Worm Culture Research Articles

A novel picorna-like virus in the flatworm Stenostomum leucops (Catenulida).

We present the genome sequence and organization and evidence of persistence of a new picorna-like virus infecting the flatworm Stenostomum leucops. The complete genome sequence belongs to a virus with a positive single-stranded RNA genome with two open reading frames (ORFs) flanked by untranslated regions and a polyadenylated C-terminus. The ORFs encode proteins with conserved motifs typical of members of the order Picornavirales. Phylogenetic analysis confirmed membership in this viral order, and it was found to be closely related to viruses found in Biomphalaria (Mollusca) in France and a virus detected in a metagenomic analysis of water sources from the USA, suggesting widespread distribution. RT-PCR analysis revealed that this virus can be detected in a laboratory-grown worm isolate for at least five years, suggesting persistent infection. However, no apparent deleterious effects were observed in the worms in culture, suggesting a possible commensal relationship between the virus and the worms.

Trichuris WAP and CAP proteins: Potential whipworm vaccine candidates?

Trichuris trichiura and T. suis are gastrointestinal dwelling roundworms that infect humans and pigs, respectively. Heavy infections cause gastrointestinal symptoms and impaired growth and development. Vaccination has the potential to reduce the disease burden of whipworm infection; however, there are currently no commercially available vaccines against these parasites and very few against other gastrointestinal-dwelling nematodes of medical and agricultural importance. The naturally occurring mouse whipworm, T. muris, has been used for decades to model human trichuriasis, and the immunogenic potential of the excretory/secretory material (E/S, which can be collected following ex vivo culture of worms) has been studied in the context of vaccine candidate identification. Despite this, researchers are yet to progress an effective vaccine candidate to clinical trials. The T. muris, T. trichiura, and T. suis genomes each encode between 10 and 27 whey acidic protein (WAP) domain-containing proteins and 15 to 34 cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) family members. WAP and CAP proteins have been postulated to play key roles in host-parasite interactions and may possess immunomodulatory functions. In addition, both protein families have been explored in the context of helminth vaccines. Here, we use phylogenetic and functional analysis to investigate the evolutionary relationship between WAP and CAP proteins encoded by T. muris, T. trichiura, and T. suis. We highlight several WAP and CAP proteins that warrant further study to understand their biological function and as possible vaccine candidates against T. trichiura and/or T. suis, based on the close evolutionary relationship with WAP or CAP proteins identified within T. muris E/S products.

Physicochemical and Microbiological Analysis of Vermiwash as an Ecofriendly Soil Conditioner

The continuous use of chemical fertilizer is a leading cause of soil contamination and hence adversely affecting biodiversity. A more suitable alternative to these chemicals are natural organic fertilizers. One of these is vermiwash, a liquid plant growth promoter known to bring about growth enhancement in a wide variety of plants. Vermiwash is a brown colored, odorless, liquid biofertilizer which is collected after it passes through a column of worm culture. It is considered as a storehouse of nutrients and microorganisms and used as a foliar spray for crops. This work was carried out to study the microbiological and physicochemical properties of vermiwash of Eisenia foetida and its application. Vermiwash was produced in a 100 L barrel by adding pre-decomposed municipal wastes along with cow dung (3:1 ratio) and composted with E. foetida. The product was collected at regular intervals of 5 d up to 15 d and subjected to various assays. Higher number of bacteria, and fungi, such as Acromobacter (1.023 × 103), Azatobacter (1.04 × 103), Bacillus baccilli (1 × 103), Bacillus subtillus (1.05 × 103), Micrococcus sp (1.02 × 103), Pseudomonas sp (2 × 103), Actinomycetes (1.24 × 103), and fungus (1.8 × 103) CFU/mL were found in the collected liquid. Nutrients such as nitrogen, phosphorous, potassium, sodium, iron, zinc, and manganese were also found in significant amounts due to earthworm activity. Field trials on Capsicum annum and Lycopersicum esculentum resulted in their enhanced growth. Hence, the high nutrient profile and microbial diversity of the vermiwash makes it a more suitable biofertilizer for enhancing plant growth and soil health.

