Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.

Molecular cytogenetic analysis of a triploid population of the human broad tapeworm, Dibothriocephalus latus (Diphyllobothriidea).

The large-sized tapeworm Dibothriocephalus latus is known as the broad or fish-borne cestode of mammals that is capable to infect humans and cause diphyllobothriosis. Recently, molecular data on D. latus has been accumulating in the literature and a complete genome sequence has been published; however, little is known about the karyotype and chromosome architecture. In this study, an in-depth karyological analysis of 2 D. latus specimens was carried out. The plerocercoids originated from a perch caught in subalpine Lake Iseo (Italy) and the tapeworms were reared in hamsters. Both specimens contained cells with a highly variable number of chromosomes ranging from18 to 27. Nevertheless, the largest portion of mitotic figures (47%) showed a number corresponding to the triploid set, 3n = 27. Accordingly, the karyotype of the analyzed specimens consisted of 9 triplets of metacentric chromosomes. Fluorescence in situ hybridization (FISH) with the 18S rDNA probe clearly demonstrated the presence of 3 clusters of hybridization signals on the triplet of chromosome 7, thus confirming the triploid status of the specimens. FISH with a telomeric (TTAGGG)n probe confined hybridization signals exclusively to the terminal chromosomal regions, supporting the earlier findings that this repetitive motif is a conserved feature of tapeworm telomeres.

Morphological and molecular characterization of Quinqueserialis (Digenea: Notocotylidae) species diversity in North America.

Estimates of trematode diversity are inaccurate due to unrecognized cryptic species and phenotypic plasticity within species. Integrative taxonomy (genetics, morphology and host use) increases the clarity of species delineation and improves knowledge of parasite biology. In this study, we used this approach to resolve taxonomic issues and test hypotheses of cryptic species in a genus of trematode, Quinqueserialis. Specimens from throughout North America were field collected from hosts and obtained from museums. We found three morphologically distinct groups and successfully sequenced specimens from two of these groups. DNA sequencing at the 28S and CO1 gene regions revealed that two of the three groups were genetically distinct. One genetic group included two morphological clusters demonstrating host-induced phenotypic plasticity within Quinqueserialis quinqueserialis. The other unique genetic group is a novel species, Quinqueserialis kinsellai n. sp., which is described herein. Our study illustrates the importance of integrating multiple sources of evidence when investigating trematode diversity to account for the influence of cryptic species or phenotypic plasticity. However, further sampling is needed to understand Quinqueserialis spp. diversity as some species have no genetic information associated with them.

Stephanoprora amurensis sp. nov., Echinochasmus milvi Yamaguti, 1939 and E. suifunensis Besprozvannykh, 1991 from the Russian southern Far East and their phylogenetic relationships within the Echinochasmidae Odhner 1910.

Mature worms of Stephanoprora amurensis sp. nov. were obtained in an experimental study of its life cycle. In the Russian southern Far East, this trematode circulates using freshwater snails Parajuga subtegulata, freshwater fish and birds as the first, second intermediate and final hosts, respectively. Stephanoprora amurensis sp. nov. differs from the well-known representatives of Stephanoprora in a number of morphometric indicators of the developmental stages. The validity of the species was also confirmed by nuclear and mitochondrial DNA markers. In addition, new genetic data were obtained for Echinochasmus suifunensis and Echinochasmus milvi. An analysis of phylogenetic relationships within Echinochasmidae based on the 28S rRNA gene and ITS2 region identified two clusters, one of which combines species of Echinochasmus with 20-22 collar spines and short-tailed cercariae, and the other which includes Stephanoprora spp. and a number of representatives of Echinochasmus with 24 collar spines and long-tailed cercariae. The results of phylogenetic analysis based on ITS2 data show interfamily level of differences between the two clusters and intergeneric differentiation between the three subclusters uniting the species of Stephanoprora and Echinochasmus.

An integrative approach assesses the intraspecific variations of Procamallanus (Spirocamallanus) inopinatus, a common parasite in Neotropical freshwater fishes, and the phylogenetic patterns of Camallanidae.

