccinated negative control group that was not challenged with L3. By the method of RT-PCR and Western-blot, transcription and expression of HcGAPDH DNA vaccine were identified at local injection sites post immunizations, respectively. After immunization with the DNA vaccine, significantly high levels of serum IgG, serum IgA, mucosal IgA, CD4+ T lymphocytes and B lymphocytes were generated. Also, increased numbers of blood eosinophils and decreased haemoglobin level after challenge were observed in the vaccinated group. Meanwhile, cumulative mean faecal worm egg counts and worm burdens in vaccinated group were reduced by 34.9% and 37.73%, respectively. In brief, recombinant HcGAPDH DNA vaccine induced partial immune response against H. contortus infection in goats.

Vaccination of goats with a glutathione peroxidase DNA vaccine induced partial protection against Haemonchus contortus infection

Due to their critical functions in worm physiology, glutathione peroxidases in Haemonchus contortus are potential candidates for vaccine to control haemonchosis. However, information on the protection potential of these molecules is lacking. In this study, recombinant HC29 antigen was tested for protection against experimental H. contortus infections in goats. Fifteen animals were allocated into three trial groups. The animals of HC29 group was vaccinated with a recombinant HC29 DNA vaccine twice at day 0 and day 14, and then challenged with 5000 infective H. contortus L3 (third larval stage) on the 28th day. An unvaccinated positive control group was challenged with L3 at the same time. An unvaccinated negative control group was not challenged with L3. The results indicated HC29 DNA vaccine was transcribed at local injection sites and expressed in vivo post immunizations, respectively. Following L3 challenge, the mean eggs per gram feces and worm burdens of HC29 group were reduced by 36.1% and 35.6% respectively compared to the positive control group. After immunization with the DNA vaccine, significantly high levels of serum IgG, serum IgA, mucosal IgA and CD4+ T lymphocytes were detected. These results suggest that recombinant H. contortus HC29 glutathione peroxidase DNA vaccine induced a partial immune response and has protective potentials against caprine haemonchosis.

Vaccination of goats against Haemonchus contortus with a recombinant cysteine protease

Abstract Due to critical functions in worm physiology, Haemonchus contortus cysteine proteases are potential candidates for immunological control of haemonchosis. However, only limited information is available on the immunoprotective potential of these molecules in goat. In the present study, HC58 DNA vaccine, a cysteine protease of adult H. contortus worms, was evaluated for protection against homologous challenge infection. Sixteen goats were allocated to four experimental groups of 4 animals each. HC58 DNA vaccine was administered 28 and 14 days before oral challenge with a dose of 5000 infective H. contortus L 3 (third larval stage). HC58 DNA vaccine was transcribed at local muscle injection sites and expressed in vivo 7 and 10 days after primary and booster vaccination, respectively. The group (mean ± S.D.) fecal eggs per gram count (FEC), percentage L 3 and worm burdens for Group 1 (HC58 DNA vaccine) dropped by 28.02, 47.6 and 28.3%, respectively. Corresponding reductions for Group 2 (recombinant HC58 protein) were 26.36, 31.6 and 16.1%, respectively ( P P 3 challenge. These findings suggested that HC58 DNA vaccine conferred some protection against H. contortus infection in goat.

A worm's life

Despite its relative anatomic simplicity, the nematode Caenorhabditis elegans (C. elegans) is a complex multicellular organism. In this review, we describe studies that have contributed to a better understanding of certain aspects of the worm's physiology. We focus on the cellular and molecular basis of the interaction between C. elegans and its environment, including its sensory capacities, the intrinsic biological clock that governs the speed of its life, and on some of the factors that control its life span. We also outline very recent findings that have demonstrated the existence of an innate immune system in C. elegans. Finally, we highlight a number of novel techniques that are transforming the worm from a largely genetic model system into an attractive organism for functional genomic studies.

Fatty acid patterns of different stages of Oesophagostomum dentatum and Oesophagostomum quadrispinulatum as revealed by gas chromatography

Total methylated fatty acid patterns of various developmental stages (third-stage larvae (L3), L3 and fourth-stage larvae (L4) cultured in vitro, L4 and female and male adults derived from intestinal contents) of the porcine nodular worms Oesophagostomum dentatum and Oesophagostomum quadrispinulatum and their cultivation medium were analysed by gas chromatography using Microbial Identification computer software. Fatty acids ranging from C-12 to C-20 could be separated. For each stage and species, characteristic patterns were found. The most prevalent fatty acids were C-18. The freshly exsheathed larvae contained the greatest variety of fatty acids (including short-chain fatty acids C-12 to C-15) with approximately equal amounts of fatty acids with odd and even chain lengths, whereas more advanced stages consisted of a lower number of fatty acids with mostly even chain lengths ≥C-16. Intestinal stages contained less odd-numbered fatty acids and less branched fatty acids than others. In contrast to intestinal L4, cultivated L4 had high amounts of C-15:0 and C-17:0. Sheathed L3 contained more C-18 than freshly exsheathed ones, and medium incubated for 7 days in the presence of parasites contained C-13 to C-15 and monounsaturated C-16, but less C-18 and C-20:4 than fresh medium or medium incubated without worms. Based on the evaluation of stage- and species-specific fatty acid patterns random samples could be assigned to the correct stage and species. In a dendrogram based on fatty acid patterns the same stages of the two species formed the closest relationships, and the intestinal stages formed a clade distinct from the cultivated larvae and L3. All stages contained considerable relative amounts of arachidonic acid, the main precursor of eicosanoids. The fixed differences between species and stages indicate genetic regulation of fatty acid patterns, while environmental influences are mirrored by differences between cultivated and intestinal stages. Regulation of fatty acid patterns probably plays a role in worm physiology and host-parasite interaction.

