Larval Motility Research Articles

Multi-targeted action of rooibos may protect against ischaemic stroke-induced neurological deficit and endothelial dysfunction

Ethnopharmacological relevanceIndigenous use communities in the Western Cape (South Africa) where Aspalathus linearis (Brum.f) R.Dahlgren - or rooibos - grows naturally, has a long history of using rooibos for medicinal purposes. Apart from its well-known antioxidant effect, the Cederberg community in particular has been using rooibos as a treatment for high blood pressure. Given the detrimental effects of high blood pressure on endothelial cells, rooibos may either directly or indirectly affect vascular health. This, together with more recent reports of neuroprotective effects, may position rooibos as complementary medicine in related vascular conditions such as ischaemic stroke. Aims of the studyThe study aimed to evaluate the potential benefit of acute administration of unfermented rooibos, on vascular health in a larval zebrafish model of stroke. Materials and methodsStroke was induced via 24-h ponatinib exposure, in the presence or absence of an aqueous solution of an ethanolic extract of unfermented Rooibos (GreenOxithin™). The magnitude of stroke was assessed by monitoring larval locomotion and thrombus formation. In terms of specific mechanisms probed, changes in redox status (MDA and TEAC), neurological markers (TH and NeuroD1) and endothelial health (tight/adhesion junction protein expression) were assessed. ResultsRooibos treatment limited thrombus formation and prevented stroke-induced deficits on larval motility. In terms of redox status, rooibos treatment prevented lipid peroxidation 3 days after initial stroke induction, reducing the need for significant upregulation of endogenous antioxidant mechanisms. Stroke-induced changes in neuronal (NeuroD1 and TH) protein expression were normalized in the presence of rooibos, suggesting a neuroprotective role. In terms of tight junction proteins, stroke-related decreases in ZO-1 expression were again prevented by rooibos treatment. In addition, rooibos treatment may beneficially modulate levels of claudin-5 and VE-cadherin, to indirectly limit stroke-associated vascular dysfunction. ConclusionsTaken together, activity data and physiological assessments suggest that unfermented rooibos may indeed have benefit in the context of stroke, via action at multiple targets. Thus, current data further our understanding of the mechanisms of actions of rooibos and warrant future research to confirm sufficient bioavailability of rooibos in target tissues, in mammalian systems.

Anthelmintic Activity of Alcoholic Leaf Extract of Tectona grandis

Background: Helminthiasis is a major concern in the livestock sector due to the immense afflictions in the production and economy of the farmer. Chemical anthelmintics are used to combat them and parasites are acquiring resistance against these drugs. Hence, the development of new drugs or alternatives is the need of the hour for combating such infestations. This study was undertaken to determine the effect of methanolic leaf extract of Tectona grandis on strongyles. Materials and Methods: The dried leaves of Tectona grandis were subjected to Soxhlet extraction using methanol, and the extract was tested for its anthelmintic activity in vitro using egg hatch assay and larval motility assay. A dose of 250, 125, 62.5, 31.25, 15.625, and 7.8125 mg/ mL was used for the study. Fresh strongyle ova were exposed to different concentrations of the extract and observed after 48 hours for the hatch. L3 larvae obtained from coproculture were subjected to treatment with extract at different concentrations, and mortality per unit time was calculated. Fourier transform infrared (FTIR) spectroscopic analysis and Gas chromatography-mass spectrometry (GC/MS) were performed to identify the chemical nature of the extract. Results: There was a dose-dependent inhibition of hatch and larval mortality with a maximum inhibition of hatch at 250 mg/mL. There was a cent percent hatch in control wells and no loss of progressive motility in the larval motility test. On exposure to the extract, the larvae progressively lost their motility, and finally, there were caesurae of movement which indicated their death. The extract at 250 mg/mL killed all the larvae by 30 min, whereas, at 31.25 mg/mL, the mortality was 66.6% after 2 hr. Conclusion: The results suggest that methanolic extract from Tectona grandis leaves has a promising anthelmintic property and further studies are required for the isolation of active molecules.

