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Characterization of the behaviour of S. mansoni miracidia in response to raw mucus of B. glabrata. (A) Arrangement of bioassay, including schematic illustration of video zone and corresponding area of observation. (B) Microscopic examination of site of application. Arrows show sites of miracidia for subsequent tracking. Asterisk indicates the pipette tip shadow. (C) Trajectories of miracidia in the presence of raw mucus. Different colours represent individual miracidium.
Source publication
The human disease schistosomiasis (or bilharzia) is caused by the helminth blood fluke parasite Schistosoma mansoni, which requires an intermediate host, the freshwater gastropod snail Biomphalaria glabrata (the most common intermediate host). The free-swimming parasite miracidia utilise an excellent chemosensory sense to detect and locate an appro...
Citations
... Two identical secreted proteins are worth pointing out: BSUD.4529 (contig 217) and BSUD.14556 (contig 559). These were identified as orthologs to the precursor protein of peptide P12 in B. glabrata (BGLB027975 [5]), which has been shown to trigger behavior modifications in S. mansoni miracidia, and thus is potentially an attractant [66,67]. Compared to the B. glabrata P12, the orthologous 13 aa region in B. sudanica, and B. pfeifferi (protein NCBI accession: KAK0045317 [8]), contains a non-synonymous change, changing the 5th amino acid from Glycine to Valine (DITSVLDPEVADD), whereas the same 13 aa orthologous region in B. straminea (protein 046859-T1 [7]) contains 6 non-synonymous amino acid changes (EVASVLDPDVADN). ...
Background
Control and elimination of schistosomiasis is an arduous task, with current strategies proving inadequate to break transmission. Exploration of genetic approaches to interrupt Schistosoma mansoni transmission, the causative agent for human intestinal schistosomiasis in sub-Saharan Africa and South America, has led to genomic research of the snail vector hosts of the genus Biomphalaria. Few complete genomic resources exist, with African Biomphalaria species being particularly underrepresented despite this being where the majority of S. mansoni infections occur. Here we generate and annotate the first genome assembly of Biomphalaria sudanica sensu lato, a species responsible for S. mansoni transmission in lake and marsh habitats of the African Rift Valley. Supported by whole-genome diversity data among five inbred lines, we describe orthologs of immune-relevant gene regions in the South American vector B. glabrata and present a bioinformatic pipeline to identify candidate novel pathogen recognition receptors (PRRs).
Results
De novo genome and transcriptome assembly of inbred B. sudanica originating from the shoreline of Lake Victoria (Kisumu, Kenya) resulted in a haploid genome size of ~ 944.2 Mb (6,728 fragments, N50 = 1.067 Mb), comprising 23,598 genes (BUSCO = 93.6% complete). The B. sudanica genome contains orthologues to all described immune genes/regions tied to protection against S. mansoni in B. glabrata, including the polymorphic transmembrane clusters (PTC1 and PTC2), RADres, and other loci. The B. sudanica PTC2 candidate immune genomic region contained many PRR-like genes across a much wider genomic region than has been shown in B. glabrata, as well as a large inversion between species. High levels of intra-species nucleotide diversity were seen in PTC2, as well as in regions linked to PTC1 and RADres orthologues. Immune related and putative PRR gene families were significantly over-represented in the sub-set of B. sudanica genes determined as hyperdiverse, including high extracellular diversity in transmembrane genes, which could be under pathogen-mediated balancing selection. However, no overall expansion in immunity related genes was seen in African compared to South American lineages.
Conclusions
The B. sudanica genome and analyses presented here will facilitate future research in vector immune defense mechanisms against pathogens. This genomic/transcriptomic resource provides necessary data for the future development of molecular snail vector control/surveillance tools, facilitating schistosome transmission interruption mechanisms in Africa.
... Miracidia were hatched in deionized water under light from SmfgfrA-repressed and control (WT, NC-treated) eggs. Miracidia were then harvested (58) and their behavior were monitored as described (67,68). Briefly, approximately 30 S. mansoni miracidia in 100 ml deionized water were distributed evenly to a microscope slide. ...
... Miracidial movement (swimming) in the field of view (FOV) was detected utilizing an Olympus-CKX41 microscope equipped with an Olympus DPI Digital Microscope Camera DP22 (25 frames per second at 2.8-megapixel image quality). Miracidial movement was recorded for 1 minute by video and followed by analyzing with the FIJI software to calculate velocity (speed) of miracidial swimming, the tortuosity (the ratio of track length to maximum displacement) of miracidial swimming and the duration (time) of miracidia staying in the FOV (67). The miracidial movement velocity was determined in pixels employing the rolling mean subtraction approach (67,68). ...