In vitro and in vivo impacts of nifedipine and diltiazem on praziquantel chemotherapy in murine Schistosoma mansoni

AimThis study was planned to evaluate the in vitro and in vivo antischistosomal effects of the widely used antihypertensive drugs, nifedipine (NIF) and diltiazem (DTZ), and their combinations with praziquantel (PZQ) on early and late Schistosoma (S.) mansoni infections 21- and 45- days old stages. MethodsIn the In vitro study, Calcium channel blockers (CCBs), NIF and DTZ were added to schistosomula and adult worm cultures in different concentrations 10, 20 and 30 mg/ml. The mortality percentage was calculated 1, 12 and 24 h after incubation. In vivo, NIF and DTZ either alone or combined with PZQ were used to treat male albino mice. The parasitological and total immunoglobulin (Ig) G and IgM anti-soluble egg antigen (SEA) were assessed to demonstrate the disease severity. ResultsIn the In vitro study, 10 mg/ml NIF induced 100% mortality percentage of both schistosomula and adult worms after 24 h incubation, while DTZ induced similar mortality percentage at 30 mg/ml concentration. In vivo results showed that early or late combination of 30 mg/kg of NIF, but not DTZ, significantly (P <0.05) enhanced the reductive efficacy of PZQ based on the parasitological data. The maximal reduction (P <0.05) of anti-SEA IgM and IgG levels was developed during NIF-PZQ administration 21- (1.12 ± 0.06 and 1.09 ± 0.04, respectively) or 45- (1.00 ± 0.03 and 0.8 ± 0.06, respectively) days post infection (PI), compared to either PZQ or NIF individual treatments. The decreased concentration of anti-SEA antibodies was correlated with the diminished granulomatous diameter and disease severity. ConclusionNifedipine improved PZQ chemotherapy targeting either early or late S. mansoni infection in mice compared to the PZQ mono-therapy. Administering NIF can be considered as a promising drug candidate for schistosomiasis chemotherapy.

Genome-wide detection of ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing.

Many conventional genetic toxicology assays require specialized cell cultures or animals and can only detect mutations that inactivate the function of a reporter gene. These limitations make such assays incompatible with many toxicological models but could be overcome by the development of techniques capable of directly detecting genome-wide somatic mutations through DNA sequencing. PacBio sequencing can generate almost error-free consensus reads by repeatedly inspecting both DNA strands from circularized molecules (a method known as PacBio HiFi). In this study, we show that PacBio HiFi can detect genome-wide ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms. The mutation frequencies (MFs) of unexposed samples in both models were ~1 × 10-7 mutations per base pair. Compared to these controls, PacBio HiFi detected MF increases of 23-fold in cultures of L5178Y cells exposed to 5 mM ethyl methanosulfonate (EMS) for 4h, and 5-, 12-, and 29-fold in cultures of C. elegans worms exposed to 12.5, 25, and 50 mM EMS for 4h, respectively. In both models, the mutation spectra of controls were diverse, while those derived from EMS-exposed samples were dominated by C:G → T:A transitions. To validate these results, clone sequencing analyses were performed on the same cultures of L5178Y cells. The results obtained by clone sequencing and PacBio HiFi were almost identical. Our results suggest that PacBio sequencing could be used for the detection, quantitation, and characterization of mutations in any DNA-containing sample, including those that are not compatible with conventional mutation detection approaches.

Studying Annelida Regeneration Using Platynereis dumerilii.

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread property in the animal kingdom. This chapter describes methods used to study this fascinating process in the annelid Platynereis dumerilii. During most of its life, this segmented worm is able to regenerate upon amputation the posterior part of its body, including its pygidium (terminal non-segmented body region bearing the anus) and a subterminal posterior growth zone which contains stem cells required for the formation of new segments. Detailed description of Platynereis worm culture and how to obtain large quantity of regenerating worms is provided. We also describe the staging system that we established and three important methods to study regeneration: whole mount in situ hybridization to study gene expression, 5-ethynyl-2'-deoxyuridine (EdU) labeling to characterize cell proliferation, and use of pharmacological treatments to establish putative roles of defined signaling pathways and processes.

The flavonoid Licochalcone A induces tegumental damages in Schistosoma mansoni and impairs its oviposition in vitro

In this study, Licochalcone A (LicoA) was investigated in in vitro and in vivo assays. The survival of worms in culture, the pattern of oviposition, the count of intact tubers and the integrity of the coat were adopted in the in vitro tests. After the animals were perfused, the number of worms recovered, their location and the oogram study were the parameters analyzed to signal the existence of potential schistosomicidal activity in vivo. We observed reduction on the survival, integument integrity and reproduction of adult worms in vitro. Murine models did not show a significant difference in the parasitological parameters analyzed that indicate activity against the worms with an oral single dose of 25 mg/ kg of LicoA or two intraperitoneal injection of 50 mg/ kg LicoA. Nevertheless, it is too early to completely exclude the schistosomicide activity of LicoA, considering that the used dosage form could not provide a regular absorption of the drug.