Integrative taxonomy was used to evaluate two component populations of Procamallanus (Spirocamallanus) inopinatus in Brazil and the phylogeny Camallanidae. Parasite populations were collected in the characiform Anostomoides passionis from River Xingu (Amazon basin) and Megaleporinus elongatus from River Miranda (Paraguay basin). Morphology was analysed using light and scanning electron microscopy (SEM). Genetic characterization was based on partial sequences of the 18S and 28S rDNA, and COI mtDNA. Phylogenies were based on 18S and COI due to data availability. Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (PTP) and *BEAST were used for species delimitation and validation. SEM revealed for the first time the presence of minute denticles and pore-like structures surrounding the oral opening, phasmids in females and confirmed other important morphological aspects. Statistical comparison between the two-component populations indicated morphometric variations, especially among males. The different component population of P. (S.) inopinatus showed variable morphometry, but uniform morphology and were validated as conspecific by the GMYC, PTP and *BEAST. Some camallanid sequences in GenBank have incorrect taxonomic labelling. Host, environment and geographic aspects seem to be related to some lineages within Camallanidae; however, their real phylogenetic meanings are still unclear.

Molecular Phylogenetic Analysis of Acotylea (Platyhelminthes: Polycladida).

Acotylea is a suborder of Polycladida (Rhabditophora, Platyhelminthes) characterized by lack of a cotyl (sucker-like structure) on the ventral surface of the body. We newly determined partial sequences of two mitochondrial (16S ribosomal RNA and cytochrome c oxidase subunit I) and two nuclear (18S and 28S ribosomal RNA) genes from 24 acotylean species (12 families and 14 genera). Based on these sequences in addition to those available in public databases, we inferred the phylogeny of 16 families and 27 genera of Acotylea from molecular phylogenetic analyses (maximum likelihood and Bayesian inference) based on concatenated gene sequences. Our analyses supported three clades corresponding to Discoceloidea, Leptoplanoidea, and Stylochoidea. The phylogenetic position of Callioplanidae remains unclear. Among family- or genus-level taxa, Gnesiocerotidae, Stylochoplanidae, and Comoplana were not monophyletic. We discuss the validities of Notocomplanidae and Koinostylochus, and the family-level assignment of Mirostylochus.

Molecular characterization of Urosporidium tapetis sp. nov., a haplosporidian hyperparasite infecting metacercariae of Parvatrema duboisi (Dollfus 1923), a trematode parasite of Manila clam Ruditapes philippinarum on the west coast of Korea.

Recently, a putative new hyperparasitic haplosporidian in the genus Urosporidium was identified from metacercariae of the trematode Parvatrema duboisi infecting Manila clam Ruditapes philippinarum on the west coast of Korea. In this study, we applied small subunit ribosomal DNA (SSU rDNA) sequences as a marker to substantiate the phylogenetic relationship of the unidentified Urosporidium within the Order Haplosporida. In our phylogenetic analysis, the 1890 bp of SSU rDNA sequences obtained were closely related to a haplosporidian parasite forming a sister clade to Urosporidium group, although the gene sequences were only 89.22-89.70% similar to Urosporidium spp. Such molecular phylogenetic distance within the genus suggested that the unidentified Urosporidium is a new member of the genus. Accordingly, we report the unidentified haplosporidian hyperparasite as Urosporidium tapetis sp. nov.

Angiostrongylosis in Cerdocyon thous (crab-eating fox) and Lycalopex gymnocercus (Pampas fox) in Southern Brazil.

The objective of this study was to identify species of Angiostrongylus spp. infecting wild carnivores in Southern Brazil, as well as to describe gross and histopathological findings associated with the infection. Necropsy was conducted in 16 wild carnivores parasitized by Angiostrongylus spp. Analysed lungs revealed multifocal dark-red areas of consolidation; in one case, multifocal firm white nodules spread in all pulmonary lobes were observed. In one animal, a focally extensive area of malacia associated with haemorrhage was noted in the encephalon. Histologically, multifocal granulomatous pneumonia or bronchopneumonia, associated with eggs and larvae in blood vessels, lung interstitium, alveoli, and sometimes in bronchi and bronchioles was observed. Adult nematodes were seen within blood vessels. The lesion observed in the brain was characterized as a focally extensive area of malacia associated with gitter cells, haemorrhage, thrombosis and a free intralesional larva. Through molecular techniques, seven positive samples of Angiostrongylus cantonensis were obtained, including the brain sample, and a positive sample of Angiostrongylus vasorum-like, all in Cerdocyon thous. The positive sample for A. vasorum showed 97% similarity with sequences deposited in GenBank, suggesting a new species or subspecies of Angiostrongylus sp. Infection of Lycalopex gymnocercus by Angiostrongylus spp. was confirmed by histological evaluation.

