Insights into the mode of action of 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89) against adult Schistosoma mansoni worms

Control of morbidity associated with schistosomiasis via chemotherapy largely relies on the drug praziquantel. Repeated therapy with praziquantel has created concerns about the possible selection of resistant worms and necessitated the search for novel drugs to treat schistosomiasis. Here, a murine model was infected with Schistosoma mansoni and treated with oral 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89), which caused a significant reduction in fecundity and egg burden and reduced morbidity when administered at 5-weeks post-infection.The analysis showed that the mode of action occurred through the ingestion of activated N-89 by the worms, and that there was no direct external effect on the S. mansoni worms. Ultrastructural analysis of the treated worms showed disruptions in the gut lumen and the presence of large volumes of material, suggestive of undigested blood meals or red blood cells. In addition, there were reduced vitelline cells in female worms and damage to sub-tegmental musculature in male worms. Eggs recovered from the treated mice showed both damage to the eggs and the production of immature eggs. Expression of mRNA responsible for gut and digestive function and egg production was also significantly affected by N-89 treatment, whereas control genes for musculature showed no significant changes.Thus, N-89 drastically affected the total digestive function and egg production of S. mansoni worms. Physiological processes requiring heme uptake such as egg production and eggshell formation were subsequently affected, suggesting that the compound could be a possible therapeutic drug candidate for schistosomiasis control.     

Developmental regulation of protein kinase A expression and activity in Schistosoma mansoni

cAMP-dependent protein kinases (PKAs) are the main transducers of cAMP signalling in eukaryotic cells. Recently we reported the identification and characterisation of a PKA catalytic subunit (SmPKA-C) in Schistosoma mansoni that is required for adult schistosome viability in vitro. To gain further insights into the role of SmPKA-C in biological processes during the schistosome life cycle, we undertook a quantitative analysis of SmPKA-C mRNA expression in different life cycle stages. Our data shows that SmPKA-C mRNA expression is developmentally regulated, with the highest levels of expression in cercariae and adult female worms. To evaluate the biological role of SmPKA-C in these developmental stages, cercariae and adult worms were treated with various concentrations of PKA inhibitors. Treatment of cercariae with H-89 or PKI 14-22 amide resulted in loss of viability suggesting that, as in adults, PKA is an essential enzyme activity in this infectious larval stage. In adult worms, in vitro exposure to sub-lethal concentrations of H-89 or PKI 14-22 amide resulted in inhibition of egg production in a dose-dependent manner. Furthermore, using a murine model of schistosome infection where S. mansoni fecundity is impaired, we show that reduced rates of egg production in vivo correlate with significant reductions in SmPKA-C mRNA expression and PKA activity. Finally, restoration of parasite egg production in vivo also resulted in normalisation of SmPKA-C mRNA expression and PKA activity. Taken together, our data suggest that PKA signalling is required for cercarial viability and may play a specific role in the reproductive activity of adult worms.     

The curvilinear relationship between worm length and fecundity of Teladorsagia circumcincta.

The variation among sheep in fecundity of Teladorsagia (Ostertagia) circumcincta was estimated by dividing the faecal egg count by the worm number following deliberate infection of mature Scottish Blackface lambs. Fecundity was skewed and ranged from 0 to 350 eggs per worm per day. Most animals had relatively low worm fecundities, but a small number of individuals had relatively high worm fecundities. However, as fecundity is a ratio of two imprecise estimates, extreme values may be statistical artefacts. Following both deliberate and natural infection, differences in worm fecundity were associated with differences in adult female worm length. In both infections, fecundity varied with worm length to the power 0.4. This relationship should assist the measurement of fecundity in studies of host immunity, in epidemiological modelling and in estimating the influence of density-dependent relationships.     