... Miracidial movement was recorded for 1 minute by video and followed by analyzing with the FIJI software to calculate velocity (speed) of miracidial swimming, the tortuosity (the ratio of track length to maximum displacement) of miracidial swimming and the duration (time) of miracidia staying in the FOV (67). The miracidial movement velocity was determined in pixels employing the rolling mean subtraction approach (67,68). The location of miracidial was monitored in each frame along an x-y axis and the trajectories were interpolated utilizing TrackMate (the plugin for FIJI software) (68,69). ...
Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function studies targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A (FGFRA). SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2’-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.
... This suggests that miracidia receptors have evolved to identify Biomphalaria-specific ligands released into the water. Recent SCW analyses demonstrated that snail-derived peptides and excretory-secretory proteins (ESPs) were the primary stimulants for miracidia behaviour change, while small molecules were less likely to be attractants (20). One such attractant peptide is P12 (-R-DITSGLDPEVADD-KR-), which is highly expressed in the central nervous system, foot, heart and kidney and produced identical changes in miracidia behaviour to naïve (uninfected) B. glabrata SCW (20). ...
... Recent SCW analyses demonstrated that snail-derived peptides and excretory-secretory proteins (ESPs) were the primary stimulants for miracidia behaviour change, while small molecules were less likely to be attractants (20). One such attractant peptide is P12 (-R-DITSGLDPEVADD-KR-), which is highly expressed in the central nervous system, foot, heart and kidney and produced identical changes in miracidia behaviour to naïve (uninfected) B. glabrata SCW (20). However, aside from P12, few attraction chemicals have been identified (21). ...
... The miracidia were resuspended in 10 mL of Milli-Q water, vortexed and 30 miracidia per 100 µL were counted. This method of SCW behavioural bioassay has been described in detail elsewhere (20). ...
Schistosomiasis, caused by infection with Schistosoma digenetic trematodes, is one of the deadliest neglected tropical diseases in the world. The Schistosoma lifecycle involves the miracidial infection of an intermediate freshwater snail host, such as Biomphalaria glabrata. Dispersing snail host-derived Schistosoma miracidia attractants has been considered a method of minimising intermediate host infections and, by extension, human schistosomiasis. The attractiveness of B. glabrata to miracidia is known to be reduced following infection; however, the relationship between duration of infection and attractiveness is unclear. Excretory-secretory proteins (ESPs) most abundant in attractive snail conditioned water (SCW) are key candidates to function as miracidia attractants. This study analysed SCW from B. glabrata that were naïve (uninfected) and at different time-points post-miracidia exposure (PME; 16h, 1-week, 2-weeks and 3-weeks PME) to identify candidate ESPs mediating Schistosoma mansoni miracidia behaviour change, including aggregation and chemoklinokinesis behaviour (random motion, including slowdown and increased turning rate and magnitude). Miracidia behaviour change was only observed post-addition of naïve and 3W-PME SCW, with other treatments inducing significantly weaker behaviour changes. Therefore, ESPs were considered attractant candidates if they were shared between naïve and 3W-PME SCW (or exclusive to the former), contained a predicted N-terminal signal peptide and displayed low identity (<50%) to known proteins outside of the Biomphalaria genus. Using these criteria, a total of 6 ESP attractant candidates were identified, including acetylcholine binding protein-like proteins and uncharacterised proteins. Tissue-specific RNA-seq analysis of the genes encoding these 6 ESPs indicated relatively high gene expression within various B. glabrata tissues, including the foot, mantle and kidney. Acetylcholine binding protein-like proteins were highly promising due to their high abundance in naïve and 3W-PME SCW, high specificity to B. glabrata and high expression in the ovotestis, from which attractants have been previously identified. In summary, this study used proteomics, guided by behavioural assays, to identify miracidia attractant candidates that should be further investigated as potential biocontrols to disrupt miracidia infection and minimise schistosomiasis.
... The egg hatching efficiency (%) in each group was calculated by dividing the number of hatched eggs with the total number of examined eggs X 100. The behavior of miracidia hatched from treated and control eggs was also monitored using a published bioassay (57,58). Briefly, around 30 S. mansoni miracidia in 100 ml deionized water were, using a pipette, evenly distributed to the centre of a microscope slide. ...
... Miracidial movement in the field of view (FOV) was recorded for 1 minute by video. Then the videoed miracidial tracks were analyzed using FIJI software to calculate three individual behavioral measurements including velocity of miracidial movement, duration (time) of miracidia staying within the FOV, and tortuosity (the ratio of track length to maximum displacement) of miracidial movement (57). The miracidial movement velocity was calculated in pixel/s using the rolling mean subtraction method (57,58). ...
