A novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae regulates inflammasome activation through the TLR4-ROS-NLRP3 pathway.

BACKGROUND: Giant pandas (Ailuropoda melanoleuca) are the obligate host of the parasitic roundworm Baylisascaris schroederi. The infection of giant pandas with B. schroederi is very common. At present, little is known about the mechanism of immune interaction between B. schroederi and the host. As an important component of innate immunity, the NOD-like receptor 3 (NLRP3) inflammasome plays an important role in host immune response and the occurrence and development of infectious diseases. METHODS: We analyzed the regulation of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) by the recombinant B. schroederi migratory larvae cysteine protease inhibitor rBsCPI-1, knowing from a previous study that the CPI-1 is highly expressed in B. schroederi migratory larvae. We first determined the effects of rBsCPI-1 and excretory-secretory products of B. schroederi migratory larvae on cell proliferation using the CCK-8 and LDH release assays. We then analyzed NLRP3 inflammasome activation, pyroptosis and pro-inflammatory cytokine release by quantitative-PCR, western blotting and enzyme-linked immunosorbent assay. The signaling pathway of rBsCPI-1 to activate NLRP3 inflammasomes was analyzed in activation and inhibition experiments. Finally, the effects of rBsCPI-1 on inflammasome activation in mice immunized with rBsCPI-1 were analyzed. RESULTS: The activation and inhibition experiments revealed that rBsCPI-1 induced inflammasome activation through the TLR4-ROS-NLRP3 signaling pathway, with reactive oxygen species (ROS) not only functioning as an activator of the NLRP3 inflammasome, but also an activation product of the NLRP3 inflammasome. rBsCPI-1 promoted the activation and assembly of the NLRP3 inflammasome, which further converted the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 into mature active forms. At the same time, caspase-1 cleaved gasdermin D to trigger cell pyroptosis. The results of animal immunization experiments further confirmed that rBsCPI-1 could induce the activation of the NLRP3 inflammasome. CONCLUSIONS: rBsCPI-1 activates the inflammasome through the TLR4-ROS-NLRP3 signaling pathway and further induces the pyroptosis of MDMs and release of pro-inflammatory factors IL-1ß and IL-18, thus promoting the occurrence and development of the inflammatory response in the host.

The P-glycoprotein repertoire of the equine parasitic nematode Parascaris univalens.

P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the major site of expression but individual gene expression patterns were not conserved between the two nematodes. In P. univalens, PunPgp-9, PunPgp-11.1 and PunPgp-16.2 consistently exhibited the highest expression level in two independent transcriptome data sets. Using RNA-Seq, no significant upregulation of any Pgp was detected following in vitro incubation of adult P. univalens with ivermectin suggesting that drug-induced upregulation is not the mechanism of Pgp-mediated ML resistance. Expression and functional analyses of PunPgp-2 and PunPgp-9 in Saccharomyces cerevisiae provide evidence for an interaction with ketoconazole and ivermectin, but not thiabendazole. Overall, this study established reliable reference gene models with significantly improved annotation for the P. univalens Pgp repertoire and provides a foundation for a better understanding of Pgp-mediated anthelmintic resistance.

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole.

BACKGROUND: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. METHODS: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10-9 M, 10-11 M, 10-13 M), pyrantel citrate (10-6 M, 10-8 M, 10-10 M), thiabendazole (10-5 M, 10-7 M, 10-9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. RESULTS: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10-9 M of ivermectin. CONCLUSIONS: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

Functional Ultrastructure of the Excretory Gland Cell in Zoonotic Anisakids (Anisakidae, Nematoda).

Excretory and secretory products are crucial for parasite infectivity and host immunomodulation, but the functioning and ultrastructure of the excretory gland cell (EC) that produces these products are still scarcely understood and described. In light of growing reports on anisakiasis cases in Europe, we aimed to characterise the EC of larval Anisakispegreffii and adult Pseudoterranovaazarasi. In the latter, EC starts 0.85 mm from the head tip, measuring 1.936 × 0.564 mm. Larval EC shows a long nucleus with thorn-like extravaginations toward the cytoplasm, numerous electron-dense and -lucent secretory granules spanning from the perinuclear to subplasmalemmal space, an elevated number of free ribosomes, small, spherical mitochondria with few cristae and a laminated matrix, small and few Golgi apparatuses, and few endoplasmic reticula, with wide cisternae complexes. Ultrastructure suggests that anaerobic glycolysis is the main metabolic pathway, obtained through nutrient endocytosis across the pseudocoelomic surface of the EC plasmalemma and its endocytic canaliculi. Thorn-like extravaginations of EC karyotheca likely mediate specific processes (Ca2+ signaling, gene expression, transport, nuclear lipid metabolism) into the extremely wide EC cytosol, enabling focal delivery of a signal to specific sites in a short time. These functional annotations of parasitic EC should help to clarify anisakiasis pathogenesis.