Schistosoma mansoni egg-derived extracellular vesicles: A promising vaccine candidate against murine schistosomiasis

Extracellular vesicles (EVs) are protein-loaded nano-scaled particles that are extracellularly released by eukaryotes and prokaryotes. Parasite’s EVs manipulate the immune system, making them probable next-generation vaccines. Schistosomal EVs carry different proteins of promising immunizing potentials. For evaluating the immune-protective role of Schistosoma mansoni (S. mansoni) egg-derived EVs against murine schistosomiasis, EVs were isolated from cultured S. mansoni eggs by progressive sequential cooling ultra-centrifugation technique. Isolated EVs were structurally identified using transmission electron microscope and their protein was quantified by Lowry’s technique. Experimental mice were subcutaneously immunized with three doses of 20 μg EVs (with or without alum adjuvant); every two weeks, then were challenged with S. mansoni cercariae two weeks after the last immunizing dose. Six weeks post infection, mice were sacrificed for vaccine candidate assessment. EVs protective efficacy was evaluated through parasitological, histopathological, and immunological parameters. Results showed significant reduction of tegumentally deranged adult worms, hepatic and intestinal egg counts reduction by 46.58%, 93.14% and 93.17% respectively, accompanied by remarkable amelioration of sizes, numbers and histopathology of hepatic granulomata mediated by high interferon gamma (IFN γ) and antibody level. Using sera from vaccinated mice, the molecular weight of EVs’ protein components targeted by the antibody produced was recognized by western immunoblot. Results revealed two bands of ~ 14 KDa and ~ 21 KDa, proving that EVs are able to stimulate specific antibodies response. In conclusion, the present study highlighted the role of S. mansoni-egg derived EVs as a potential vaccine candidate against murine schistosomiasis mansoni.     

Synergy of Omeprazole and Praziquantel In Vitro Treatment against Schistosoma mansoni Adult Worms

BackgroundTreatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.MethodologyWe used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay.ConclusionsFunctional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.     

Population stability in relation to resource availability in an introduced population of an herbivorous lady beetle

Mechanisms responsible for population stability in relation to resource availability were studied in an introduced herbivorous lady beetle,Epilachna niponica. The introduced population was relatively constant over a seven-year study period. Egg density was related to the variation in host-plant abundance in different years, and was highly stabilized during the period from reproductive adult to egg stage. Two density-dependent processes were identified in the reproductive season: (1) density-dependent reduction in fecundity and (2) density-dependent increase in female mortality and/or emigration, all of which operated early in the season. As a result, temporal variability in cumulative egg density was greatly reduced by mid-May, by which time approximately40% of total eggs were laid. A field cage experiment demonstrated that egg-laying of individual females was largely limited by resource availability even at low levels of leaf herbivory. Since movement activity of ovipositing females increased in a density-dependent manner, inter-plant movement is more likely to cause density-dependent reduction in fecundity and female loss, due to enhanced energy expendiditure. The introduced population was less stable than the source population, probably because of decreased inter-plant movement of females and the unlikelihood of egg resorption, both of which contribute significantly to the temporal stability ofE. niponica population densities.     

In vitro and in vivo studies on the bioactivity of a ginger (Zingiber officinale) extract towards adult schistosomes and their egg production

The bioactivity of an ethyl acetate extract of ginger (Zingiber officinale) towards Schistosoma mansoni adult pairs, both cultured in vitro and in vivo in laboratory mice, was investigated by monitoring worm mortality and fecundity. In vitro, a concentration of 200 mg l(-1) of extract killed almost all worms within 24 h. Male worms seemed more susceptible than female under these conditions. Cumulative egg output of surviving worm pairs in vitro was considerably reduced when exposed to the extract. For example, after 4 days of exposure to 50 mg l(-1), cumulative egg output was only 0.38 eggs per worm pair compared with 36.35 for untreated worms. In vivo efficacy of the extract was tested by oral and subcutaneous delivery of 150 mg kg(-1) followed by assessment of worm survival and fecundity. Neither delivery route produced any significant reduction in worm numbers compared with untreated controls. Worm fecundity was assessed in vivo by cumulative egg counts per liver at 55 days post infection with mice treated subcutaneously. Such infections showed egg levels in the liver of about 2000 eggs per worm pair in 55 days, in both treated and control mice, with no significant difference between the two groups. To ensure that density-dependent effects did not confound this analysis, a separate experiment demonstrated no such influence on egg output per worm pair, at intensities between 1 and 23 worms per mouse.     