... Then the videoed miracidial tracks were analyzed using FIJI software to calculate three individual behavioral measurements including velocity of miracidial movement, duration (time) of miracidia staying within the FOV, and tortuosity (the ratio of track length to maximum displacement) of miracidial movement (57). The miracidial movement velocity was calculated in pixel/s using the rolling mean subtraction method (57,58). Miracidial location was tracked in each frame along an x-y axis and the trajectories were interpolated using the plugin for FIJI software, known as TrackMate (58,59). ...
Stem cells play significant roles in driving the complex life cycle of Schistosoma mansoni. Fibroblast growth factor (FGF) receptor A (SmFGFRA) is essential for maintaining the integrity of schistosome stem cells. Using immunolocalization, we demonstrated that SmFGFRA was distributed abundantly in germinal/stem cells of different S. mansoni life stages including eggs, miracidia, cercariae, schistosomula and adult worms. Indeed, SmFGFRA was also localized amply in embryonic cells and in the perinuclear region of immature eggs; von Lichtenberg’s layer and the neural mass of mature eggs; the ciliated surface and neural mass of miracidia; the tegument cytosol of cercariae, schistosomula and adult worms; and was present in abundance in the testis and vitellaria of adult worms of S. mansoni. The distribution pattern of SmFGFRA illustrates the importance of this molecule in maintaining stem cells, development of the nervous and reproductive system of schistosomes, and in the host-parasite interplay. We showed SmFGFRA can bind human FGFs, activating the mitogen activated protein kinase (MAPK) pathway of adult worms in vitro. Inhibition of FGF signaling by the specific tyrosine kinase inhibitor BIBF 1120 significantly reduced egg hatching ability and affected the behavior of miracidia hatched from the treated eggs, emphasizing the importance of FGF signaling in driving the life cycle of S. mansoni. Our findings provide increased understanding of the complex schistosome life cycle and host-parasite interactions, indicating components of the FGF signaling pathway may represent promising targets for developing new interventions against schistosomiasis.
... One study implicated "miracidia-attracting glycoproteins" present within the snail mucus 12 , while in silico analysis from B. glabrata snail conditioned-water proteins predicted interactions of uncharacterized S. mansoni proteins with B. glabrata proteins 43 . Peptides have also been implicated, whereby a snail-derived novel peptide (named P12) stimulated changes in the behaviour of the S. mansoni miracidia 47 . ...
Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections. A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia. Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.
... Temperature influences the velocity and longevity of S. mansoni miracidia. High temperatures increase their velocity and reduce their lifespan, while low temperatures decrease their velocity and increase their lifespan (Benex and Deschiens, 1963;Samuelson et al., 1984) and mortality is compensated by an increased probability of successfully finding their intermediate host, especially at temperatures greater than 20°C (Wang et al., 2019). High water salinity and acidity also reduce egg hatching and motility (Donnelly et al., 1984). ...
... When miracidia are present at high concentrations and are exposed to snail-conditioned water, an increase in both angular and linear velocities of miracidia is observed (Samuelson et al., 1984). At this higher velocity, miracidia exhibit a random circulatory movement which increases their probability of finding a snail (Wang et al., 2019). ...
Schistosoma mansoni is the main causative agent of intestinal schistosomiasis which affects millions of people worldwide. At the larval stage, miracidia are released into bodies of water where they utilize their motility to successfully infect their intermediate host, snails. Here, we revisit the motility and survival of S. mansoni miracidia throughout its life span. Briefly, miracidia motility was monitored at 30-min and 60-min intervals under the presence/absence of natural/artificial light. Based on a subjective evaluation of activity, body shape and transparency, 6 categories of miracidia activity were established from its fully active stage to its immobile larva stage. The estimated life span of miracidia was 5.8 and 3.5 h in the experiments with 60-min and 30-min observation intervals, respectively. Death was defined by an absence of cilia and body movement. When mobility was used as a proxy for infectivity, infective miracidia were detected at 2.5 and 4.5 h, respectively. The present miracidia motility and survival re-evaluation supports parameters optimization for computational modelling of schistosomiasis transmission dynamics. Target control interventions, especially at late stages next to transmission interruption, may greatly benefit from improved modelling studies.
... Thus, selection is expected to finetune the parasites' ability to discriminate between compatible and incompatible hosts (Johnson et al., 2019). Previous studies have shown that the recognition behaviors of a miracidium are largely mediated by snail chemical cues, such as glycoproteins, kairomones, or other peptides (Chernin, 1970;Fogarty et al., 2019;Haberl et al., 1995;Haberl & Haas, 1992;Theron et al., 1998;Wang et al., 2019). However, the degree of conservation of these chemical cues among and within host species is unknown. ...