Sodium Hypochlorite: Optimization of Application as an Exsheathing Agent for Nematode Larvae to Minimize the Risk of Reduced Viability

Objective: Nematode larvae exsheathing is a crucial procedure employed in experimental studies such as nematode viability assays, evaluating nematodes' resistance to anthelmintics, and evaluating the efficacy of anthelmintic medications. The exsheathment procedure allows for quantifying the direct impact on larvae when the protective sheath is removed. Sodium hypochlorite (NaOCl) is a commonly used artificial exsheathment medium. However, concerns have been raised about its potential impact on the viability of exsheathed larvae. A previous study utilized Strongyloides papillosus larvae, a nematode species without a protective sheath, to investigate the effects of NaOCl on parasite viability. The findings revealed that there are specific concentration (C) and exposure-time (T) thresholds (0.3% > C > 0.2%; 10 min > T > 5 min) beyond which viability decreases, and excessive exposure can cause significant damage. The current study aims to determine the optimal concentration of NaOCl that can induce exsheathment in ensheathed-type larvae without compromising their viability. Methods: Third-stage strongylid larvae of donkey origin were utilized for this study, with chemical concentrations of 3%, 0.3%, 0.2%, 0.1%, or 0.05%. Larval motility served as the criterion for evaluating viability. Results: NaOCl can exsheath larvae at any of the five concentration levels evaluated, depending on the duration of exposure to the compound. A concentration of 0.2% is considered optimal, inducing exsheathment as early as 2 minutes. Extended exposure for up to 10 minutes did not impact larval viability. A 3% solution causes larval damage. Conclusions: Regarding the effect of NaOCl on nematode larvae, the results validate the earlier findings with Strongyloides papillosus and those by other authors employing nematodes such as Haemonchus contortus and Cooperia curticei, and the entomopathogenic nematode Heterorhabditis bacteriophora.

Investigation of nonclinical toxicity of cosmetic nail gel in Caenrohabditis elegans model

The study investigates the non-clinical toxicity of a cosmetic nail gel using the nematode model Caenorhabditis elegans. The research was motivated by the need for alternative methods to vertebrate animal testing due to ethical and legal considerations. The main objective was to evaluate the influence of cosmetic gel ingredients on the nematodes' behavioral and viability parameters. The N2 Bristol strain of C. elegans was used, exposed to different gel formulations. The methodology included analysis of egg hatching and L4 larval motility, comparing control and treated groups. The results showed no significant difference in egg hatching between groups, indicating that the gel components are not toxic to nematode reproduction. However, a decrease in the motility of L4 larvae exposed to the gel was observed, suggesting possible behavioral interference. The research concludes that while the formulation did not affect reproductive viability, the influence on motility requires further investigation to understand the nature of these behavioral changes. These findings underscore the relevance of using C. elegans as an alternative model for cosmetic safety evaluation.

Synergistic Enhancement of Motor Function by Curcumin and Piperine in Alpha-Synuclein Expressing Drosophila Models

Parkinson’s disease (PD) is a neurodegenerative disease characterized by Lewy body formation mainly phosphorylated and aggregated α-synuclein, with subsequent neurotoxicity and death of dopaminergic neurons in the basal ganglia. The neuroprotective action of curcumin and piperine has been reported. We used the GAL4/UAS bipartite system for targeted expression of α-synuclein pan-neuronal and dopamine neurons to create genetically induced PD. Hence, in this study we assessed the ameliorative effect of curcumin and piperine on α-synuclein-induced dopaminergic neuron degeneration and photoreceptor degeneration in the Drosophila melanogaster model of PD. Male Drosophila melanogaster (upstream activating sequence (UAS) linked to synuclein (SNCA) flies were crossed with virgin wild-type Can-ton-S, DDC-GAL4 or GMR-GAL4 female flies) cultured on either medium containing graded concentrations of curcumin and/or piperine (5, 10, and 50 μM) or vehicle. The parameters measured included fecundity, larval motility, negative geotaxis, and lifespan. Vehicle, curcumin, or piperine supplementation in SNCA>Cs, SNCA>DDC, or SNCA>GMR flies did not affect fecundity or larval motility, but piperine (5 μM and 10 μM but not 50 μM) supplementation reduced fecundity. SNCA>DDC flies significantly reduced climbing performance and lifespan, peaked in week 4, ameliorated by the curcumin and piperine combination. Moreover, the increased aggregation of synuclein, as evidenced in ommatidial degeneration in SNCA>GMR flies’ eyes, was attenuated by the curcumin and piperine combination. Findings from the present study further reinforced the potential benefit of curcumin and piperine in the management of PD.