Evidence for in vitro and in vivo activity of the antimalarial pyronaridine against Schistosoma

BackgroundSchistosomiasis is highly prevalent in Africa. Praziquantel is effective against adult schistosomes but leaves prepatent stages unaffected—which is a limit to patient management and elimination. Given the large-scale use of praziquantel, development of drug resistance by Schistosoma is feared. Antimalarials are promising drugs for alternative treatment strategies of Schistosoma infections. Development of drugs with activity against both malaria and schistosomiasis is particularly appealing as schistosome infections often occur concomitantly with malaria parasites in sub-Saharan Africa. Therefore, antiplasmodial compounds were progressively tested against Schistosoma in vitro, in mice, and in a clinical study.ResultsAmongst 16 drugs and 1 control tested, pyronaridine, methylene blue and 5 other antimalarials were highly active in vitro against larval stage schistosomula with a 50% inhibitory concentration below 10 μM. Both drugs were lethal to ex vivo adult worms tested at 30 μM with methylene blue also active at 10 μM. Pyronaridine treatment of mice infected with S. mansoni at the prepatent stage reduced worm burden by 82% and cured 7 out of 12 animals, however in mice adult stages remained viable. In contrast, methylene blue inhibited adult worms by 60% but cure was not achieved. In an observational pilot trial in Gabon in children, the antimalarial drug combination pyronaridine-artesunate (Pyramax) reduced S. haematobium egg excretion from 10/10 ml urine to 0/10 ml urine, and 3 out of 4 children were cured.ConclusionPyronaridine and methylene blue warrant further investigation as candidates for schistosomiasis treatment. Both compounds are approved for human use and evidence for their potential as antischistosomal compounds can be obtained directly from clinical testing. Particularly, pyronaridine-artesunate, already available as an antimalarial drug, calls for further clinical evaluation.Trial registrationClinicalTrials.gov Identifier {"type":"clinical-trial","attrs":{"text":"NCT03201770","term_id":"NCT03201770"}}NCT03201770.     

Schistosoma japonicum translationally controlled tumour protein,which is associated with the development of female worms,as a target for control of schistosomiasis

Schistosomiasis is a globally important helminthic disease of both humans and animals, and is the second most common parasitic disease after malaria. Although praziquantel is extensively used for treatment of parasitic diseases, drug resistance has been reported. Therefore, new drugs and effective vaccines are needed for continuous control of schistosomiasis. Eggs produced by schistosomes are responsible for the occurrence and spread of schistosomiasis. Revealing the reproductive mechanism of schistosomes will help to control this disease. In this study, the proteomic profiles of single-sex infected female worms and bisexual infected mature female worms of Schistosoma japonicum at 18, 21, 23 and 25 days p.i. were identified with isobaric tags for relative quantitation-coupled liquid chromatography–tandem mass spectrometry. Differentially expressed proteins were subsequently used for bioinformatic analysis. Six highly expressed differentially expressed proteins in mature female worms were selected and long-term interference with small interfering RNA (siRNA) was conducted to determine biological functions. SiRNA against S. japonicum translationally controlled tumour protein (SjTCTP) resulted in the most significant effect on the growth and development of MF worms. Sjtctp mRNA expression gradually increased over time with a high level of expression maintained at 25–42 days p.i., while levels were significantly higher in mature female worms than male and SF worms. The subsequent animal immune protection experiments showed that recombinant SjTCTP (rSjTCTP) reduced the number of adults by 44.7% (P < 0.01), average egg burden per gram of liver by 57.94% (P < 0.01), egg hatching rate by 47.57% (P < 0.01), and oviposition of individual females by 43.16%. rSjTCTP induced higher levels of serum IgG, IL-2, and IL-10 in mice. Collectively, these results show that SjTCTP is vital to reproduction of female worms and, thus, is a candidate antigen for immune protection.     