Epidemiology of Sulcascaris sulcata (Nematoda: Anisakidae) ulcerous gastritis in the Mediterranean loggerhead sea turtle (Caretta caretta).

Sulcascaris sulcata Rudolphi 1819 is a gastric nematode parasite of sea turtles. Here, we report the occurrence and describe for the first time the pathological changes caused by S. sulcata in the Mediterranean loggerhead sea turtle (Caretta caretta) stranded along the Tyrrhenian coast and northern Adriatic coast of Italy. Prevalence of infection was significantly higher in loggerhead sea turtles from the Adriatic Sea. Both prevalence and abundance of infection showed an increasing trend along with host age classes from both geographical localities. Nevertheless, while many small loggerhead sea turtles were found infected from the Adriatic Sea, only bigger individuals were infected from the Tyrrhenian Sea. The most common gross pathological change was a mucous gastritis with focal to multifocal raised ulcerous lesions roundish to irregular in shape ranging from 1 to over 20 cm in length, and cream-yellowish to greenish in color. The severity grade of gastritis increased with higher number of S. sulcata individuals. Microscopic pathological changes ranged from atrophic gastritis with heterophilic infiltration in the lamina propria to the destruction of the mucosal and sub-mucosal surfaces and necrosis. Results here obtained demonstrate that S. sulcata may cause ulcerous gastritis in both samples of loggerhead sea turtles studied from the Mediterranean Sea. Observed differences in S. sulcata infection among the different host age classes and between the two studied basins are likely linked to the differences of regional habitat and intermediate prey host availability.

Fatty-binding protein and galectin of Baylisascaris schroederi: Prokaryotic expression and preliminary evaluation of serodiagnostic potential.

Baylisascaris schroederi is a common parasite of captive giant pandas. The diagnosis of this ascariasis is normally carried out by a sedimentation-floatation method or PCR to detect eggs in feces, but neither method is suitable for early diagnosis. Fatty acid-binding protein (FABP) and galectin (GAL) exist in various animals and participate in important biology of parasites. Because of their good immunogenicity, they are seen as potential antigens for the diagnosis of parasitic diseases. In this study, we cloned and expressed recombinant FABP and GAL from B. schroederi (rBs-FABP and rBs-GAL) and developed indirect enzyme-linked immunosorbent assays (ELISAs) to evaluate their potential for diagnosing ascariasis in giant pandas. Immunolocalization showed that Bs-FABP and Bs-GAL were widely distributed in adult worms. The ELISA based on rBs-FABP showed sensitivity of 95.8% (23/24) and specificity of 100% (12/12), and that based on rBs-GAL had sensitivity of 91.7% (22/24) and specificity of 100% (12/12).

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.

Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans.

P-glycoproteins (Pgps) are suspected to mediate drug extrusion in nematodes contributing to macrocyclic lactone resistance. This association was recently shown for Parascaris Pgp-11. Ivermectin resistance was correlated with the presence of three pgp-11 single nucleotide polymorphisms and/or increased pgp-11 mRNA levels. In the present study, the ability of Pgp-11 to modulate ivermectin susceptibility was investigated by its expression in a pgp-11-deficient Caenorhabditis elegans strain. Expression of Parascaris pgp-11 in two transgenic lines significantly decreased ivermectin susceptibility in a motility (thrashing) assay conducted in liquid medium. The EC50 values increased by 3.2- and 4.6-fold in the two lines relative to a transgenic control strain. This is the first report on the successful functional analysis of a parasitic nematode Pgp in the model organism C. elegans.

Deep amplicon sequencing of preselected isolates of Parascaris equorum in ß-tubulin codons associated with benzimidazole resistance in other nematodes.

BACKGROUND: The development of anthelmintic resistance (AR) to macrocyclic lactones in the equine roundworm Parascaris equorum has resulted in benzimidazoles now being the most widely used substance to control Parascaris infections. However, over-reliance on one drug class is a risk factor for the development of AR. Consequently, benzimidazole resistance is widespread in several veterinary parasites, where it is associated with single nucleotide polymorphisms (SNPs) in drug targets encoded by the ß-tubulin genes. The importance of these SNPs varies between different parasitic nematodes, but it has been hypothesised that they occur, at low allele frequencies, even in unselected populations. This study investigated whether these SNPs exist in the P. equorum population and tested the hypothesis that BZ resistance can develop from pre-existing SNPs in codons 167, 198 and 200 of the ß-tubulin isotype 1 and 2 genes, reported to be associated with AR in strongylids. The efficacy of the oral paste formula fenbendazole on 11 farms in Sweden was also assessed. METHODS: Two isotype-specific primer pairs were designed, one on either side of the codon 167 and one on either side of codons 198 and 200. A pool of 100,000 larvae was sequenced using deep amplicon sequencing by Illumina HiSeq. Faecal egg count reduction test was used to assess the efficacy of fenbendazole. RESULTS: No SNPs were observed in codons 167, 198 or 200 of the ß-tubulin isotype 1 or 2 genes of P. equorum, even though 100,000 larvae were sequenced. Faecal egg count reduction testing of fenbendazole showed that this anthelmintic was still 100% effective, meaning that the likelihood of finding high allele frequency of SNPs associated with benzimidazoles resistance in P. equorum was low. Unexpectedly, the allele frequencies observed in single worms were comparable to those in pooled samples. CONCLUSIONS: We concluded that fenbendazole does not exert selection pressure on the ß-tubulin genes of isotypes 1 and 2 in P. equorum. The fact that no pre-existing SNPs were found in codons 167, 198 and 200 in P. equorum also illustrates the difficulties in generalising about AR mechanisms between different taxonomic groups of nematodes.

Comparative study of the metal accumulation in Hysterothalycium reliquens (nematode) and Paraphilometroides nemipteri (nematode) as compared with their doubly infected host, Nemipterus peronii (Notched threadfin bream).

In February 2013, forty-seven Notched threadfin bream, the Nemipterus peronii, were sampled from the eastern coastal waters of the South China Sea. The concentration of various elements, namely cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), strontium (Sr), manganese (Mn), selenium (Se), Lead (Pb), nickel (Ni), aluminum (Al), arsenic (As), iron (Fe), and Zinc (Zn) were analyzed in the liver, muscle, and kidney organs of the host, as well as in their parasites Hysterothalycium reliquens (nematode) and the Paraphilometroides nemipteri (nematode), using inductively coupled plasma mass spectrometry (ICP-MS). The former group of parasites showed highest accumulation capacity for Cr, Cu, Fe, Mn, Se, Ni, and Zn while the latter group had high accumulation potential of As, Hg, Cd, Al, Pb, and Sr. The divergence in heavy-metal accumulation profiles of both nematodes is linked with the specificity of microhabitats, cuticle morphology, and interspecific competition. The outcome of this study indicates that both parasite models can be used for biomonitoring of metal pollution in marine ecosystems.

Potential of recombinant inorganic pyrophosphatase antigen as a new vaccine candidate against Baylisascaris schroederi in mice.

The intestinal nematode Baylisascaris schroederi is an important cause of death for wild and captive giant pandas. Inorganic pyrophosphatases (PPases) are critical for development and molting in nematode parasites and represent potential targets for vaccination. Here, a new PPase homologue, Bsc-PYP-1, from B. schroederi was identified and characterized, and its potential as a vaccine candidate was evaluated in a mouse challenge model. Sequence alignment of PPases from nematode parasites and other organisms show that Bsc-PYP-1 is a nematode-specific member of the family I soluble PPases. Immunohistochemistry revealed strong localization of native Bsc-PYP-1 to the body wall, gut epithelium, ovary and uterus of adult female worms. Additionally, Bsc-PYP-1 homologues were found in roundworms infecting humans (Ascaris lumbricoides), swine (Ascaris suum) and dogs (Toxocara canis). In two vaccine trials, recombinant Bsc-PYP-1 (rBsc-PYP-1) formulated with Freund complete adjuvant induced significantly high antigen-specific immunoglobulin (Ig)G but no IgE or IgM responses. Analysis of IgG-subclass profiles revealed a greater increase of IgG1 than IgG2a. Splenocytes from rBsc-PYP-1/FCA-immunized mice secreted low levels of T helper (Th)1-type cytokines, interferon-γ and interleukin (IL)-2, while producing significantly high levels of IL-10 and significantly elevated levels of IL-4 (Th2 cytokines) after stimulation with rBsc-PYP-1 in vitro. Finally, vaccinated mice had 69.02-71.15% reductions (in 2 experiments) in larval recovery 7 days post-challenge (dpc) and 80% survival at 80 dpc. These results suggest that Th2-mediated immunity elicited by rBsc-PYP-1 provides protection against B. schroederi, and the findings should contribute to further development of Bsc-PYP-1 as a candidate vaccine against baylisascariasis.

Mercury in parasitic nematodes and trematodes and their double-crested cormorant hosts: bioaccumulation in the face of sequestration by nematodes.

Endoparasites can alter their host's heavy metal concentrations by sequestering metals in their own tissues. Contracaecum spp. (a nematode), but not Drepanocephalus spathans (a trematode), were bioaccumulating mercury to concentrations 1.5 times above cormorant hosts. Nematodes did not have significantly greater stable nitrogen isotope values (δ(15)N) than their hosts, which is contradictory to prey-predator trophic enrichment studies, but is in agreement with other endoparasite-host relationships. However, Contracaecum spp. δ(13)C values were significantly greater than their hosts, which suggest that nematodes were consuming host tissues. Nematodes were accumulating and thus sequestering some of their cormorant hosts' body burden of methyl mercury; however, they were not dramatically reducing their hosts' accumulation of methyl mercury.

Accumulation of some heavy metals seasonally in Hysterotylacium aduncum (Nematoda) and its host Red Sea Bream, Pagellus erythrinus (Sparidae) from Gulf of Iskenderun (North-eastern Mediterranean).

The Red Sea Bream's nematode and Sparus aurata, sampled from the Iskenderun Bay, North-eastern Mediterranean in March 2008 were analysed by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) for their some heavy metal (Cd, Cr, Cu, Fe, Hg, Mn, Mg, Pb and Zn) levels. The metal concentrations of the parasites were compared to different organs (liver, muscle, swimbladder, intestine and skin) of the fish hosts. The highest Cd (0.303 mg/kgg ww) concentrations were found in the muscle, highest Cr (4.932 mg/kg ww), Hg (2.350 mg/kg ww) Pb (22.82 mg/kg ww) concentrations were found in the parasite, highest Cu (7.608 mg/kg ww) and Fe (176.7 mg/kg ww) concentrations were found in the liver, highest Mn (31.24 mg/kg ww) Zn (78.51 mg/kg ww) concentrations were found in the swimbladder for parasitized fish. The highest Cd (0.612 mg/kg ww), Cu (8.261 mg/kg ww) Fe (261.1 mg/kg ww) concentrations were found in the liver, highest Cr (6.123 mg/kg ww) and Pb (9.125 mg/kg ww) concentrations were found in the intestine, highest Hg (2.013 mg/kg ww) Zn (83.30 mg/kg ww) and Mn (41.24 mg/kg ww) concentrations were found in the swimbladder for un-parasitized fish.

CO2-fixing enzymes and phosphoenolpyruvate metabolism in the fish parasite Hysterothylacium aduncum (Ascaridoidea, Anisakidae).

CO2 stimulates the development of many of the intestinal helminths that are able to fix CO2 by means of phosphoenolpyruvate carboxykinase (PEPCK), such as Hysterothylacium aduncum. We determined the activity of CO2-fixing enzymes such as PEPCK and phosphoenolpyruvate carboxylase (PEPC), although no significant activity was detected for pyruvate carboxylase or carboxylating-malic enzyme. The former act on phosphoenolpyruvate (PEP) to yield oxalacetate. In the helminths studied, PEP has a vital role in glucidic metabolism. Consequently, we determined the activity of other enzymes involved in the crossroad of PEP, such as pyruvate kinase (PK), lactate dehydrogenase and malate dehydrogenase. All enzymes detected showed significant variations in activity during the in vitro development of the parasite from the third larval stage to mature adult. Fixing of CO2 by PEPCK decreased during development (from 228 to 115 nmol min(-1) mg(-1) protein), while that by PEPC increased (from 19 to 46 nmol min(-1) mg(-1) protein). This enzyme, which is rare in animals, could play a part in detecting levels of free phosphate, releasing it from PEP when required for processes such as glycogenolysis, glycolysis and adenosine 5'-triphosphate (ATP) synthesis. PK, which showed increasing activity during development up to immature adult (from 56 to 82 nmol min(-1) mg(-1) protein), could act in combination with PEPC to obtain energy in the cytosol (in the form of ATP) and in the mitochondria (possible destination of the pyruvate formed), compensating for the decrease in activity of PEPCK.