Preliminary screening of plant essential oils from an oceanic climate zone (NW Spain) for the control of equine cyathostomins

Cyathostomins (Strongylida: Cyathostominae) are gastrointestinal nematodes (GIN) that affect equines in many parts of the world. Due to anthelmintic resistance (AR), it is necessary to search for control alternatives. The objective of the study was to carry out a screening of 26 essential oils (EOs) from plants to determine in vitro their action on cyathostomins. Essential oils were obtained by hydro-distillation from leaves, flowers, fruits, and seeds of the selected species and used against the eggs of cyathostomins by means of the tests of egg hatching and larval motility inhibition. For each EO, different concentrations were prepared (250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 2.0, 1.0, 0.5 and 0.24 mg/mL). Oxfendazole at 2.5% mixed with closantel 5% was used as positive control, and PBS served as negative control. Cyathostomin eggs were obtained from feces of infected horses and infective larvae were recovered after coproculture. The in vitro test was carried out 36 h after the challenge with the EOs. The highest production of EOs was obtained from leaves of bay, eucalyptus, mallow, and basil, as well as from seeds of black pepper, coriander, cinnamon, anise, and peel of orange and cloves, cumin, and Lawson’s cypress. The oils extracted from laurel leaves, ground cinnamon, anise, cumin, and coriander seeds had a very high effect on cyathostomins eggs up to concentrations lower than 3.9 mg/mL. Therefore, in addition to the uses against other pathogens such as fungi and bacteria, some EOs might attain great importance as an alternative control strategy in the control of nematode eggs.

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

Diet modulates strongyle infection and microbiota in the large intestine of horses.

The use of anthelminthic drugs has several drawbacks, including the selection of resistant parasite strains. Alternative avenues to mitigate the negative effects of helminth infection involve dietary interventions that might affect resistance and/or tolerance by improving host immunity, modulating the microbiota, or exerting direct anthelmintic effects. The aim of this study was to assess the impact of diet on strongyle infection in horses, specifically through immune-mediated, microbiota-mediated, or direct anthelmintic effects. Horses that were naturally infected with strongyles were fed either a high-fiber or high-starch diet, supplemented with either polyphenol-rich pellets (dehydrated sainfoin) or control pellets (sunflower and hay). When horses were fed a high-starch diet, they excreted more strongyle eggs. Adding sainfoin in the high-starch diet reduced egg excretion. Additionally, sainfoin decreased larval motility whatever the diet. Moreover, the high-starch diet led to a lower fecal bacterial diversity, structural differences in fecal microbiota, lower fecal pH, lower blood acetate, and lower hematocrit compared to the high-fiber diet. Circulating levels of Th1 and Th2 cytokines, lipopolysaccharides, procalcitonin, and white blood cells proportions did not differ between diets. Overall, this study highlights the role of dietary manipulations as an alternative strategy to mitigate the effect of helminth infection and suggests that, in addition to the direct effects, changes in the intestinal ecosystem are the possible underlying mechanism.

Green synthesis and anthelmintic activity of silver nanoparticles using Morus Alba Fruit extract against different stages of equine strongyles.

The current study was designed to green synthesize silver nanoparticles (GRAgNPs) using Morus alba fruit extract and evaluate their nematicidal effects against strongyle nematodes compared to commercial silver nanoparticles (CAgNPs) in vitro. The nanoparticles were characterized by Ultraviolet-visual absorption spectrography, transmission electron microscopy, and X-ray diffraction. Next, uptake of AgNPs by the first stage larvae (L1), egg hatch inhibition (EHI) and the motility of infectious larvae (L3s), and the ultrastructural analysis of the eggs and worms were conducted. Moreover, some of oxidative/nitrosative stress indicators, including total antioxidant status content (TAC), protein carbonylation (PCO), lipid peroxidation (MDA), and DNA damage were assessed in the homogenized samples of strongyle L3s. We found that the GRAgNPs had spherical shape, 20-30nm in diameter with rough surface. Following incubation with GRAgNPs at concentrations of 43.40, 21.70 and 10.85 ppm and CAgNPs at concentrations of 43.40 and 21.70, EHI was more than 90%. In addition, concentrations of 43.40 and 21.70 ppm of GRAgNPs led to inhibition of larval motility by more than 90%. The LC50 at 24h of treatment for GRAgNPs and CAgNPs was determined to be 8.62 and 10.34 ppm, respectively. GRAgNPs and CAgNPs, in a concentration-dependent manner, resulted in the induction of oxidative/nitrosative stress evidenced by decreased TAC levels, and increased levels of MDA and PCO, together with increased DNA damage. The uptake of AgNPs by the L1 larvae revealed that FITC labeled GRAgNPs fluoresced with high intensity largely in the intestinal area. Scanning Electron Microscopy analysis of eggs and larvae revealed that GRAgNPs penetrated the cuticle of larvae, changed the tegmentum, and ultimately killed the worm. In conclusion, GRAgNPs had more robust anthelminthic effects than the standard antiparasitic and CAgNPs. They could be considered as a promising antiparasitic agent.