Discovery and Characterization of Novel Anti-schistosomal Properties of the Anti-anginal Drug,Perhexiline and Its Impact on Schistosoma mansoni Male and Female Reproductive Systems

BackgroundSchistosomiasis, one of the world’s greatest human neglected tropical diseases, is caused by parasitic trematodes of the genus Schistosoma. A unique feature of schistosome biology is that the induction of sexual maturation as well as the maintenance of the differentiation status of female reproductive organs and egg production, necessary for both disease transmission and pathogenesis, are strictly dependent on the male. The treatment and most control initiatives of schistosomiasis rely today on the long-term application of a single drug, praziquantel (PZQ), mostly by campaigns of mass drug administration. PZQ, while very active on adult parasites, has much lower activity against juvenile worms. Monotherapy also favors the selection of drug resistance and, therefore, new drugs are urgently needed.Conclusions/SignificanceOverall, our data indicate that PHX could represent a promising starting point for novel schistosomicidal drug discovery programmes.     

Evolutionary epidemiology of schistosomiasis: linking parasite genetics with disease phenotype in humans

Here we assess the role of parasite genetic variation in host disease phenotype in human schistosomiasis by implementing concepts and techniques from environmental association analysis in evolutionary epidemiology. Schistosomiasis is a tropical disease that affects more than 200 million people worldwide and is caused by parasitic flatworms belonging to the genus Schistosoma. While the role of host genetics has been extensively studied and demonstrated, nothing is yet known on the contribution of parasite genetic variation to host disease phenotype in human schistosomiasis. In this study microsatellite genotypes of 1561 Schistosoma mansoni larvae collected from 44 human hosts in Senegal were linked to host characteristics such as age, gender, infection intensity, liver and bladder morbidity by means of multivariate regression methods (on each parasite locus separately). This revealed a highly significant association between allelic variation at the parasite locus L46951 and host infection intensity and bladder morbidity. Locus L46951 is located in the 3′ untranslated region of the cGMP-dependent protein kinase gene that is expressed in reproductive organs of adult schistosome worms and appears to be linked to egg production. This putative link between parasite genetic variation and schistosomiasis disease phenotype sets the stage for further functional research.     

The effects of histamine and an antihistamine on Trichinella spiralis and on trichinous enteritis in the host

The effects of histamine and an antihistamine on the number and fecundity of adult Trichinella spiralis and on trichinous enteritis measured by determining myeloperoxidase activity in the small intestine of the host on days 7, 9 and 11 postinfection (PI) were examined during primary infections of the CD-1 Swiss white mouse. In mice receiving oral doses of histamine the fecundity of adult worms decreased, the number of adult worms was unaffected and enteritis was elevated above that seen with untreated mice or mice receiving oral doses of saline alone. In mice injected intramuscularly with antihistamine after day 7 PI fecundity of adult worms increased, the number of adult worms remained the same and enteritis decreased compared to untreated mice receiving intramuscular injections of saline. As the concentration of histamine present in the incubation medium (0.01, 0.1, 10, 25, 50 or 100 mg%) was increased above 0.01 mg% the fecundity of adult worms during culture in vitro decreased. As the concentration of antihistamine present in incubation medium (0.01, 0.1, 10 or 100 mg%) was increased above 0.1 mg% the fecundity of adult worms during culture in vitro decreased.     

Determinants of female fecundity in a simultaneous hermaphrodite: the role of polyandry and food availability

Classical sexual selection theory assumes that the reproductive success of females is primarily limited by the resources available for egg production rather than by the number of mating partners. However, there is now accumulating evidence that multiple mating can entail fitness costs or benefits for females. In this study we investigated the effect of polyandry (i.e., the mating with different mating partners) and food availability on the reproductive output of the female sex function in an outcrossing simultaneous hermaphrodite, the free-living flatworm Macrostomum lignano. We exposed virgin worms to different group sizes, a treatment that has previously been shown to affect the level of polyandry in this species. Moreover, we manipulated the food availability throughout the subsequent egg laying period, during which the worms were kept in isolation. The number of offspring produced was used as an estimate of female fecundity. We found that food availability, but not group size, had a significant effect on female fecundity. Additionally, female fecundity was positively correlated with the number of stored sperm in the female sperm-storage organ at the time of isolation, but it was not correlated with body or ovary size of the worms. Our results suggest that female fecundity in M. lignano is primarily determined by the resources available for egg production, and not by the level of polyandry, confirming classic sexual selection theory for simultaneous hermaphrodites.     