Accumulation of some heavy metals in Raphidascaris acus (Bloch, 1779) and its host (Esox lucius L., 1758).

Concentrations of some heavy metals (Fe, Zn, Cu, Mn and Cr) in liver of pike (Esox lucius L., 1758) and its endoparasite [Raphidascaris acus (Bloch, 1779)] inhabiting Isikli Lake (Turkey) were analyzed using atomic absorption spectrophotometry. Only Fe and Zn were detected in R. acus and liver of fish, while levels of Cu, Mn and Cr were below detection limit (<0.028). The Fe and Zn level in R. acus were 68.4 and 86.9 times higher than in the liver. Nematodes could provide reliable information about the heavy metal pollution of the lakes.

Treatment of Baylisascaris procyonis infections in dogs with milbemycin oxime.

An examination was made as to the ability of Sentinel Flavor Tabs (milbemycin oxime/lufenuron) to treat Baylisascaris procyonis infections in dogs. The study was designed as a critical trial and included five naturally infected dogs and two dogs that were experimentally infected. Another dog from a prior clinical trial that was treated with Sentinel Flavor Tabs as part of the original FDA submission package for intestinal nematode infections was also included with the treated dogs. Of the five naturally infected dogs treated as part of the critical trial, three were cleared of their infections. These five dogs passed a total of 52 worms after treatment; one dog retained 23 worms and the other retained 1 worm at necropsy 7 days after treatment. Two of five experimentally infected Beagle dogs that had been given mice that had been fed 200 infectious eggs, developed patent infections with the parasite. These dogs were treated, and one of the dogs passed one worm and the other passed two worms after treatment with no worms being detected at necropsy 7 days after treatment. The one dog that was treated with milbemycin oxime as part of the FDA submission was clear of worms at necropsy. Overall, the mean efficacy of Sentinel Flavor Tabs was found to be 91.0%. Of the eight dogs that were treated, six were totally cleared of their infections, a cure rate of 75%. The two dogs that did not clear their infections had very large numbers of adult B. procyonis within their intestinal tracts at the time of treatment, one dog had 40 worms (23 remaining) and the other had 26 worms (1 remaining). It is suggested that the treatment of dogs with monthly Sentinel Flavor Tabs could markedly reduce the chance of infected dogs contaminating the environment. Also, additional monthly treatments are highly likely to clear dogs of any worms not killed with the initial treatment.

Thienylvinylindoles as inhibitors of mitochondrial NADH dehydrogenase.

In connection with a previous study, new phenylindoles bearing a 2- or 3-thienyl group were synthesized and tested as specific inhibitors of mitochondrial NADH dehydrogenase. The position of the phenyl ring and the geometrical configuration play an important role in the activity and specificity of these derivatives. In order to study the mechanism of action of these thienylvinylindoles, their activity was compared with that of known inhibitors in a new test employing exogenous quinones.

Identification of ecdysone 25-O-beta-D-glucopyranoside as a new metabolite of ecdysone in the nematode Parascaris equorum.

A major metabolite of [3H]ecdysone injected into adults of the nematode Parascaris equorum maintained in vitro for 48 h was secreted into the culture medium. The compound could be hydrolysed with a crude hydrolase preparation from Helix pomatia, yielding ecdysone. Sufficient quantity of this material for identification was produced by administration of ecdysone to the parasites. The resulting compound was purified by h.p.l.c. and identified as ecdysone 25-O-beta-D-glucopyranoside by n.m.r. spectroscopy and by fast atom bombardment mass spectrometry of the conjugate and of the sugar released by enzymic hydrolysis. The significance of formation of the glucoside is uncertain.