Composite substrates for coral larval settlement and reef restoration based on natural hydraulic lime and inorganic strontium and magnesium compounds

Coral reefs face unprecedented threats from climate change and human activities, making reef restoration increasingly important for the preservation of marine biodiversity and the sustainability of coastal communities. One promising restoration method relies on coral breeding and larval settlement, but this approach requires further innovation to achieve high rates of settlement and survival. In this study, we built on our previous work engineering lime mortar-based coral settlement substrates by investigating three different compositions of a natural hydraulic lime (NHL) base material as well as composite NHL substrates containing alkaline earth metals. These materials were tested with larvae of three reef-building Caribbean coral species: Orbicella faveolata (Mountainous star coral), Diploria labyrinthiformis (Grooved brain coral), and Colpophyllia natans (Boulder brain coral). We found that the base material composition, including its silicate and calcium carbonate (CaCO3) content, as well as the addition of the inorganic additives strontium carbonate (SrCO3), magnesium carbonate (MgCO3), and magnesium sulfate (MgSO4), all influenced coral larval settlement rates. Overall, NHL formulations with lower concentrations of silicate and higher concentrations of calcium, strontium, and magnesium carbonates significantly increased coral settlement. Further, when dissolved ions of magnesium and strontium were added to seawater, both had a significant effect on larval motility, with magnesium promoting settlement and metamorphosis in C. natans larvae, supporting the observation that these additives are also bioactive when incorporated into substrates. Our results demonstrate the potential benefits of incorporating specific inorganic ion additives such as Mg2+ and Sr2+ into substrates to facilitate early coral life history processes including settlement and metamorphosis. Further, our results highlight the importance of optimizing multiple aspects of coral substrate design, including material composition, to promote settlement and survival in coral propagation and reef restoration.

A phenotypic screen of the Global Health Priority Box identifies an insecticide with anthelmintic activity

BackgroundInfection with parasitic nematodes (helminths), particularly those of the order Strongylida (such as Haemonchus contortus), can cause significant and burdensome diseases in humans and animals. Widespread drug (anthelmintic) resistance in livestock parasites, the absence of vaccines against most of these nematodes, and a lack of new and effective chemical entities on the commercial market demands the discovery of new anthelmintics. In the present study, we searched the Global Health Priority Box (Medicines for Malaria Venture) for new candidates for anthelmintic development.MethodsWe employed a whole-organism, motility-based phenotypic screening assay to identify compounds from the Global Health Priority Box with activity against larvae of the model parasite H. contortus, and the free-living comparator nematode Caenorhabditis elegans. Hit compounds were further validated via dose–response assays, with lead candidates then assessed for nematocidal activity against H. contortus adult worms, and additionally, for cytotoxic and mitotoxic effects on human hepatoma (HepG2) cells.ResultsThe primary screen against H. contortus and C. elegans revealed or reidentified 16 hit compounds; further validation established MMV1794206, otherwise known as ‘flufenerim’, as a significant inhibitor of H. contortus larval motility (half-maximal inhibitory concentration [IC50] = 18 μM) and development (IC50 = 1.2 μM), H. contortus adult female motility (100% after 12 h of incubation) and C. elegans larval motility (IC50 = 0.22 μM). Further testing on a mammalian cell line (human hepatoma HepG2 cells), however, identified flufenerim to be both cytotoxic (half-maximal cytotoxic concentration [CC50] < 0.7 μM) and mitotoxic (half-maximal mitotoxic concentration [MC50] < 0.7 μM).ConclusionsThe in vitro efficacy of MMV1794206 against the most pathogenic stages of H. contortus, as well as the free-living C. elegans, suggests the potential for development as a broad-spectrum anthelmintic compound; however, the high toxicity towards mammalian cells presents a significant hindrance. Further work should seek to establish the protein–drug interactions of MMV1794206 in a nematode model, to unravel the mechanism of action, in addition to an advanced structure–activity relationship investigation to optimise anthelmintic activity and eliminate mammalian cell toxicity.Graphical

Embryonic exposures to cadmium and PAHs cause long-term and interacting neurobehavioral effects in zebrafish

Developmental exposure to either polycyclic aromatic hydrocarbons (PAHs) or heavy metals has been shown to cause persisting and overlapping neurobehavioral effects in animal models. However, interactions between these compounds have not been well characterized, despite their co-occurrence in a variety of environmental media. In two companion studies, we examined the effects of developmental exposure to cadmium (Cd) with or without co-exposure to prototypic PAHs benzo[a]pyrene (BaP, Exp. 1) or fluoranthene (FA, Exp. 2) using a developing zebrafish model. Zebrafish embryos were exposed to Cd (0–0.3 μM), BaP (0–3 μM), FA (0–1.0 μM), or binary Cd-PAH mixtures from 5 to 122 h post fertilization (hpf). In Exp. 1, Cd and BaP produced independent effects on an array of outcomes and interacting effects on specific outcomes. Notably, Cd-induced deficits in dark-induced locomotor stimulation were attenuated by BaP co-exposure in the larval motility test and BaP-induced hyperactivity was attenuated by Cd co-exposure in the adolescent novel tank test. Likewise, in Exp. 2, Cd and FA produced both independent and interacting effects. FA-induced increases on adult post-tap activity in the tap startle test were attenuated by co-exposure with Cd. On the predator avoidance test, FA- and 0.3 μM Cd-induced hyperactivity effects were attenuated by their co-exposure. Taken together, these data indicate that while the effects of Cd and these representative PAHs on zebrafish behavior were largely independent of one another, binary mixtures can produce sub-additive effects for some neurobehavioral outcomes and at certain ages. This research emphasizes the need for detailed risk assessments of mixtures containing contaminants of differing classes, and for clarity on the mechanisms which allow cross-class toxicant interactions to occur.

Developmental exposure to pesticides that disrupt retinoic acid signaling causes persistent retinoid and behavioral dysfunction in zebrafish.

Early developmental exposure to environmental toxicants may play a role in the risk for developing autism. A variety of pesticides have direct effects on retinoic acid (RA) signaling and as RA signaling has important roles in neurodevelopment, such compounds may cause developmental neurotoxicity through an overlapping adverse outcome pathway. It is hypothesized that a pesticide's embryonic effects on retinoid function may correspond with neurobehavioral disruption later in development. In the current studies, we determined the effects of RA-acting pesticides on neurobehavioral development in zebrafish. Buprofezin and imazalil caused generalized hypoactivity in the larval motility test, whereas chlorothalonil and endosulfan I led to selective hypoactivity and hyperactivity, respectively. With buprofezin, chlorothalonil, and imazalil, hypoactivity and/or novel anxiety-like behaviors persisted in adulthood and buprofezin additionally decreased social attraction responses in adulthood. Endosulfan I did not produce significant adult behavioral effects. Using qPCR analyses of adult brain tissue, we observed treatment-induced alterations in RA synthesis or catabolic genes, indicating persistent changes in RA homeostasis. These changes were compound-specific, with respect to expression directionality, and potential patterns of homeostatic disruption. Results suggest the likely persistence of disruptions in RA signaling well into adulthood and may represent compensatory mechanisms following early life stage exposures. This study demonstrates that early developmental exposure to environmental toxicants that interfere with RA signaling causes short as well as long-term behavioral disruption in a well-established zebrafish behavioral model and expand upon the meaning of the RA adverse outcome pathway, indicating that observed effects likely correspond with the nature of underlying homeostatic effects.

Anthelmintic Activity of Ethanolic and Aqueous Extracts of Khaya grandifoliola Stem Bark against Heligmosomoides polygyrus: In Vitro and In Silico Approaches.

