No abstract

Lymphatic filariasis (LF) afflicts over 60 million people worldwide and leads to severe pathological outcomes in chronic cases. The nematode parasites (Nematoda: Filarioidea) that cause LF require both arthropod (mosquito) intermediate hosts and mammalian definitive hosts for their propagation. The invasion and migration of filarial worms through host tissues are complex and critical to survival, yet little is known about the receptors and signaling pathways that mediate directed migration in these medically important species. In order to better understand the role of chemosensory signaling in filarial worm taxis, we employ comparative genomics, transcriptomics, reverse genetics, and chemical approaches to identify putative chemosensory receptor proteins and perturb chemotaxis phenotypes in filarial worms. We find that chemoreceptor family size is correlated with the presence of environmental (extrahost) stages in nematode life cycles, and that filarial worms contain compact and highly diverged chemoreceptor complements and lineage-specific ion channels that are predicted to operate downstream of chemoreceptor activation. In Brugia malayi, an etiological agent of LF, chemoreceptor expression patterns correspond to distinct parasite migration events across the life cycle. To interrogate the role of chemosensation in the migration of larval worms, arthropod and mammalian infectious stage Brugia parasites were incubated in nicotinamide, an agonist of the nematode transient receptor potential (TRP) channel OSM-9. Exposure of microfilariae to nicotinamide alters intramosquito migration, and exposure of L3s reduces chemotaxis toward host-associated cues in vitro. Nicotinamide also potently modulates thermosensory responses in L3s, suggesting a polymodal sensory role for Brugia osm-9. Reverse genetic studies implicate both Brugia osm-9 and the cyclic nucleotidegated (CNG) channel subunit tax-4 in larval chemotaxis toward host serum, and these ion channel subunits partially rescue sensory defects in Caenorhabditis elegans osm-9 and tax-4 knock-out strains. Together, these data reveal genetic and functional diversification of chemosensory signaling proteins in filarial worms and encourage a more thorough investigation of clade-and parasite-specific facets of nematode sensory receptor biology.

RNA interference (RNAi) is a powerful tool to silence endogenous mosquito and mosquito-borne pathogen genes in vivo. As the number of studies utilizing RNAi in basic research grows, so too does the arsenal of physiological targets that can be developed into products that interrupt mosquito life cycles and behaviors and, thereby, relieve the burden of mosquitoes on human health and well-being. As this technology becomes more viable for use in beneficial and pest insect management in agricultural settings, it is exciting to consider its role in public health entomology. Existing and burgeoning strategies for insecticide delivery could be adapted to function as RNAi trigger delivery systems and thereby expedite transformation of RNAi from the lab to the field for mosquito control. Taken together, development of RNAi-based vector and pathogen management techniques & strategies are within reach. That said, tools for successful RNAi design, studies exploring RNAi in the context of vector control, and studies demonstrating field efficacy of RNAi trigger delivery have yet to be honed and/or developed for mosquito control.

Mosquito-borne diseases continue to remain major threats to human and animal health and impediments to socioeconomic development. Increasing mosquito resistance to chemical insecticides is a great public health concern, and new strategies/technologies are necessary to develop the next-generation of vector control tools. We propose to develop a novel method for mosquito control that employs nanoparticles (NPs) as a platform for delivery of mosquitocidal dsRNA molecules to silence mosquito genes and cause vector lethality. Identifying optimal NP chemistry and morphology is imperative for efficient mosquitocide delivery. Toward this end, fluorescently labeled polyethylene glycol NPs of specific sizes, shapes (80 nm x 320 nm, 80 nm x 5000 nm, 200 nm x 200 nm, and 1000 nm x 1000 nm) and charges (negative and positive) were fabricated by Particle Replication in Non-Wetting Templates (PRINT) technology. Biodistribution, persistence, and toxicity of PRINT NPs were evaluated in vitro in mosquito cell culture and in vivo in Anopheles gambiae larvae following parenteral and oral challenge. Following parenteral challenge, the biodistribution of the positively and negatively charged NPs of each size and shape was similar; intense fluorescence was observed in thoracic and abdominal regions of the larval body. Positively charged NPs were more associated with the gastric caeca in the gastrointestinal tract. Negatively charged NPs persisted through metamorphosis and were observed in head, body and ovaries of adults. Following oral challenge, NPs were detected in the larval mid- and hindgut. Positively charged NPs were more efficiently internalized in vitro than negatively charged NPs. Positively charged NPs trafficked to the cytosol, but negatively charged NPs co-localized with lysosomes. Following in vitro and in vivo challenge, none of the NPs tested induced any cytotoxic effects.