Genetic and morphometric categorization of Taenia ovis from Sheep in Iran.

Little is known about the genetic and morphological characters of Taenia ovis. The purpose of the present study was to characterize sheep isolates of T. ovis using rostellar hook morphometry as well as mitochondrial genes sequence analysis. Ninety sheep specimens of Cysticercus ovis were collected from 18 slaughterhouses in Iran. The mean ± s.d. for total length of large and small hooks were 174.1 ± 6.4 and 116.7 ± 5.4 µm, respectively. CO1 and 12S rRNA sequence analysis showed 11 and nine haplotypes, respectively. The level of pairwise nucleotide variations between individual haplotypes of CO1 and 12S rRNA genes were 0.3-1.1 and 0.2-1.0%, respectively. Level of nucleotide variation in CO1 and 12S rRNA between T. ovis haplotypes from present study and eight other Taenia species was found to be 11.3-17.8 and 5.3-16.3%, respectively. Phylogenetic analysis clustered all T. ovis isolates into a single clade comprised of the all CO1 and 12S rRNA haplotypes. CO1 nucleotide difference between T. ovis ovis and T. asiatica was 13.6% that is lesser than the corresponding difference between T. ovis ovis and T. ovis krabbei, warranting the designation of two separate species as T. ovis and T. krabbei. Interclass correlation coefficients showed that there was no significant association between rostellar hook length variation and the variability of the mitochondrial genes.

New information on morphology and molecular data of camallanid nematodes parasitising Xenopus laevis (Anura: Pipidae) in South Africa.

Three species of nematodes from the Camallanidae that are known to infect Xenopus laevis Daudin (Anura: Pipidae) were collected from several localities across South Africa. New data on morphology, partial 28S and cox1 genes, infection levels and distribution are presented herein. The most common species, Batrachocamallanus slomei Southwell et Kirshner, 1937, from the stomach and less often oesophagus, was found in eight localities. Camallanus kaapstaadi Southwell et Kirshner, 1937, also from the oesophagus, was found in two localities and C. xenopodis Jackson et Tinsley, 1995, from the intestine, at a single locality. New localities for both C. kaapstaadi and C. xenopodis provide a geographical range extension. Males of C. xenopodis are described for the first time herein. The existence of a left spicule in the males of both the species of Camallanus Railliet and Henry, 1915 is confirmed and measurements are provided. Although C. xenopodis is distinguished from C. mazabukae Kung, 1948 in the present study, we suggest greater sampling effort in other African amphibians to confirm the species status of the latter taxon. Finally, the new molecular data showed distant relationships between collected species of Camallanus and species parasitising fish and freshwater turtles.

Two new species of Stillabothrium (Cestoda: Rhinebothriidea) from stingrays of the genus Fontitrygon from Senegal.

Morphological and molecular analyses of cestode specimens collected during survey work of batoid elasmobranchs and their parasites in Senegal revealed two new species of the rhinebothriidean cestode genus Stillabothrium Healy et Reyda 2016. Stillabothrium allisonae Dedrick et Reyda sp. n. and Stillabothrium charlotteae Iwanyckyj, Dedrick et Reyda sp. n. are both described from Fontitrygon margaritella (Compagno et Roberts) and Fontitrygon margarita (Günther). Both new cestode species overlap in geographic distribution, host use and proglottid morphology, but are distinguished from each other, and from the other seven described species of Stillabothrium, on the basis of their pattern of bothridial loculi. Phylogenetic analyses based on sequence data for 1,084 bp from the D1-D3 region of 28S rDNA that included multiple specimens of both new species and eight other species of Stillabothrium corroborated the morphologically-determined species boundaries. The phylogenetic analyses indicate that S. allisonae sp. n. and S. charlotteae sp. n. are sister species, a noteworthy pattern given that the two species of the stingray genus Fontitrygon they both parasitise, F. margaritella and F. margarita, are also sister species. Although species of Stillabothrium vary widely in their patterns of facial loculi, the variation does not appear to correlate with phylogeny. Most species of Stillabothrium parasitise myliobatiform elasmobranch genera of the Dasyatidae Jordan. This study brings the number of described species of Stillabothrium to nine, three of which occur in the eastern Atlantic, two of which occur off the northern coast of Australia, and four of which are from coastal Borneo.