Parasitic infection remains a serious health trade for humans and livestock. The purpose of this study was to present scientific proof of the anthelmintic properties of Khaya grandifoliola, which the native population uses to cure helminthiasis. Fresh Heligmosomoides polygyrus eggs were isolated from faecal samples of experimentally infected mice. The faecal material was cultured, and L1 and L2 larval stages were recovered after 48 and 120 hours, respectively. Using the worm microtracker, the anthelminthic efficacy of the extracts against H. polygyrus was assessed. Two different extracts (aqueous and ethanol extracts) were prepared. For the ovicidal and larvicidal activities, 100 µL of various concentrations of plant extracts, levamisole and 1.5% dimethyl sulfoxide (DMSO), were introduced into a 96-well microplate titer followed by the addition of 100 µL of embryonated eggs (60 eggs) for the ovicidal activity and 100 µL of L 1 or L 2 larvae (50 larvae) for the larvicidal activity. The movement of the worm was monitored for 24 hours in the worm microtracker at 27°C. The Glide module of the Schrodinger Maestro software was used to perform docking studies. For the aqueous extracts, the highest percentage of inhibition of hatching was 42.77 ± 12% at 7.5 mg/mL. The IC50 values for the ethanol (0.36 mg/mL) extract showed that the ethanol extract had a good inhibitory effect on the ability of parasites to hatch from eggs. The inhibition percentage of L1 larvae motility at 7.5 mg/mL was 98.0 ± 1.66% and 83.33 ± 1.66% for ethanol and aqueous extracts, respectively. The negative controls, distilled water and 1.5% DMSO, had no inhibitory impact on larvae. On L1-larvae, the drug of choice levamisole (positive control) had the highest percentage effect (100.0%). Six compounds had the highest docking score and their interactions with the receptor as well. Grandiamide A interacts most with tyrosine, glycine, phenylalanine, asparagine, and serine, and its benzene ring and oxygens inhibit these receptors. Carbonyl and hydroxyl (OH) groups connect grandiamide D to asparagine, isoleucine, and phenylalanine, respectively. By donating hydrogen to the receptor through OH groups, D-glucopyranose-6-phosphate also forms relatively strong hydrogen bonds with its oxygen-bound phosphorus and the receptor. 1-O-deacetylkhayanolide E interacts most with serine and glutamic acid. The carbamic acid benzyl ester of carbamic acid [(1S)-1-phenyl-2-[(4-methylphenyl) sulfinyl] ethyl] interacts most with the receptor with carbonyl groups and with asparagine and serine. With its abundant hydroxide, D-mannitol acts as a hydrogen donor and acceptor and interacts most strongly with amino acids such as glycine, asparagine, aspartic acid, alanine, and glutamic acid. K. grandifoliola extracts possess anthelminthic properties. However, in vivo studies are still necessary to demonstrate the effectiveness of this plant for the treatment of helminthiasis.

In vitro anthelmintic activity of ethanol and aqueous extracts of Terminalia macroptera and Bridelia micrantha against Heligmosomoides polygyrus, Caenorhabditis elegans and in-silico molecular docking evaluation of some isolated phytoconstituents

BackgroundIn tropical and subtropical African nations, helminthiases are widespread and devastating to school-aged children. This study was undertaken in order to objectively support the ethnobotanical use of the stem bark of Terminalia macroptera and the leaves of Bridelia micrantha as anthelminthic agent, using Heligmosomoides polygyrus and Caenorhabditis elegans. MethodThe flotation technique was used to obtain fresh eggs of H. polygyrus from the feces of experimentally infected mice and culture to different stages of the larvae. Caenorhabditis elegans L4 larvae were obtained after bleaching adult worms to create a synchronized medium and then was cultured until the L4 larval stage. By tracking the larvae's movement, the worm micro tracker was used to assess the anthelminthic activity in a 96-well microplate. The extract was tested at various concentrations and distilled water served as the negative control, and albendazole as the positive control. The percentage of larvae motility inhibition was calculated. The Glide module of the Schrodinger Maestro software was used to conduct the docking studies. The software's score function was used to rank and classify several potentials adduct patterns produced by molecular docking. ResultsFor the ethanol and aqueous extracts of T. macroptera, the IC50 of the ovicidal activity was 0.013 and 0.042 mg/ml, respectively, whereas for the ethanol and aqueous extracts of B. micrantha, it was 0.38 and 0.63 mg/ml. This demonstrates that ethanol extracts are more effective than water extracts in preventing hatching inside the host. T. macroptera larvicidal activity against L1, L2, and L3 larvae resulted in IC50 values of 0.47, 0.29, and 0.004 mg/ml for the ethanol extract, respectively, while B. micrantha's had IC50 values of 0.154, 1.4, and 0.047 mg/ml. Caenorhabditis elegans was very sensitive with IC50 values of, 4.733, and 2.8 µg̸ mL, respectively, for the ethanol and aqueous extract. In B. micrantha, Isoquercetin has the most interaction with amino acids, Asparagine, Phenylalanine, Glycine, Glutamic acid, and Tyrosine, because of its abundant hydroxyl functional group as well as carbonyl group and benzene ring, which makes it have a better inhibitory effect on the receptor protein while that of T. macroptera was Orientin. ConclusionThese findings imply that B. micrantha and T. macroptera may be effective as an anthelminthic agent. However, additional in vivo research is required to verify this biological activity. In addition, the hydroxyl groups as well as the flavonoid backbone of these compounds are the reason for the good effect of these proteins on the receptor protein, and the in silico studies of these compounds on the receptor protein and its interactions with amino acids confirm this claim.