Schistosome parasites cause a chronic inflammatory disease in humans, and recent studies have emphasized the importance of control programs for understanding the aquatic phases of schistosomiasis transmission. The host‐seeking behavior of larval schistosomes (miracidia) for their snail intermediate hosts plays a critical role in parasite transmission. Using field‐derived strains of Kenyan snails and parasites, we tested two main hypotheses: (1) Parasites prefer the most compatible host, and (2) parasites avoid hosts that are already infected. We tested preference to three Biomphalaria host snail taxa (B. pfeifferi, B. sudanica, and B. choanomphala), using allopatric and sympatric Schistosoma mansoni isolates and two different nonhost snail species that co‐occur with Biomphalaria, Bulinus globosus, and Physa acuta. We also tested whether schistosomes avoid snail hosts that are already infected by another trematode species and whether competitive dominance played a role in their behavior. Preference was assessed using two‐way choice chambers and by visually counting parasites that moved toward competing stimuli. In pairwise comparisons, we found that S. mansoni did not always prefer the more compatible snail taxon, but never favored an incompatible host over a compatible host. While parasites preferred B. pfeifferi to the nonhost species B. globosus, they did not significantly prefer B. pfeifferi versus P. acuta, an introduced species in Kenya. Finally, we demonstrated that parasites avoid infected snails if the resident parasite was competitively dominant (Patagifer sp.), and preferred snails infected with subordinates (xiphidiocercariae) to uninfected snails. These results provide evidence of “fine tuning” in the ability of schistosome miracidia to detect hosts; however, they did not always select hosts that would maximize fitness. Appreciating such discriminatory abilities could lead to a better understanding of how ecosystem host and parasite diversity influences disease transmission and could provide novel control mechanisms to improve human health.
... One study implicated "miracidia-attracting glycoproteins" present within the snail mucus [12], while in silico analysis from B. glabrata snail conditioned-water proteins predicted interactions of uncharacterized S. mansoni proteins with B. glabrata proteins [46]. Peptides have also been implicated, whereby a snail-derived novel peptide (named P12) stimulated changes in the behaviour of the S. mansoni miracidia [47]. ...
Background: Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections.
Results: A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia.
Conclusions: Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.
... One study implicated "miracidia-attracting glycoproteins" present within the snail mucus [12], while in silico analysis from B. glabrata snail conditioned-water proteins predicted interactions of uncharacterized S. mansoni proteins with B. glabrata proteins [46]. Peptides have also been implicated, whereby a snail-derived novel peptide (named P12) stimulated changes in the behaviour of the S. mansoni miracidia [47]. ...
Background: Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections.
Results: A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia.
Conclusions: Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.
... The exo-siRNA pathway is antiviral in nature and represents a major pathogen defense mechanism in invertebratesif this has been hindered in spiny lobster species, they should be highly susceptible to viral infection. Interestingly, however, only a single virus has ever been isolated from any spiny lobster, which suggests a powerful antiviral immune system [57,58]. The contradiction of powerful antiviral mechanisms and a lack of an RNAi response indicates that other mechanisms that deal with viruses must exist within spiny lobsters. ...
... The exo-siRNA pathway is antiviral in nature and represents a major pathogen defense mechanism in invertebrates-if this has been hindered in spiny lobster species, they should be highly susceptible to viral infection. Interestingly, however, only a single virus has ever been isolated from any spiny lobster, which suggests a powerful antiviral immune system [57,58]. The contradiction of powerful antiviral mechanisms and a lack of an RNAi response indicates that other mechanisms that deal with viruses must exist within spiny lobsters. ...
RNA interference (RNAi) has become a widely utilized method for studying gene function, yet despite this many of the mechanisms surrounding RNAi remain elusive. The core RNAi machinery is relatively well understood, however many of the systemic mechanisms, particularly double-stranded RNA (dsRNA) transport, are not. Here, we demonstrate that dsRNA binding proteins in the serum contribute to systemic RNAi and may be the limiting factor in RNAi capacity for species such as spiny lobsters, where gene silencing is not functional. Incubating sera from a variety of species across phyla with dsRNA led to a gel mobility shift in species in which systemic RNAi has been observed, with this response being absent in species in which systemic RNAi has never been observed. Proteomic analysis suggested lipoproteins may be responsible for this phenomenon and may transport dsRNA to spread the RNAi signal systemically. Following this, we identified the same gel shift in the slipper lobster Thenus australiensis and subsequently silenced the insulin androgenic gland hormone, marking the first time RNAi has been performed in any lobster species. These results pave the way for inducing RNAi in spiny lobsters and for a better understanding of the mechanisms of systemic RNAi in Crustacea, as well as across phyla.