Probable role of Cutibacterium acnes in the gut of the polychaete Capitella teleta

Capitella teleta, a marine polychaete that feeds on a refractory diet consisting of sediment, was shown to contain unique gut microbiota comprised of microbial functional groups involved in fermentation. Results of our previous studies showed that C. teleta's core gut microbiota were dominated by propionibacteria, and that these bacteria were more abundant in worms than in sediment and feces. In order to test the hypothesis that the worm nutritionally benefits from its gut microbiota, we identified, and genetically and biochemically characterized Cutibacterium acnes strains (formerly Propionibacterium acnes) that were isolated from the gut of C. teleta. Here we show that 13 worm-isolated Cutibacterium acnes strains primarily belonged to phylotype group IB, likely as a clonal population. We also provide evidence that all tested strains produced propionate and vitamin B12, which are essential host-requiring microbial metabolites. The presence of C. acnes in C. teleta was not unique to our worm culture and was also found in those obtained from geographically distant laboratories located in the U.S. and Europe. Moreover, populations of worm gut-associated C. acnes increased following antibiotic treatment. Collectively, results of this study demonstrated that C. acnes is a member of the worm's core functional microbiota and is likely selectively favored by the physiology and chemistry of the host gut environment. To our knowledge, this is the first report of the presence of C. acnes in the C. teleta gut. Our data strongly suggest that C. acnes, a bacterium previously studied as an opportunistic pathogen, can likely act as a symbiont in C. teleta providing the host essential nutrients for survival, growth, and reproduction.

Identification and Probiotic Potency Screening of Cellulolytic Bacilli Isolated from Fecal Pellets and Gastrointestinal Tract of Nypha Worm (Namalycastis rhodochorde), West Kalimantan, Indonesia

The bacterial isolates NrLtF1, NrLtF4, NrLtF5, and NrLtG2 isolated from fecal pellets and gastrointestinal tract of nypha worms (Namalycastis rhodochorde) have cellulolytic, proteolytic activity and produce organic acids. The four isolates have the potency to be developed as probiotics in nypha worm cultivation feed. This study aims to determine the probiotics potency and identify the species of NrLtF1, NrLtF4, NrLtF5, and NrLtG2 isolate based on 16srDNA sequence. The probiotic potency was carried out by the acid tolerance assays on distilled water and 0.3% acid bile media, and the antimicrobial testing against Escherichia coli (MF exp21.12). Bacterial identification was carried out by sequencing of 16sDNA sequence based on GeneBank data. The results showed that the bacterial isolates of NrLtF1, NrLtF4, NrLtF5, and NrLtG2 were able to grow on 0.3% distilled water and acid bile media. However, only the NrLtF4 and NrLtF5 inhibited E. coli (MF exp21.12) with halo zones 30 mm and 18 mm, respectively. Blasting results of the 16srDNA sequences showed that the NrLtF1, NrLtF4, NrLtF5, and NrLtG2 were closely related to Bacillus wiedmannii, Brevibacterium sediminis, Bacillus proteolyticus, and Bacillus paramycoides. The nypha worm bacterial isolates have the potency to be developed as probiotics in nypha worm culture.

Identification of hit compounds with anti-schistosomal activity on in vitro generated juvenile worms in cell-free medium.

BackgroundAnthelminthic treatment options against schistosomiasis are limited. The current treatment relies almost exclusively on a single drug, praziquantel (PZQ). As a consequence, the development of resistance to PZQ and limited activity of PZQ against earlier development stages are respectively a risk and a limitation to achieving the goals of the new WHO roadmap towards elimination. For the discovery of new chemical starting points, the in vitro drug screening on Schistosoma mansoni (S. mansoni) against newly transformed schistosomula (NTS) is still the most predominant approach. The use of only NTS in the initial screening limits sensitivity to potential new compounds which are predominantly active in later developmental stages. Using our recently described highly standardized, straightforward and reliable culture method that generates high rates of juvenile worms, we aimed to repurpose a subset of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (340 compounds) to identify new hits with an in vitro worm culture assay.Methodology/Principal findingsCercariae were mechanically transformed into skin-stage (SkS) schistosomula and continuously cultured for 3–6 weeks to the liver stage (LiS). A commercial source of serum was identified, and decrease of NTS/well along with optimal drug testing conditions was established to test compounds on early and late LiS worms. The library was screened in 96-well format assays using praziquantel (PZQ) as a positive control. Primary screening allowed a 5.9% hit rate and generated two confirmed hits on adult worms; a prophylactic antianginal agent and an antihistaminic drug.ConclusionWith this standardized and reliable in vitro assay, important S. mansoni developmental stages up to LiS worms can be generated and cultured over an extended period. When exposed to a subset of the NCATS Pharmaceutical Collection, 3 compounds yielded a defined anti-schistosomal phenotype on juvenile worms. Translation of activity on perfused adult S. mansoni worms was achieved only for perhexiline (a prophylactic antianginal agent) and astemizole (an antihistaminic drug).

Budidaya Cacing Lumbricus Rubellus dengan Media Limbah Jamur sebagai Bahan Dasar Kosmetik dan Obat-Obatan

Merjosari Village in Malang, which used to be still an agricultural area, has gradually narrowed due to the large number of housing developments by developers. This has an impact on reduced agricultural output, especially chili and tomatoes. Farmers and laborers are unemployed because of the decreasing number of their agricultural lands. The program aims to provide training on the use of mushroom waste in the form of grajen into worm breeding media, program implementation methods by providing outreach programs, training, and providing worm culture facilities and infrastructure for farmers. The results of the program show that farmers and laborers in Merjosari Village can carry out their own cultivation of the lumbricus rubellus worm by utilizing mushroom baglog waste (grajen). The results of the worm cultivation can be sold in the form of worms that can be used as animal feed, birds / poultry, fish, shrimp and so on, the basic ingredients of cosmetics and medicines, and worm feces (vermicompost) used for high-quality organic fertilizers, especially for plants such as flowers and fruit. For farmers and other communities, this program can create self-employment opportunities (entrepreneurship), improve the economy and family welfare, participate in maintaining the balance of ecosystems and the environment. DOI: https://doi.org/10.26905/abdimas.v5i2.4407

Effects of Wetting Media Cattle Blood, Rice Gruel and Water on the Yield and Amino Acid Composition of Tubificid Worms

The effects of three wetting media (cattle blood, rice gruel and subsurface clean water) of the culture media ingredients (a mixture of mustard oil cake, soybean meal and mud) on the yield and amino acid composition of tubificid worms cultured on a pilot scale in a fish farm for 42 days were evaluated. The worm yields were in the order: cattle blood wetted media (683.68 ± 3.86 mg/cm2), rice gruel (615.63 ± 3.66 mg/cm2), water (584. 38 ± 1.41 mg/cm2). Lysine, arginine and leucine were the most prevalent of the eight essential amino acids in worms cultured in the media wetted in cattle blood medium. The results suggested that a mixture of 40% mustard oil cake, 30% soybean meal and 30% mud can be used for the commercial culture of tubificid worms as live fish food. The tubificid worms showed high level of amino acids suitable for fish that require a high content of dietary amino acids.&#x0D; Asiat. Soc. Bangladesh, Sci. 45(2): 229-239, December 2019

Automated Platform for Long-Term Culture and High-Content Phenotyping of Single C. elegans Worms

The nematode Caenorhabditis elegans is a suitable model organism in drug screening. Traditionally worms are grown on agar plates, posing many challenges for long-term culture and phenotyping of animals under identical conditions. Microfluidics allows for ‘personalized’ phenotyping, as microfluidic chips permit collecting individual responses over worms’ full life. Here, we present a multiplexed, high-throughput, high-resolution microfluidic approach to culture C. elegans from embryo to the adult stage at single animal resolution. We allocated single embryos to growth chambers, for observing the main embryonic and post-embryonic development stages and phenotypes, while exposing worms to up to 8 different well-controlled chemical conditions. Our approach allowed eliminating bacteria aggregation and biofilm formation-related clogging issues, which enabled us performing up to 80 hours of automated single worm culture studies. Our microfluidic platform is linked with an automated phenotyping code that registers organism-associated phenotypes at high-throughput. We validated our platform with a dose-response study of the anthelmintic drug tetramisole by studying its influence through the life cycle of the nematodes. In parallel, we could observe development effects and variations in single embryo and worm viability due to the bleaching procedure that is standardly used for harvesting the embryos from a worm culture agar plate.

Doxycycline hyclate: A schistosomicidal agent in vitro with immunomodulatory potential on granulomatous inflammation in vivo

We investigated the effect in vitro and in vivo of doxycycline hyclate (Dx), a broad-spectrum antibiotic inhibitor of matrix metaloproteinases (MMPs), on adult Schistosoma mansoni worms and granulomatous liver inflammation in infected mice. Adult S. mansoni worms in culture treated with different concentrations of Dx (50–180 μg/mL) were studied for eight days to assess its morphology, eggs production, and mortality. Uninfected mice and those infected with S. mansoni, untreated and treated with praziquantel (Pz; 200 mg/kg) or Dx (50 mg/kg), were evaluated for 60 days. Our results indicated that Dx induced dose-dependent integumentary lesions (bubbles, tubercle collapse, spicule disappearance, peeling, and erosion), reduced mating rate and eggs-laying in adult S. mansoni worms. The effective lethal dose required to kill 50% of worms was 112.0 μg/mL Dx (DL50). In mice, S. mansoni infection induced hepatomegaly, intense IL-4, IL-10, TNF-α and TGF-β production, granulomatous inflammation and hepatic glycogen depletion. The number and size of the granulomas was similar in untreated and Dx-treated animals. Untreated animals showed a predominance of productive granulomas, and intense MMP-2 and MMP-9 activities. Dx-treated mice exhibited a significant increase in IL-4 levels, tissue inflammation, proportion of involutive granulomas, and hepatic collagenogenesis, as well as attenuated MMP-2 and MMP-9 activities. Our findings indicated that Dx is toxic to adult S. mansoni worms in vitro. However, in vitro beneficial effects were not reproduced in vivo, since Dx treatment increased liver granulomatous inflammation and collagenogenesis in S. mansoni-infected mice by a process potentially associated with Dx-mediated hepatic MMP-2 and MMP-9 inhibition.

Budi daya cacing sutra (Tubifex sp.) dengan sistim air mengalir di Balai Perikanan Budidaya Air Tawar Tatelu (BPBAT), Propinsi Sulawesi Utara

The field work training of silk worm culture was done in one month at Balai Perikanan Budidaya Air Tawar Tatelu (BPBAT), Province of North Sulawesi, from May to June, 2016. The media used are poultry dung (chicken manure) and EM4 with a flowing water system and fed with additional tofu waste. The initial spread of silk worm seeds was 75 g / m2, and the results obtained were silk worm biomass of 150 g / m2, with the measurement of water quality parameters for DO, temperature and pH are respectively 1.61 ppm, 24.4-27.7° C and 6.1-7.3.Keywords: aquaculture, life feed, silk worm, Tubifex sp., water quality

The use of primary murine fibroblasts to ascertain if Spirocerca lupi secretory/excretory protein products are mitogenic ex vivo

BackgroundSpirocerca lupi is a nematode that parasitizes vertebrates in particular canids, by forming nodules in the thoracic cavity specifically in the oesophagus. In 25% of Spirocerca infections of the domestic dog, nodules progress from inflammatory to pre-neoplastic to sarcomatous neoplasia. With the mechanism of neoplastic transformation being incompletely understood, this study investigates if S. lupi parasite proteinaceous secretory/excretory products (ESPs) play a role in the neoplastic transformation.MethodsTo facilitate collection of ESPs, we maintained naturally harvested adult parasites in the laboratory under artificial conditions. Media in which the parasites were grown was subsequently evaluated for the presence of proteinaceous compounds using a mass spectroscopy library as well as for their ability to be mitogenic in primary murine fibroblastic cells.ResultsChromatrography of the ethyl acetate extracted incubation media showed the presence of 9 protein compounds, of which three were identified as non-specific proteins isolated from Nematostella vectensis, Caenorhabditis brenneri and Sus scrofa, with the rest being unknown. Acetone, methanol, hexane and ethylacetate extracted culture media were unable to induce a mitogenic change in primary murine fibroblasts in comparison to the controls.ConclusionWhile no mitogenic effect was evident, further studies are required to understand the role of worm excretory/secretory products on clastogenesis under chronic exposure. In addition, while not of primary importance for this study, the observed duration of parasite survival indicates that ex vivo studies on S. lupi are possible. For the latter we believe that the worm culture method can be further optimized if longer survival times are required.

Production and nutritional composition of white worms Enchytraeus albidus fed different low-cost feeds

The marine oligochaete, white worm, Enchytraeus albidus (Henle, 1837), was evaluated as a potential live feed by exploring its production capacity and nutritional composition fed coffee grounds, spent brewing grains, stale bread, mixed produce, or sugar kelp over the course of 6-, 9-, or 12-week production cycles. Feed type and production cycle duration affected white worm biomass, reproductive potential, and proximate and fatty acid composition. In general, white worm cultures fed coffee grounds, stale bread, and spent brewing grains had higher production yields than cultures fed mixed produce or sugar kelp. Dependent on feeds and production cycle duration, white worms were high in protein (49–69%) and lipids (10–27%) and low in ash (5–8%), indicating that they would meet the dietary needs of species requiring a high protein, relatively high lipid, low ash diet. Compared to fatty acid profiles reported for standard live feeds like rotifers, Artemia, and copepods, white worms provided less n-3 long-chain polyunsaturated fatty acid content (DHA 0–0.5%, EPA 2–18%, total LC-PUFAs 4–25%), with the highest levels in worms fed mixed produce or sugar kelp. White worms exhibit many attractive characteristics as feeds, but commercialization will require improved culture techniques to produce greater worm biomass while reducing production costs. Depending on the target species, white worms may need enrichment to increase n-3 LC-PUFA levels.

Logarithmic bacterial gradient chip for analyzing the effects of dietary restriction on C. elegans growth

C. elegans is a commonly used model organism for studying the basic mechanisms of lifespan extension by dietary restriction (DR). Progress has been limited, however, by the lack of an automated system for quantitative analysis of this effect of bacterial diet. In this work, multifunctional logarithmic gradient-customizing microfluidic device, which takes advantage of hydrodynamics, was developed to long-term maintain worm culture on the chip with parallel live imaging and generate logarithmic concentration gradient of bacteria. We successfully employed this platform to investigate the longevity and stress response of the worms in response to various concentrations of bacterial food. The results indicated that the lifespan extension of the worms was induced by DR when bacteria density was 108cellsmL−1 and further dilutions would reduce the lifespan, which proposed a new dietary restriction regime in C. elegans. In addition, imaging analysis showed that DR regulated the subcellular localization DAF-16 in a way different from starvation that reduced lifespan of the worms. The platform allows not only horizontal studies of C. elegans in response to various environments over a wide range of concentrations, but also multiparameter evaluation of longevity in the worms with high temporal resolution, providing clues to better understand the aging process and help age-related drug screen.

Structure of a pentameric virion-associated fiber with a potential role in Orsay virus entry to host cells.

Despite the wide use of Caenorhabditis elegans as a model organism, the first virus naturally infecting this organism was not discovered until six years ago. The Orsay virus and its related nematode viruses have a positive-sense RNA genome, encoding three proteins: CP, RdRP, and a novel δ protein that shares no homology with any other proteins. δ can be expressed either as a free δ or a CP-δ fusion protein by ribosomal frameshift, but the structure and function of both δ and CP-δ remain unknown. Using a combination of electron microscopy, X-ray crystallography, computational and biophysical analyses, here we show that the Orsay δ protein forms a ~420-Å long, pentameric fiber with an N-terminal α-helical bundle, a β-stranded filament in the middle, and a C-terminal head domain. The pentameric nature of the δ fiber has been independently confirmed by both mass spectrometry and analytical ultracentrifugation. Recombinant Orsay capsid containing CP-δ shows protruding long fibers with globular heads at the distal end. Mutant viruses with disrupted CP-δ fibers were generated by organism-based reverse genetics. These viruses were found to be either non-viable or with poor infectivity according to phenotypic and qRT-PCR analyses. Furthermore, addition of purified δ proteins to worm culture greatly reduced Orsay infectivity in a sequence-specific manner. Based on the structure resemblance between the Orsay CP-δ fiber and the fibers from reovirus and adenovirus, we propose that CP-δ functions as a cell attachment protein to mediate Orsay entry into worm intestine cells.

Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models.

BackgroundWhile many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions – e.g. neurodegenerative diseases – often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field.ResultsWe describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms’ tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system’s ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model.ConclusionOur microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0083-6) contains supplementary material, which is available to authorized users.