Post-mortem tissue migration of Anisakis simplex (s.s.) larvae (Nematoda: Anisakidae) in three commercially harvested fish species from the Northeast Atlantic: The role of storage time and temperature

Anisakis simplex sensu stricto is a parasite infecting several commercial fish species in the Northeast (NE) Atlantic, known to be the aetiological agent of the human zoonosis anisakiasis. The present study investigated the response of A. simplex (s. s.) third stage larvae (identified to species level by mtDNA cox2 sequencing) to the storage time and temperature of Atlantic herring (Clupea harengus), Atlantic mackerel (Scomber scombrus) and blue whiting (Micromesistius poutassou) from NE Atlantic fishing areas. A total of 300 fish per species were divided in batches of 50 individuals straight after catch. Each batch was stored at different temperature conditions (2 °C, 5 °C, 15 °C) for different time intervals (24h and 48h). A batch of 50 fish of each species was frozen immediately after catch and used as control (time zero). All fish were inspected by the UV-press method. Blue whiting was the most infected fish species while mackerel harboured the highest proportion of intra-vitam A. simplex (s. s.) larvae in the musculature. In blue withing there was a significant increase in the proportion of larvae in the muscle with increasing storage temperatures (5 °C < 15 °C) and time (24h < 48h). Herring showed a weak trend of increasing parasite infection in the muscle with increasing temperature/time. In contrast, no significant differences of muscle/viscera larval distribution were observed between batches of mackerel stored at different temperatures for different time intervals. Storage temperature and time seem to play a role in the post-mortem motility of A. simplex (s. s.) larvae in herring and blue whiting. Keeping the temperature at ≤ 2 °C seems to prevent post-mortem larval migration into the flesh during fish storage, handling, and transport. Besides abiotic variables, the differences observed in larval post-mortem motility in the different fish species are biologically determined, and attributable to species-specific host-parasite interactions.

Systematic analysis of proximal midgut- and anorectal-originating contractions in larval zebrafish using event feature detection and supervised machine learning algorithms.

Zebrafish larvae are translucent, allowing in vivo analysis of gut development and physiology, including gut motility. While recent progress has been made in measuring gut motility in larvae, challenges remain which can influence results, such as how data are interpreted, opportunities for technical user error, and inconsistencies in methods. To overcome these challenges, we noninvasively introduced Nile Red fluorescent dye to fill the intraluminal gut space in zebrafish larvae and collected serial confocal microscopic images of gut fluorescence. We automated the detection of fluorescent-contrasted contraction events against the median-subtracted signal and compared it to manually annotated gut contraction events across anatomically defined gut regions. Supervised machine learning (multiple logistic regression) was then used to discriminate between true contraction events and noise. To demonstrate, we analyzed motility in larvae under control and reserpine-treated conditions. We also used automated event detection analysis to compare unfed and fed larvae. Automated analysis retained event features for proximal midgut-originating retrograde and anterograde contractions and anorectal-originating retrograde contractions. While manual annotation showed reserpine disrupted gut motility, machine learning only achieved equivalent contraction discrimination in controls and failed to accurately identify contractions after reserpine due to insufficient intraluminal fluorescence. Automated analysis also showed feeding had no effect on the frequency of anorectal-originating contractions. Automated event detection analysis rapidly and accurately annotated contraction events, including the previously neglected phenomenon of anorectal contractions. However, challenges remain to discriminate contraction events based on intraluminal fluorescence under treatment conditions that disrupt functional motility.

Condensed tannins, novel compounds and sources of variation determine the antiparasitic activity of Nordic conifer bark against gastrointestinal nematodes

The antiparasitic potential of plants could offer a vital solution to alleviating the costs of gastrointestinal nematode (GIN) infections in ruminant production globally. Leveraging known bioactive molecules, however, is complex, where plant species, extraction processes and seasonality impact bioavailability and efficacy. This study assessed the impact of a comprehensive set of factors on the antiparasitic activity of Norwegian conifers to identify bark compounds specific against GIN. Antiparasitic activity was determined using in vitro assays targeting morphologically distinct life stages of ovine GIN: the egg hatch assay and larval motility assay. In depth characterisation of the chemical composition of the bark extracts was carried out using chromatographic separation, UV-absorbance, and molecular mass profiles to identify compounds implicated in the activity. Three key findings emerged: (1) the activity of bark extracts varied markedly from 0 to 100% antiparasitic efficacy, owing to tree species, extraction solvent and seasonality; (2) the GIN exhibited species-and stage-specific susceptibility to the bark extracts; (3) the presence of condensed tannins, amongst other compounds, was associated with anthelmintic activity. These findings add new insights into urgently needed alternative parasite control strategies in livestock.

Optimizing the xWORM assay for monitoring hookworm larvae motility

Parasitic worms (helminths) infect almost all taxa across the animal kingdom, and pose significant challenges to public health and economies, particularly in developing countries. To address this problem, researchers have developed various tools to measure the motility and viability of helminths. However, the conditions used in anthelmintic screening assays are often not optimized, and can vary considerably between research teams. These unoptimized conditions may impact novel drug screens, as little is known about the effects of different conditions on the health of the target parasites. To improve future research, this study determined the effects of key assay parameters including, media type, media concentration, in-well parasite density, and assay duration on the infective third-stage larva (L3) of two types of hookworms, namely Nippostrongylus brasiliensis in rodents, and Necator americanus in humans. Conditions were screened over several days using the xCELLigence worm real-time motility assay (xWORM); a real-time impedance-based helminth motility assay using the xCELLigence system with 96-well microplates. While results varied depending on the species and media used, the study found that 500–1,000 L3/200-µL and a media concentration of 3.13–25% generally produced good to excellent assay conditions. The findings of this study can guide the future selection of xWORM assay parameters for novel drug trials involving these parasite species and serve as a suggested model for optimizing trial conditions for alternative parasite targets and assays.

UPLC-QToF-ESI-MS identification and anthelmintic activity of Mitragyna inermis (Willd.) Kuntze (Rubiaceae)

Medicinal plants attract the attention of many researchers to find natural and safe remedies for various resistant diseases. Leaves of Mitragyna inermis are widely used in traditional veterinary medicine for the treatment of gastrointestinal strongyles of small ruminants. The aim of the current study is to estimate the antioxidant, anthelmintic and the larval toxicity of the aqueous and hydroethanolic extracts of this plant in addition to the hexane, dichloromethane and ethanol fractions of the hydroethanolic extract. Investigation of the most active extract using Ultra Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight Electrospray Ionization Mass Spectrometry (UPLC-QToF-ESI-MS). Both plant extracts showed good antioxidant activity by scavenging the 2,2′-diphényl-1-picrylhydrazyl (DPPH) radical and reducing the ferric ion. Similarly, they were no-toxic to Artemia salina larvae (CL50 > 0.1 μg/mL). Also, they significantly reduced larval migration and motility of Haemonchus contortus adult worms (p < 0.001). The hexane, dichloromethane and ethanolic fractions of the hydroethanolic extract showed low activity compared to crude extracts except for the hexane fraction on H. contortus adult worms (p < 0.001) while it showed a poor result on larvae. It thus appears that the anthelmintic activity of the extract may be linked to the synergistic action of these compounds. The UPLC-QToF-ESI-MS analysis revealed the tentative identification of 15 compounds including 7 alkaloids. The results of the present study confirm the anthelmintic activity of M. inermis in traditional veterinary medicine.