Clinical diagnostic value of viable Schistosoma japonicum eggs detected in host tissues.

BACKGROUND: Schistosomiasis, one of the neglected tropical diseases, is endemic in more than 70 countries. However, the clinical diagnosis of patients with a low degree of infection is an unsolved technical problem. In areas endemic for schistosomiasis japonica, proctoscopy detection of eggs has been one method used for clinical diagnosis. However, it is often a challenge to find typical live eggs and it is difficult to distinguish live eggs from large numbers of partially degraded and/or completely degraded eggs within colon biopsy tissue. To address this problem, we tested six different morphological and biochemical/molecular markers (ALP; morphological characteristics of egg; CalS (calcified substance); AOS (antioxidase); SDHG (succinic dehydrogenase) and SjR2 mRNA (retrotransposons 2 of S.japonicum genome mRNA)), including four new markers (CalS; AOS; SDHG and SjR2 mRNA.), to determine the viability of S. japonicum eggs deposited in human and mouse colon tissues. Our ultimate aim is to obtain a new method that is more sensitive, practical and accurate to clinically diagnose schistosomiasis. METHODS: Tissue samples were collected from mice at six different time points during S. japonicum infection with or without treatment with praziquantel (PZQ). Four new biochemical or molecular markers were used for the detection of egg viability from mouse liver and intestinal samples: CalS; AOS; SDHG and SjR2 mRNA. Subsequently, all markers were employed for the detection and analysis of eggs deposited in biopsy materials from patients with suspected schistosomiasis japonica for clinical evaluation. Microscopic examination of the egg morphology, worm burden in vivo and ALP (alkaline phosphatase) levels were used as a reference standard to evaluate the sensitivity and reliability of four new markers detecting egg viability. RESULTS: The results of the study showed that the morphology of S. japonicum eggs deposited in tissues of hosts with schistosomiasis, especially cases with chronic schistosomiasis, is complex and egg viability is difficult to judge morphologically, particularly eggs with a fuzzy structure or partially modified eggs. We found that the majority of the viable schistosome eggs determined by four new markers (CalS, AOS, SDHG and SjR2 mRNA) were morphologically difficult to identify. CONCLUSIONS: Among the markers, the most sensitive and specific method was the detection of SjR2 mRNA and the most simple, rapid and practical method was the detection of SDHG. Therefore, the detection of SDHG is the most practical for clinical application and its use could improve the accuracy in diagnosing active schistosome infection.

Characterization and expression analysis of a new small heat shock protein Hsp20.4 from Eimeria tenella.

Small heat shock proteins (sHsps) are ubiquitous and diverse molecular chaperones. Found in almost all organisms, they regulate protein refolding and protect cells from stress. Until now, no sHsp has been characterized in Eimeria tenella. In this study, the novel EtsHsp20.4 gene was cloned from E. tenella by rapid amplification of cDNA ends based on a previously identified expressed sequence tag. The full-length cDNA was 1019bp in length and contained an open reading frame of 558bp that encoded a 185-amino acid polypeptide with a calculated molecular weight of 20.4 kDa. The EtsHsp20.4 protein contained a distinct HSP20/alpha-crystallin domain that is the key determinant of their function as molecular chaperones and belongs to the HSP20 protein family. EtsHsp20.4 mRNA levels were higher in sporulated oocysts than in sporozoites or second-generation merozoites by real-time quantitative PCR, the transcription of EtsHsp20.4 was barely detectable in unsporulated oocysts. Immunolocalization with EtsHsp20.4 antibody showed that EtsHsp20.4 was mainly located on the surface of sporozoites, first-generation merozoites and second-generation merozoites. Following the development of parasites in DF-1 cells, EtsHsp20.4 protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts. Malate dehydrogenase thermal aggregation assays indicated that recombinant EtsHsp20.4 had molecular chaperone activity in vitro. These results suggested that EtsHsp20.4 might be involved in sporulation in external environments and intracellular growth of the parasite in the host.

Variability in gene expression underlies incomplete penetrance.