Effects of chronic metal exposure and sediment organic matter on digestive absorption efficiency of cadmium by the deposit‐feeding polychaete Capitella species I

AbstractOrganic matter such as humic acid and bacterial slime exopolymer are common in estuarine and coastal sediments, where they are ingested by animals that process particulate detritus. Both humic acid (HA; refractory) and exopolymer (EPS; easily digestible) bind metals and therefore might represent a source of particulate‐bound metals to deposit‐feeding organisms. This study examined how cadmium preexposure (3 and 30 μg Cd/g dry wt. sed.), gut passage time (GPT), and quality and quantity of the organic coating on sediment particles interact to determine cadmium absorption efficiency (Cd‐AE) in Capitella sp. I. Pulse‐chase experiments using 109Cd and 51Cr were used to determine Cd‐AE in individual worms. Worms were given a pulse of carbon‐cleaned, HA‐coated or EPS‐coated sediment particles. The third treatment was divided into three EPS concentrations (high, medium, and low). A 5‐d preexposure to cadmium did not affect the egestion rates during either the preexposure period or the chase phase. Worms given a pulse of carbon‐cleaned particles exhibited higher egestion rates during the chase phase than worms given a pulse of organic‐coated particles, and no differences were seen in egestion rate between worms exposed to HA‐ and high‐EPS‐coated particles. Egestion rates decreased with increasing EPS concentration. The presence of refractory organic material decreased the absorption efficiency of cadmium from sediment relative to Cd‐AE from carbon‐cleaned sediment but not relative to Cd‐AE from sediment coated with a high concentration of EPS. The Cd‐AE increased linearly with increasing exopolymer coating on sediment particles. Overall, Cd‐AE increased with increasing gut passage time in worms that were not preexposed, although Cd‐AE from HA‐coated particles was independent of gut passage time. Preexposure to cadmium reversed the relationship between gut passage time and cadmium absorption efficiency. Thus, the implications are that sedimentary organic matter and worm physiology might be especially important in controlling metal bioavailability in deposit‐feeding organisms and should be considered in sediment quality approaches.

EFFECTS OF CHRONIC METAL EXPOSURE AND SEDIMENT ORGANIC MATTER ON DIGESTIVE ABSORPTION EFFICIENCY OF CADMIUM BY THE DEPOSIT-FEEDING POLYCHAETE CAPITELLA SPECIES I

Organic matter such as humic acid and bacterial slime exopolymer are common in estuarine and coastal sediments, where they are ingested by animals that process particulate detritus. Both humic acid (HA; refractory) and exopolymer (EPS; easily digestible) bind metals and therefore might represent a source of particulate-bound metals to deposit-feeding organisms. This study examined how cadmium preexposure (3 and 30 μg Cd/g dry wt. sed.), gut passage time (GPT), and quality and quantity of the organic coating on sediment particles interact to determine cadmium absorption efficiency (Cd-AE) in Capitella sp. I. Pulse-chase experiments using 109Cd and 51Cr were used to determine Cd-AE in individual worms. Worms were given a pulse of carbon-cleaned, HA-coated or EPS-coated sediment particles. The third treatment was divided into three EPS concentrations (high, medium, and low). A 5-d preexposure to cadmium did not affect the egestion rates during either the preexposure period or the chase phase. Worms given a pulse of carbon-cleaned particles exhibited higher egestion rates during the chase phase than worms given a pulse of organic-coated particles, and no differences were seen in egestion rate between worms exposed to HA- and high-EPS-coated particles. Egestion rates decreased with increasing EPS concentration. The presence of refractory organic material decreased the absorption efficiency of cadmium from sediment relative to Cd-AE from carbon-cleaned sediment but not relative to Cd-AE from sediment coated with a high concentration of EPS. The Cd-AE increased linearly with increasing exopolymer coating on sediment particles. Overall, Cd-AE increased with increasing gut passage time in worms that were not preexposed, although Cd-AE from HA-coated particles was independent of gut passage time. Preexposure to cadmium reversed the relationship between gut passage time and cadmium absorption efficiency. Thus, the implications are that sedimentary organic matter and worm physiology might be especially important in controlling metal bioavailability in deposit-feeding organisms and should be considered in sediment quality approaches.