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).     

Morphological and genomic characterisation of the Schistosoma hybrid infecting humans in Europe reveals admixture between Schistosoma haematobium and Schistosoma bovis

Schistosomes cause schistosomiasis, the world’s second most important parasitic disease after malaria in terms of public health and social-economic impacts. A peculiar feature of these dioecious parasites is their ability to produce viable and fertile hybrid offspring. Originally only present in the tropics, schistosomiasis is now also endemic in southern Europe. Based on the analysis of two genetic markers the European schistosomes had previously been identified as hybrids between the livestock- and the human-infective species Schistosoma bovis and Schistosoma haematobium, respectively. Here, using PacBio long-read sequencing technology we performed genome assembly improvement and annotation of S. bovis, one of the parental species for which no satisfactory genome assembly was available. We then describe the whole genome introgression levels of the hybrid schistosomes, their morphometric parameters (eggs and adult worms) and their compatibility with two European snail strains used as vectors (Bulinus truncatus and Planorbarius metidjensis). Schistosome-snail compatibility is a key parameter for the parasites life cycle progression, and thus the capability of the parasite to establish in a given area. Our results show that this Schistosoma hybrid is strongly introgressed genetically, composed of 77% S. haematobium and 23% S. bovis origin. This genomic admixture suggests an ancient hybridization event and subsequent backcrosses with the human-specific species, S. haematobium, before its introduction in Corsica. We also show that egg morphology (commonly used as a species diagnostic) does not allow for accurate hybrid identification while genetic tests do.     

Population dynamics ofMalacosoma neustria testacea (Lepidoptera: Lasiocampidae): Stabilizing process in a field population

Stabilizing mechanism in population of Malacosoma neustria testacea was investigated in a central part of Japan based on eight year survey. Population fluctuation in each developmental stage in the experimental field was rather small, i.e., 5.9 times in egg and 85.0 times in female adult. Pupal weight negatively correlated with the densities of 5th instar larvae and prepupae (cocoons) and correlation coefficient was highly significant in females. Population density was stabilized by density-dependent dispersal of female moths in preovipositional period. Comparison between fecundity of emerged moths and that of actually oviposited ones in the experimental field suggested that density-dependent dispersal took place as the result of density-dependent size variation, i.e., small-sized female months have higher flying ability. This hypothesis was supported by the experiment in which flying ability of newly emerged female moth was measured. Similar stabilizing mechanism is expected to occur in semelparous or pro-ovigenic insects.     

In vitro effects of amodiaquine on paired Schistosoma mansoni adult worms at concentrations of less than 5 μg/mL

In this study, the in vitro effects of amodiaquine (AQ) monotherapy on the egg output of paired adult Schistosoma mansoni worms and their survival during in vitro culture were assessed. In addition, the gross morphological alterations of male and female worms caused by AQ were visually observed under a dissecting microscope. AQ significantly reduced the daily egg output of paired adult S. mansoni worms following incubation for 14 days at 1-5 µg/mL, but not at 0.5 µg/mL, compared with the control group. AQ also reduced the survival of male and female worms at concentrations of 2 and 5 µg/mL, respectively. Moreover, exposure to 5 µg/mL AQ caused severe swelling and/or localisation of black content in the body of all male and female worms within one or two days of incubation; subsequently, shrinkage in the male worms and elongation in the female worms were observed. The initial morphological alterations caused by AQ occurred along the intestinal tract of the male and female worms. To our knowledge, this is the first study to report not only the efficacy of AQ at concentrations lower than 5 µg/mL on paired adult S. mansoni worms, but also the effects of AQ on the intestinal tracts of worms in in vitro culture.