The phenotypic differences between individual organisms can often be ascribed to underlying genetic and environmental variation. However, even genetically identical organisms in homogeneous environments vary, indicating that randomness in developmental processes such as gene expression may also generate diversity. To examine the consequences of gene expression variability in multicellular organisms, we studied intestinal specification in the nematode Caenorhabditis elegans in which wild-type cell fate is invariant and controlled by a small transcriptional network. Mutations in elements of this network can have indeterminate effects: some mutant embryos fail to develop intestinal cells, whereas others produce intestinal precursors. By counting transcripts of the genes in this network in individual embryos, we show that the expression of an otherwise redundant gene becomes highly variable in the mutants and that this variation is subjected to a threshold, producing an ON/OFF expression pattern of the master regulatory gene of intestinal differentiation. Our results demonstrate that mutations in developmental networks can expose otherwise buffered stochastic variability in gene expression, leading to pronounced phenotypic variation.

Digital gene expression approach over multiple RNA-Seq data sets to detect neoblast transcriptional changes in Schmidtea mediterranea.

BACKGROUND: The freshwater planarian Schmidtea mediterranea is recognised as a valuable model for research into adult stem cells and regeneration. With the advent of the high-throughput sequencing technologies, it has become feasible to undertake detailed transcriptional analysis of its unique stem cell population, the neoblasts. Nonetheless, a reliable reference for this type of studies is still lacking. RESULTS: Taking advantage of digital gene expression (DGE) sequencing technology we compare all the available transcriptomes for S. mediterranea and improve their annotation. These results are accessible via web for the community of researchers. Using the quantitative nature of DGE, we describe the transcriptional profile of neoblasts and present 42 new neoblast genes, including several cancer-related genes and transcription factors. Furthermore, we describe in detail the Smed-meis-like gene and the three Nuclear Factor Y subunits Smed-nf-YA, Smed-nf-YB-2 and Smed-nf-YC. CONCLUSIONS: DGE is a valuable tool for gene discovery, quantification and annotation. The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

Host ABC transporter proteins may influence the efficacy of ivermectin and possibly have broader implications for the development of resistance in parasitic nematodes.

ABC transporter proteins function to extrude compounds from the cell. These proteins present an obstacle for treatment and for overcoming drug resistance as they are expressed by both host and parasite, and function similarly. The contribution of host ABC proteins to drug efficacy was examined using ivermectin and a Brugia malayi model system. Parallel in vitro and in vivo experiments were conducted using equal concentrations of ivermectin. The motilities and fecundity of B. malayi exposed to ivermectin in vitro were significantly lower than those treated in vivo. The higher motilities were correlated with low concentrations of ivermectin in worms extracted from treated hosts. The expression of ABC proteins was significantly higher in worms treated in vitro compared to those treated in vivo as well as in gerbils treated with ivermectin than in non-treated controls. The results suggest that host ABC transporters may influence the efficacy of ivermectin.

RNAscope: a novel method for the detection of Heterobilharzia americana ova in canine liver.

Canine schistosomiasis caused by Heterobilharzia americana can lead to severe morbidity and eventual mortality, in part due to the deposition of fluke ova in the liver and gastrointestinal tract, which promotes an influx of peri-ova inflammatory cells. Although fluke eggs can be identified in H&E-stained histologic sections, cases exist in which only fragments of the ova persist, or the egg is obscured by inflammatory infiltrates, which can confound definitive histologic diagnosis. Unfortunately, antibodies specific to Heterobilharzia are not commercially available for immunohistochemical labeling. Therefore, we aimed to use an RNA in situ hybridization strategy to fluorescently label Heterobilharzia ova. Using the H. americana 18S rRNA sequence, we developed an RNA probe and validated its performance on archival formalin-fixed, paraffin-embedded canine tissue. A positive signal was observed for all identifiable ova, fragmented and whole. Use of this methodology could aid understanding of the pathogenesis of H. americana infection in dogs. This technique augments standard diagnostic methodology, enabling spatial colocalization of fluke ova and inflammatory infiltrates when using fluorescent techniques.

Fasciola hepatica - where is 28S ribosomal RNA?

Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis.

Functional exploration of the C. elegans genome using DNA microarrays.

Global changes in gene expression underlie developmental processes such as organogenesis, embryogenesis and aging in Caenorhabditis elegans. Recently developed methods allow gene expression profiles to be determined selectively for individual tissues and cell types. Results from both whole-animal and tissue-specific expression profiling have provided an unprecedented view into genome organization and gene function. Integration of these results with other types of functional genomics data gathered from RNA-mediated interference and yeast two-hybrid analyses will allow rapid identification and exploration of the complex functional gene networks that govern C. elegans development.

Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi.

Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi) is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA) and double stranded RNA (dsRNA) into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1) is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed.