Next generation sequencing technologies have facilitated a shift from a few targeted loci in population genetic studies to whole genome approaches. Here, we review the types of questions and inferences regarding the population biology and evolution of parasitic helminths being addressed within the field of population genomics. Topics include parabiome, hybridization, population structure, loci under selection and linkage mapping. We highlight various advances, and note the current trends in the field, particularly a focus on human-related parasites despite the inherent biodiversity of helminth species. We conclude by advocating for a broader application of population genomics to reflect the taxonomic and life history breadth displayed by helminth parasites. As such, our basic knowledge about helminth population biology and evolution would be enhanced while the diversity of helminths in itself would facilitate population genomic comparative studies to address broader ecological and evolutionary concepts.

The free-living infectious stages of macroparasites, specifically, the cercariae of trematodes (flatworms), are likely to be significant (albeit underappreciated) vectors of nutritionally important polyunsaturated fatty acids (PUFA) to consumers within aquatic food webs, and other macroparasites could serve similar roles. In the context of de novo omega-3 (n-3) PUFA biosynthesis, it was thought that most animals lack the fatty acid (FA) desaturase enzymes that convert stearic acid (18:0) into ɑ-linolenic acid (ALA; 18:3n-3), the main FA precursor for n-3 long-chain PUFA. Recently, novel sequences of these enzymes were recovered from 80 species from six invertebrate phyla, with experimental confirmation of gene function in five phyla. Given this wide distribution, and the unusual attributes of flatworm genomes, we conducted an additional search for genes for de novo n-3 PUFA in the phylum Platyhelminthes. Searches with experimentally confirmed sequences from Rotifera recovered nine relevant FA desaturase sequences from eight species in four genera in the two exclusively endoparasite classes (Trematoda and Cestoda). These results could indicate adaptations of these particular parasite species, or may reflect the uneven taxonomic coverage of sequence databases. Although additional genomic data and, particularly, experimental study of gene functionality are important future validation steps, our results indicate endoparasitic platyhelminths may have enzymes for de novo n-3 PUFA biosynthesis, thereby contributing to global PUFA production, but also representing a potential target for clinical antihelmintic applications.

Specific antisera, directed against the highly conserved C-terminal hexapeptide amide of mammalian pancreatic poly-peptide (PP) and the invertebrate peptide FMRFamide, have been used in conjunction with post-embedding, IgG-conjugated colloidal gold immunostaining to demonstrate peptide immunoreactivity at subcellular level in the nervous system of adult Diclidophora merlangi. Gold labelling revealed that immunoreactivity for PP and FMRFamide was localized exclusively in dense-cored vesicles occupying the majority of axons in the central nervous system. Double-labelling demonstrated an apparent co-localization of PP and FMRFamide in the same dense-cored vesicles. Antigen pre-absorption experiments indicated cross-reactivity of the two antisera as unlikely, and that some if not all of the PP/FMRFamide immunostaining in the parasite was due to a neuropeptide F-like peptide.

An electron microscopical examination has been made of the fine structure and disposition of pancreatic polypeptide immunoreactive cells associated with the egg-forming apparatus in Diclidophora merlangi. The cell bodies are positioned in the parenchyma surrounding the ootype and taper to axon-like processes that extend to the ootype wall. The terminal regions of these processes branch and anastomose and, in places, the swollen endings or varicosities form synaptic appositions with the muscle fibres in the ootype wall. The cells are characterized by an extensive GER-Golgi system that is involved in the assembly and packaging of dense-cored vesicles. The vesicles accumulate in the axons and terminal varicosities, and their contents were found to be immunoreactive with antisera raised to the C-terminal hexapeptide amide of pancreatic polypeptide. It is concluded that the cells are neurosecretory in appearance and that, functionally, their secretions may serve to regulate ootype motility and thereby help co-ordinate egg production in the worm.

The effects of each of the known platyhelminth neuropeptides were determined on muscle-strip preparations from the liver fluke, Fasciola hepatica. The activity of synthetic replicates of the C-terminal nonapeptide of neuropeptide F (NPF9, Moniezia expansa), and the FMRFamide-related peptides (FaRPs), GNFFRFamide, RYIRFamide, GYIRFamide and YIRFamide, were examined. Muscle-strip activity was recorded from 1 mm segments of muscle prepared from 28 to 32-day-old worms, using a photo-optic transducer system. None of the peptides (≤ 10 μM) altered baseline tension significantly; however, each of the peptides increased the amplitude and frequency of muscle contraction. The threshold for activity of each of the peptides examined was, respectively, 1 nM (RYIRFamide), 0·3 μM (GYIRFamide and YIRFamide), and 10 μM (GNFFRFamide and NPF9). All of the effects were reversible and repeatable, following wash-out. Muscle-strip integrity was tested following experimentation, using arecoline (10 μM) and high-K+ bathing medium (90 mM K+).

Muscle fibres isolated from adult Schistosoma mansoni contracted in a dose-dependent manner when exposed to elevated K+ with a maximum response obtained with 25 mM K+. These contractions were dependent on extracellular Ca2+ since Co2+ (5 mM) or nicardipine (1 μM) blocked the high K+ contractions. Serotonin (300 nM or higher) was required for maintenance of high K+ contractions. With concentrations of serotonin less than 300 nM the response was dose dependent. 5-Methoxytryptamine or α-methylserotonin at 1 μM as well as 10 μM tryptamine were able to substitute for serotonin, but 1 μM 5-carboxyamidotryptamine was ineffective. The order of potency for antagonists (10 μM) was: methiothepin > metergoline > Ly-278,584 = ketanserin. This pattern of responsiveness does not fit well with any known mammalian serotonin receptor subtype. Since forskolin, an adenylate cyclase activator, is able to mimic the action of serotonin and H89, a protein kinase inhibitor, is able to block the effect of serotonin, the effect of serotonin on contractility of the muscle may be via a cAMP-dependent pathway.

The localization and distribution of SALMFamide immunoreactivity (IR), SI(GFNSALMFamide), in the nervous system of both the adult and larval stages of the trematode Schistosoma mansoni has been determined by an indirect immunofluorescent technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the nervous system of adult male and female S. mansoni. In the central nervous system (CNS), IR was evident in nerve cells and fibres in the anterior ganglia, cerebral commissure and dorsal and ventral nerve cords. In the peripheral nervous system (PNS), IR was apparent in nerve plexuses associated with the subtegmental musculature, oral and ventral suckers, the lining of the gynaecophoric canal, and in fine nerve fibres innervating the dorsal tubercles of the male worm. In the reproductive system of male and female worms, Sl-IR was only observed around the ootype/Mehlis' gland complex in the female. Immunostaining was also evident in the nervous system of both miracidium and cercarial larval stages. A post-embedding, IgG-conjugated colloidal gold immunostaining technique was employed to examine the subcellular distribution of SALMFamide-IR in the CNS of S. mansoni. Gold labelling of peptide was localized over dense-cored vesicles within nerve cell bodies and fibres constituting the neuropile of the anterior ganglia, cerebral commissure and nerve cords of the CNS. Antigen pre-absorption studies indicated that the results obtained do suggest S1-like immunostaining and not cross-reactivity with other peptides, in particular FMRFamide.

Three morphologically distinct types of muscle fibres isolated from the platyhelminth Schistosoma mansoni have been studied with whole cell current- and voltage-clamp techniques. Fibres showed a marked time-dependent decrease in membrane resistance in response to depolarizing current injections. Voltage-clamp experiments revealed the presence of two distinct voltage-gated outward currents. The most prominent current is a slowly activating, slowly and incompletely inactivating potassium current similar to delayed rectifier currents which have been described in a variety of cell types from a variety of organisms. Also present is a faster activating, quickly and completely inactivating potassium current that shares functional characteristics with ‘A’-currents. All three of the cell types studied possess a delayed rectifier current, but only two of the three types have ‘A’-currents. Though depolarization with high K+ leads to contraction of the dispersed fibres, no voltage-gated inward currents could be detected by whole cell voltage-clamp under any of our conditions.

We have isolated a cDNA (SKvl. 1) encoding a Shaker-related K+ channel from an adult cDNA library of the human parasitic trematode Schistosoma mansoni. The deduced amino acid sequence (512 aa, 56·5 kDa) contains 6 putative membrane-spanning domains (S1–S6) and a pore-forming domain (H5). SKv1.1 is grouped in the Shaker family, but forms a unique branch within this family, on the basis of dendrogram analysis. SKv1.1 shows significant sequence identity with most other Shaker channels, with 64—74% identity in the core region (S1–S6). It has the highest sequence identity with the K+ channel (Ak01a) from Aplysia. Northern blot analysis detected a single primary transcript of 2·8 kb. Southern blot analysis indicated that SKv1.1 is present as a single copy in the genomic DNA of S. mansoni. Expression of SKv1. 1 in Xenopus oocytes produced a rapidly activating and inactivating outward K+ current which is highly sensitive to 4-aminopyridine, but is insensitive to tetraethylammonium, mast cell degranulating peptide, dendrotoxin and charybdo-toxin. The presence of a Shaker homologue in Schistosoma suggests that Sh subfamilies may exist in other lower invertebrates as well as platyhelminths.

Molluscan FMRFamide and two recently discovered platyhelminth FMRFamide-related peptides (FaRPs), GNFFRFamide from the cestode Moniezia expansa and RYIRFamide from the terrestrial turbellarian Artioposthia triangulata, cause dose-dependent contractions of individual muscle fibres from Schistosoma mansoni in vitro. The most potent FaRP tested was the turbellarian peptide RYIRFamide, which produced a concentration-dependent effect between 10−9 and 10−7 M. FMRFamide and GNFFRFamide were less potent, inducing contractions between 10−8–10−6 M and 10−7–10−5 M respectively. The contractile effect of each of these peptides was blocked by the presence of 1 µM FMR-DFamide. FMRF free acid did not elicit contraction of the muscle fibres. The FaRP-induced contractions did not occur if the Ca2+ was omitted and 0·5 µM EGTA was added to the extracellular medium. The FaRP-induced contractions were not blocked by the Ca2+ channel blockers nicardipine, verapamil or diltiazem, although high K+-induced contractions of these fibres were blocked by nicardipine. These data indicate the presence of FaRP receptors on schistosome muscle fibres and demonstrate their ability to mediate muscle contraction. The action of these endogenous flatworm peptides on schistosome muscle is the first demonstration of a direct excitatory effect of any putative neurotransmitter on the muscle of a flatworm, and establishes a role for FaRPs in neuromuscular transmission in trematodes. In addition, it provides the first evidence that the peptidergic nervous system is a rational target for chemotherapeutic attack in parasitic platyhelmiths.

As we have recently shown that GABA should be considered a putative neurotransmitter in Schistosoma mansoni, the present work aimed to search for GABAA receptors in adult worms using [3H]-flunitrazepam to label the allosteric benzodiazepine binding site which is classically present on GABAA receptor complexes. We detected a large population (Bmax=8·25±1·1 pmol . mg protein−1) of high affinity (Kd=33·6±1·5 nm) binding sites for flunitrazepam. These sites harboured a singular pharmacological modulation that does not fit well with a mammalian central benzodiazepine receptor, mainly due to a very high affinity for Ro5-4864 and a very low affinity for clonazepam. We also detected a second population of benzodiazepine binding sites labelled with high affinity (IC50=85 nm) by [3H]-PK11195, a selective ligand of the mammalian peripheral benzodiazepine receptor. In conclusion, this work describes the pharmacological properties of a large population of central-like benzodiazepine receptors supporting their study as putative new targets for the development of anti-parasitic agents. We also describe, for the first time, the presence of peripheral benzodiazepine receptors in this parasite.

The protonephridium of platyhelminths including Schistosoma mansoni plays a pivotal role in their survival by excretion of metabolic wastes as well as xenobiotics, and can be revealed in the living adult parasite by certain fluorescent compounds which are concentrated in excretory tubules and collecting ducts. To determine the presence of the multidrug resistance-associated protein (MRP) as a possible transporter in protonephridial epithelium, adult schistosomes were exposed to a fluorescent Ca2+ indicator, fluo-3 acetyloxymethyl ester, which is a potential substrate of mammalian MRP. Specific fluorescence related to fluo-3/Ca2+ chelate delineated the whole length of the protonephridial system. Simultaneously, a fluorescent substance was accumulated in the posterior part of collecting ducts and the excretory bladder. Similarly, when other fluorogenic substrates for mammalian MRP such as monoclorobimane, fluorescein diacetate, and 5(6)-carboxyfluorescein diacetate were applied to adult schistosomes, these fluorescent markers were observed in the excretory tubules through to the excretory bladder. The excretory system of mechanically-transformed schistosomula was not labelled with any of these 4 fluorescent markers. These findings suggest that the protonephridial epithelium of adult schistosomes, but not schistosomula, might express the homologue of the mammalian MRP transporting organic anionic conjugates with glutathione, glucuronate or sulphate as well as unconjugated amphiphilic organic anions.

Excretion of metabolic wastes as well as xenobiotics is a major concern of all living organisms, and the Platyhelminthes including Schistosoma mansoni possess the protonephridial excretory system for their survival. Except for some ultrastructural and biochemical information, little is known about the protonephridium of platyhelminths due to a lack of established techniques for exploring the excretory activity. This study describes a new technique to assess the excretory activity of S. mansoni by use of the fluorescent marker resorufin, which is a potential substrate of the drug efflux pump, P-glycoprotein. After simple diffusion into the schistosome body, fluorescent resorufin was concentrated in the excretory tubules by an energy-dependent mechanism and excreted via the nephridiopore. The present technique of labelling functionally the excretory system was applicable to adult worms, but not schistosomula or cercariae. A variety of modulators known to interfere with mammalian P-glycoprotein function perturbed resorufin excretion from male adult schistosomes, including cyclosporin A, Ro11-2933, verapamil, or nifedipine. This technique of labelling the excretory system with fluorescent resorufin has enabled us to study aspects of the physiological function, hitherto unknown, of the protonephridial system of S. mansoni.

Viviparous gyrodactylid monogeneans which are detached from their host may form an important source of infection in fish communities. This is particularly significant for these ectoparasites which have no specific free-living stage and where transmission usually occurs through transfer of adult parasites when fish come into close contact. In this study, in vitro survival of Gyrodactylus gasterostei was correlated with changes in tissue ultrastructure of parents and their embryos during detachment and following return to a host. At 10 °C, detached worms survived for up to 89 h (50% survival = 20 h) with mortality influenced by both age-independent and age-dependent factors. The gastrodermis of attached, fed parasites is syncytial and contains a variety of vesicles and feeding vacuoles. However, following detachment and starvation, degenerative changes occur within the gastrodermis, eventually leading to marked damage to the embryo/s. When starved worms reattached to a fish, they began browsing on host epithelial cells within 1 min and ultrastructural evidence for phagocytic activity in the gastrodermis was detected after 5 min. Putative waste vacuoles increased in abundance after 5–30 min on the new host, coinciding with the reappearance of electron-dense vesicles and possibly indicating completion of the first intracellular cycle of digestion. Parental feeding directly influenced the normal cyclical maturation and regression of the uterine lining between births. In extreme cases, starvation led to detached parasites (6·1%) aborting their embryos. However, even short periods off the host influenced development and survival of embryos, suggesting that temporary interruption of nutrient flow to the embryo can significantly affect gyrodactylid reproductive rates.

The Zn2+-chelating metalloprotease inhibitor 1,10-phenanthroline (phenanthroline, 5–150 μM) elicited dose-dependent contraction of the longitudinal and circular (transverse) musculature of adult male schistosomes. At the same concentrations, phenanthroline did not cause contraction of dispersed individual muscle fibres. The phenanthroline-induced contractions were reduced by the inclusion of 100 or 300 μM Zn2+ in the extracellular medium. Phenanthroline (0·5–150 μM) also inhibited the egg production of adult worm pairs in vitro, with a 98% reduction at 50 μM. When worm pairs were exposed to phenanthroline, the males detached from the dish and released the females, resulting in unpaired worms. At the higher concentrations (50 and 150 μM), the worms were killed in vitro. Worm burdens were reduced by over 50% in infected mice injected with phenanthroline (20 mg/kg/day for 4 days), but twice the dose resulted in only a 25% reduction. Phenanthroline injections also induced an hepatic shift and an unpairing of adult worms in infected mice, and the female worms appeared degenerate and lacked gut pigmentation. Mice fed a diet containing 0·3% phenanthroline received significant protection from infection when challenged with schistosome cercaria, where phenanthroline-fed mice had 94% fewer adult worms than control mice. The broad range of phenanthroline effects on schistosomes suggests broad and important functions for metalloproteases in these worms.

The neuromuscular sodium currents of early invertebrates such as platyhelminths display distinctive kinetic and pharmacological properties. We have cloned a cDNA from the horseshoe crab flatworm Bdelloura candida that encodes a protein homologous to the primary subunit of voltage-gated sodium channels. The B. candida protein, named BdNa1, exhibits amino acid identity of 40–47% to sodium channels of vertebrates and higher invertebrates. BdNa1 has the multidomain structure characteristic of sodium channels, and is most highly conserved in the hydrophobic transmembrane segments and the regions that form the pore of the channel. Northern blot analysis confirms the presence of a 5·4 kb BdNa1 transcript in B. candida tissue. The information provided by analysis of the BdNa1 sequence offers insight into the physiology of platyhelminth sodium currents.