Single Worm Research Articles

TRICHINELLA MURRELLI POZIO AND LA ROSA, 2000 IN A GRAY FOX (UROCYON CINEREOARGENTEUS) FROM PENNSYLVANIA: A NEW HOST RECORD FOR THIS ZOONOTIC NEMATODE.

Trichinella murrelli Pozio and La Rosa, 2000, is the primary sylvatic trichinellid encountered in temperate North America. During a survey for Sarcocystis in wild canids, a single worm matching the morphology of encapsulated Trichinella was observed in a muscle tissue squash from a gray fox male originating from Pennsylvania. The worm was photographed and then separated from the host tissue by artificial digestion, and genomic DNA was extracted from the worm. This DNA was subjected to species-specific multiplex PCR and short-read genomic sequencing. The banding pattern of the multiplex PCR indicated that the worm was T. murrelli, and the sequence of the mitochondrial Cytochrome c oxidase 1 gene and the ribosomal 18S ribosomal RNA, Internal Transcribed Spacer 1, 5.8S ribosomal RNA, Internal Transcribed Spacer 2, and 28S ribosomal RNA confirmed the diagnosis. This is the first report of T. murrelli in gray foxes that includes assays for assigning parasite species. This report confirms suspected data from surveys conducted over 30 yr ago and establishes a new host record for T. murrelli.

Molecular identification and genetic variability of equine and bovine ocular setariasis in india: molecular profiling by mitochondrial genes.

Ocular setariasis is an ectopic infection caused by a parasite under the genus Setaria. Adult worms belong to the Setariidae family and typically reside in the peritoneal cavity of ungulates. However, immature forms of these species may aberrantly migrate to the eyes of cattle, buffalo, goats, horses and several other hosts, leading to corneal opacity and blindness. Here, we have distinguished the Setaria digitata collected from both equine and buffalo hosts based on the morphology, molecular profiling of mitochondrial cytochrome c oxidase subunit 1 (Cox1), cytochrome c oxidase subunit 3 (Cox3) and, Nicotinamide Adenine Dinucleotide dehydrogenase subunit 1 (NAD1) genes. A single filarial worm was collected from the eye of one equine and one bovine host. These worms were then processed for morphological examination and DNA isolation. Cox1, Cox3 and NAD1 genes were amplified using specific primers and subjected to custom sequencing. The sequences were then used for multiple sequence alignment, assessment of entropy, similarity and haplotype diversity analysis. Key morphological features confirmed the worms collected were male and female Setaria digitata from equine and buffalo hosts, respectively. Cox1, Cox3 and NAD1 gene sequence analysis showed a close association of S.digitata Indian isolates with its counterparts from Sri Lanka and China isolates. The phylogram of bovine S. digitata sequences shows a close relationship to other equine S. digitata sequences, indicating the need for further in-depth studies on the prevalence of infection across various host species and intermediate hosts. Although the sequence results suggest that S. digitata is likely the causative agent of ocular setariasis in India, additional samples are needed to confirm this conclusion. Comprehensive analysis of the transcriptome and proteome of S. digitata from both bovine and equine hosts is necessary to explore variations in host-parasite interactions. These findings will aid in future parasite identification, investigations into vector prevalence in India, and the development of control measures against ocular setariasis.

Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.

Autochthonous Ascariasis, Mississippi, USA.

We describe a case of a 2-year-old child who expelled a single adult female Ascaris lumbricoides worm. The patient is from a rural county in Mississippi, USA, with no reported travel outside of the United States. The caregivers in the home practice good sanitation. Exposure to domestic pigs is the likely source of infection.

Single Worm PCR: A Method to Extract and Amplify Genomic DNA

Single Worm PCR: A Method to Extract and Amplify Genomic DNA

Juggling cadmium detoxification and zinc homeostasis: A division of labour between the two C. elegans metallothioneins

The chemical properties of toxic cadmium and essential zinc are very similar, and organisms require intricate mechanisms that drive selective handling of metals. Previously regarded as unspecific “metal sponges”, metallothioneins (MTLs) are emerging as metal selectivity filters. By utilizing C. elegans mtl-1 and mtl-2 knockout strains, metal accumulation in single worms, single copy fluorescent-tagged transgenes, isoform specific qPCR and lifespan studies it was possible to demonstrate that the handling of cadmium and zinc by the two C. elegans metallothioneins differs fundamentally: the MTL-2 protein can handle both zinc and cadmium, but when it becomes unavailable, either via a knockout or by elevated cadmium exposure, MTL-1 takes over zinc handling, leaving MTL-2 to sequester cadmium. This division of labour is reflected in the folding behaviour of the proteins: MTL-1 folded well in presence of zinc but not cadmium, the reverse was the case for MTL-2. These differences are in part mediated by a zinc-specific mononuclear His3Cys site in the C-terminal insertion of MTL-1; its removal affected the entire C-terminal domain and may shift its metal selectivity towards zinc. Overall, we uncover how metallothionein isoform-specific responses and protein properties allow C. elegans to differentiate between toxic cadmium and essential zinc.

Optimal growth and feeding behaviour of the valuable bait Halla parthenopeia (Polychaeta: Oenonidae) in small-scale rearing systems

Commercial farming of polychaetes used as baits in recreational fishing and feed supplements for aquaculture has been regrettably limited to a few species. The rearing conditions and diet that optimize the survival and growth remain largely unknown for most species. This study aimed at improving the current knowledge on the rearing of the large bivalve-feeder polychaete Halla parthenopeia, one of the most appreciated and expensive baits by Mediterranean fishermen. We set up a laboratory rearing system for H. parthenopeia to address several management procedures and solutions aimed at improving the rearing method that has been employed so far. We also provide a detailed reconstruction of feeding behaviour and prey preferences which were unknown for the Mediterranean populations. Finally, we assessed growth when fed on commercial, intact clams or artificially opened clams. The latter fed has been never tested in previous studies on Halla species and simulates commercial waste clams, a potential low-cost resource for polychaete maintenance. The setting up of a grow-out system based on single worms maintained in small PET containers on the bottom of recirculating aquaria permitted the non-stressful manipulation of H. parthenopeia and an easy management of the aquarium system. We observed that polychaetes consumed both C. gallina and R. philippinarum, showing a nocturnal foraging behaviour consisting in three phases: searching, handling, and feeding. The duration of handling + feeding period (1–4 h) depended on bivalve size. Offering intact R. philippinarum clam, a significant positive correlation between the predator and prey size occurred i.e. the smaller the predator, the smaller the prey-size selected, probably because less time and energy were required for handling. The same behaviour was observed also for the congeneric Halla okudai and in H. parthenopeia from the Suez Canal. Polychaetes preferred feeding on open clams than intact clams, independently from clam and worm size. An increase in the daily predation rate (up to 300%) and growth increment (up to 250%) was found for H. parthenopeia feeding on open clam, maybe for the lack of energetic cost for prey handling. These results suggest that an indoor small-scale production of H. parthenopeia fed with waste clams could meet the demand of the angling community and support further biotechnological applications of this polychaete.

Elegant Nematodes Improve Our Understanding of Human Neuronal Diseases, the Role of Pollutants and Strategies of Resilience.

The prevalence of neurodegenerative disorders such as Alzheimer's and Parkinson's disease are rising globally. The role of environmental pollution in neurodegeneration is largely unknown. Thus, this perspective advocates exposome research in C. elegans models of human diseases. The models express amyloid proteins such as Aβ, recapitulate the degeneration of specifically vulnerable neurons and allow for correlated neurobehavioral phenotyping throughout the entire life span of the nematode. Neurobehavioral traits like locomotion gaits, rigidity, or cognitive decline are quantifiable and carefully mimic key aspects of the human diseases. Underlying molecular pathways of neurodegeneration are elucidated in pollutant-exposed C. elegans Alzheimer's or Parkinson's models by transcriptomics (RNA-seq), mass spectrometry-based proteomics and omics addressing other biochemical traits. Validation of the identified disease pathways can be achieved by genome-wide association studies in matching human cohorts. A consistent One Health approach includes isolation of nematodes from contaminated sites and their comparative investigation by imaging, neurobehavioral profiling and single worm proteomics. C. elegans models of neurodegenerative diseases are likewise well-suited for high throughput methods that provide a promising strategy to identify resilience pathways of neurosafety and keep up with the number of pollutants, nonchemical exposome factors, and their interactions.

Experimental investigation for fault diagnosis of a single stage worm gearbox using response surface methodology

Gearbox transmission system is a fundamental factor in the industrial applications. If gearbox stop working due to different faults, it may break normal machine operation and cause a production loss. This research paper focused on fault analysis of combination of worm wheel and bearing by using root mean square (RMS) and response surface method (RSM). Faults considered on outer race and inner race of worm wheel bearing, on worm wheel tooth. These faults and load were considered as an important independent factor to understand their effects on RMS response of worm gearbox. Worm gearbox experimental setup is for laboratory experimentation and RSM for analysis. Twenty-seven experimental trial conducted for three level of parameters based on design of experiment (DOE). Vibration based response measured in frequency domain and root mean square parameter extracted for the fault analysis. Box behnken design RSM is implemented to investigate independent parameters effect on output parameter i.e. RMS. The result shows that effect of faulty inner race bearing is more on worm wheel as compared to faulty outer race bearing and fault parameter influence RMS than load parameter.

Experimental Investigation on Churning Power Loss of Splash Lubricated Worm Gear

Churning power losses are a complex phenomenon that produces critical power losses when considering the splash lubrication of gear units. This article describes the method to investigate the churning power loss in a worm gearbox. A particular test rig was designed and fabricated to experiment on single start worm gear incompletely submerged in an oil bath. The direct torque measurement technique was used to determine the churning power losses. Experiments have been conducted to determine the impact of a variety of operating conditions on churning power losses, including worm speeds, gear immersion depth, lubricant temperatures, and lubricant type (mineral and synthetic). It was found that the churning losses were significantly affected by the worm shaft orientation, speed of gear, and the depth of immersion (static head). The lubricant's temperature is more essential than the type of lubricant in terms of churning power loss.

Physical evidence of direct antagonistic interactions between trematodes in the host gut: the kiss of death?

Although within-host competition among parasites if often assumed to occur based on statistical patterns, actual physical evidence of direct antagonistic interactions between parasites, either intraspecific or interspecific, is very rare. Here, we report such evidence between and within two species of hemiurid trematodes infecting the deep-sea grenadier fish Coryphaenoides subserrulatus. We found pairs of worms attached together, with one worm using its ventral sucker against another worm, and sucking out a large protuberance on the victim. We also found single worms showing clear signs of past attacks. There was no evidence that these interactions were more common at high intensities of infection, where the conditions would be expected to be more conducive to competitive interactions. Our findings provide evidence that trematodes may cause some harm to co-occurring individuals, suggesting a direct form of interference competition among intestinal helminths.

Comparison of two molecular barcodes for the study of equine strongylid communities with amplicon sequencing.

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

Successful Removal of Angiostrongylus cantonensis Larvae from the Central Nervous System of Rats 7- and 14-Days Post-Infection Using a Product Containing Moxidectin, Sarolaner and Pyrantel Embonate (Simparica Trio™) in Experimental Infections

Angiostrongylus cantonensis is a nematode with an indirect lifecycle, using molluscs as intermediate hosts. Rats are the definitive host. By administering a suitable anthelmintic, at an appropriate interval, the risk of clinical neuroangiostrongyliasis occurring in paratenic hosts (e.g., dogs, man) can be eliminated. We wanted to determine if infective larvae (L3) of A. cantonensis can be safely killed during their migration through the central nervous system (CNS) by oral administration of an anthelmintic combination containing moxidectin (480 µg/kg, Simparica Trio™; M-S-P), thereby preventing patent infections in rats. Eighteen rats were used: ten received oral M-S-P every four weeks; eight rats were used as controls. Rats were initially given M-S-P as a chew to eat, but an acquired food aversion meant that subsequent doses were given by orogastric lavage. All 18 rats were challenged once or twice with approximately 30 L3 A. cantonensis larvae via orogastric lavage. Infection status was determined by faecal analysis using the Baermann technique and necropsy examination of the heart, pulmonary arteries and lungs. Eight out of ten rats dosed with M-S-P had zero lungworms at necropsy; a single female worm was detected in each of the remaining two rats. No treated rats had L1 larvae in faeces. In contrast, all eight controls were infected with patent infections, with a median of 14.5 worms per rat detected at necropsy. The difference in infection rates was significant (two tailed Fishers Exact; p = 0.0011). Moxidectin given orally once every month killed migrating larvae before they reached the pulmonary arteries in 80% of treated rats, while in 20%, only a single female worm was present. Considering the short half-life of moxidectin in the rat, it is likely that the effectiveness of moxidectin is due to larvicidal action on migrating L3, L4 and L5 larvae in the brain parenchyma or subarachnoid space, either 7 days (L3/L4 in cerebrum and spinal cord) or 14 days (L4/L5 in cerebrum and subarachnoid space) after inoculation. This study is a prelude for future research to determine if monthly moxidectin administration orally as M-S-P could prevent symptomatic neuroangiostrongyliasis in dogs.

Diversity, Distribution, and Development of Hyperparasitic Microsporidia in Gregarines within One Super-Host.

Metchnikovellids (Microsporidia: Metchnikovellida) are poorly studied hyperparasitic microsporidia that live in gregarines inhabiting the intestines of marine invertebrates, mostly polychaetes. Our recent studies showed that diversity of metchnikovellids might be significantly higher than previously thought, even within a single host. Four species of metchnikovellids were found in the gregarines inhabiting the gut of the polychaete Pygospio elegans from littoral populations of the White and Barents Seas: the eugregarine Polyrhabdina pygospionis is the host for Metchnikovella incurvata and M. spiralis, while the archigregarine Selenidium pygospionis is the host for M. dogieli and M. dobrovolskiji. The most common species in the White Sea is M. incurvata, while M. dobrovolskiji prevails in the Barents Sea. Gregarines within a single worm could be infected with different metchnikovellid species. However, co-infection of one and the same gregarine with several species of metchnikovellids has never been observed. The difference in prevalence and intensity of metchnikovellid invasion apparently depends on the features of the life cycle and on the development strategies of individual species.

Contact between Mesocestoides vogae tetrathyridia induces their division.

Mesocestoides vogae is a cestode of the order Cyclophyllidea, and its second intermediate hosts are mammals, birds, amphibians, and reptiles. The parasite forms a tetrathyridium in the second intermediate host and multiplies asexually, sometimes to the point of filling the host's abdominal cavity. Proliferated tetrathyridium may cause lethal conditions in the host. During the asexual multiplication period, the scolex first replicates into two and then divides into two worms. In this study, to investigate the factors that promote the replication, tetrathyridia were cultured under various in vitro conditions. When several worms which already had two scolexes were cultured together, the division into two worm bodies was complete, but when single worm was cultured, the division hardly proceeded. The result indicates that the division progression of tetrathyridia with two scolexes requires the presence of other worms. In contrast, tetrathyridia with only one scolex did not initiate the division process, whether cultured together or alone. Then, the necessity of direct contact between the bodies of the worms to promote the division of tetrathyridia with two scolexes was assessed. For this, the well of the culture plate was partitioned into upper and lower parts using a mesh, and 20 worms in the upper part and single worm in the lower part were cultured. In all examined wells, worms in the upper part showed complete division, whereas the worms in the lower part rarely completed the division. Thus, direct contact between tetrathyridium promotes the division of tetrathyridia bearing two scolexes.

Automatically tracking feeding behavior in populations of foraging C. elegans.

Caenorhabditis elegans feeds on bacteria and other small microorganisms which it ingests using its pharynx, a neuromuscular pump. Currently, measuring feeding behavior requires tracking a single animal, indirectly estimating food intake from population-level metrics, or using restrained animals. To enable large throughput feeding measurements of unrestrained, crawling worms on agarose plates at a single worm resolution, we developed an imaging protocol and a complementary image analysis tool called PharaGlow. We image up to 50 unrestrained crawling worms simultaneously and extract locomotion and feeding behaviors. We demonstrate the tool's robustness and high-throughput capabilities by measuring feeding in different use-case scenarios, such as through development, with genetic and chemical perturbations that result in faster and slower pumping, and in the presence or absence of food. Finally, we demonstrate that our tool is capable of long-term imaging by showing behavioral dynamics of mating animals and worms with different genetic backgrounds. The low-resolution fluorescence microscopes required are readily available in C. elegans laboratories, and in combination with our python-based analysis workflow makes this methodology easily accessible. PharaGlow therefore enables the observation and analysis of the temporal dynamics of feeding and locomotory behaviors with high-throughput and precision in a user-friendly system.

Exposome, Molecular Pathways and One Health: The Invertebrate Caenorhabditis elegans.

Due to its preferred habitats in the environment, the free-living nematode Caenorhabditis elegans has become a realistic target organism for pollutants, including manufactured nanoparticles. In the laboratory, the invertebrate animal model represents a cost-effective tool to investigate the molecular mechanisms of the biological response to nanomaterials. With an estimated number of 22,000 coding genes and short life span of 2–3 weeks, the small worm is a giant when it comes to characterization of molecular pathways, long-term low dose pollutant effects and vulnerable age-groups. Here, we review (i) flows of manufactured nanomaterials and exposition of C. elegans in the environment, (ii) the track record of C. elegans in biomedical research, and (iii) its potential to contribute to the investigation of the exposome and bridge nanotoxicology between higher organisms, including humans. The role of C. elegans in the one health concept is taken one step further by proposing methods to sample wild nematodes and their molecular characterization by single worm proteomics.

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus

Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA2004) isolates of H. contortus. Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae.

Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates movement. Additionally, gravity sensation, which occurs in the mammalian inner ear, is closely related to hearing - thus, understanding this process has implications for auditory and vestibular research. Gravitaxis assays exist for some model organisms, including Drosophila. Single worms have previously been assayed for their orientation preference as they settle in solution. However, a reliable and robust assay for Caenorhabditis gravitaxis has not been described. The present protocol outlines a procedure for performing gravitaxis assays that can be used to test hundreds of Caenorhabditis dauers at a time. This large-scale, long-distance assay allows for detailed data collection, revealing phenotypes that may be missed on a standard plate-based assay. Dauer movement along the vertical axis is compared with horizontal controls to ensure that directional bias is due to gravity. Gravitactic preference can then be compared between strains or experimental conditions. This method can determine molecular, cellular, and environmental requirements for gravitaxis in worms.

Calpain inhibitor suppresses both extracellular vesicle-mediated secretion of miRNAs and egg production from paired adults of Schistosoma japonicum.

Extracellular vesicles (EVs) have been reported to be secreted from Schistosoma japonicum at all developmental stages. However, the reproduction and communication mechanisms between the paired adults through the EVs in dioecious Trematoda have not been reported. In this study, EVs containing many exosome-like vesicles and microvesicles were observed in the supernatants of paired adults cultured in vitro, and abundant selected miRNAs were contained in them. In particular, the female-specific miR-bantam was present only in vesicles and was hardly secreted outside the vesicles. In this study, we found that male-female pairing induced secretion of miR-3479 and miR-bantam in EVs, but not of male-specific miR-61. Furthermore, ingestion of mouse erythrocytes also increased the production of miRNAs in paired adult and single female worms. Vesicles were found in the tegument of females treated with erythrocytes under electron microscopy. After the paired worms were treated with several inhibitors against the secretion of EVs, only calpain inhibitor (calpeptin) significantly reduced the amount of miRNA in EVs. Furthermore, the worms treated with only calpeptin inhibited egg production in vitro. Together, these results indicate that qualitative miRNA production through EVs regulated by calpain plays a role in egg production in S. japonicum.