BackgroundNanotechnology offers great potential for molecular genetic investigations and potential control of medically important arthropods. Major advances have been made in mammalian systems to define nanoparticle (NP) characteristics that condition trafficking and biodistribution of NPs in the host. Such information is critical for effective delivery of therapeutics and molecules to cells and organs, but little is known about biodistribution of NPs in mosquitoes.Methodology/Principal FindingsPRINT technology was used to construct a library of fluorescently labeled hydrogel NPs of defined size, shape, and surface charge. The biodistribution (organ, tissue, and cell tropisms and trafficking kinetics) of positively and negatively charged 200 nm x 200 nm, 80 nm x 320 nm, and 80 nm x 5000 nm NPs was determined in adult Anopheles gambiae mosquitoes as a function of the route of challenge (ingestion, injection or contact) using whole body imaging and fluorescence microscopy. Mosquitoes readily ingested NPs in sugar solution. Whole body fluorescence imaging revealed substantial NP accumulation (load) in the alimentary tracts of the adult mosquitoes, with the greatest loads in the diverticula, cardia and foregut. Positively and negatively charged NPs differed in their biodistribution and trafficking. Following oral challenge, negatively charged NPs transited the alimentary tract more rapidly than positively charged NPs. Following contact challenge, negatively charged NPs trafficked more efficiently in alimentary tract tissues. Following parenteral challenge, positively and negatively charged NPs differed in tissue tropisms and trafficking in the hemocoel. Injected NPs were also detected in cardia/foregut, suggesting trafficking of NPs from the hemocoel into the alimentary tract.Conclusions/SignificanceHerein we have developed a tool box of NPs with the biodistribution and tissue tropism characteristics for gene structure/function studies and for delivery of vector lethal cargoes for mosquito control.

Lymphatic filariasis (LF) afflicts over 60 million people worldwide and leads to severe pathological outcomes in chronic cases. The filarial nematode parasites that cause LF require both arthropod (mosquito) intermediate hosts and mammalian definitive hosts for their propagation. The invasion and migration of filarial parasites through host tissues are complex and critical to survival, yet little is known about the receptors and signaling pathways that mediate taxis in these medically important species.To better understand filarial chemosensation we employ comparative genomics, transcriptomics, reverse genetics, and chemical approaches to identify putative chemosensory receptor proteins and perturb chemosensory phenotypes in filarial nematode parasites. We find that chemoreceptor family size is correlated with the presence of environmental (extra-host) stages in nematode life cycles, and that filarial parasites contain a compact and highly-diverged chemoreceptor complement and lineage-specific ion channels that are predicted to operate downstream of chemoreceptor activation. In Brugia malayi, an etiological agent of LF, chemoreceptor expression patterns correspond to distinct parasite migration events across the life cycle. To interrogate the role of chemosensation in host migration, arthropod infectious stage (microfilariae) and vertebrate infectious stage (L3) Brugia parasites were incubated in nicotinamide, an agonist of the nematode transient receptor potential (TRP) channel osm-9 . Exposure of microfilariae to nicotinamide alters intra-mosquito migration while exposure of L3s reduces chemotaxis towards host-associated cues in vitro . Nicotinamide exposure also modulates thermosensory responses in L3s, suggesting a polymodal sensory role for Brugia osm-9 . Reverse genetic studies implicate both osm-9 and the cyclic nucleotide-gated (CNG) channel subunit tax-4 in larval chemotaxis towards host serum, while these ion channel subunits do not rescue chemosensory defects in C. elegans osm-9 and tax-4 knock-out strains. Together, these data reveal genetic and functional diversification of chemosensory signaling proteins in filarial nematode parasites, and encourage a more thorough investigation of clade and parasite-specific facets of nematode sensory receptor biology.

Wild ruminants are susceptible to infection from generalist helminth species, which can also infect domestic ruminants. A better understanding is required of the conditions under which wild ruminants can act as a source of helminths (including anthelmintic-resistant genotypes) for domestic ruminants, and vice versa, with the added possibility that wildlife could act as refugia for drug-susceptible genotypes and hence buffer the spread and development of resistance. Helminth infections cause significant productivity losses in domestic ruminants and a growing resistance to all classes of anthelmintic drug escalates concerns around helminth infection in the livestock industry. Previous research demonstrates that drug-resistant strains of the pathogenic nematode Haemonchus contortus can be transmitted between wild and domestic ruminants, and that gastro-intestinal nematode infections are more intense in wild ruminants within areas of high livestock density. In this article, the factors likely to influence the role of wild ruminants in helminth infections and anthelmintic resistance in livestock are considered, including host population movement across heterogeneous landscapes, and the effects of climate and environment on parasite dynamics. Methods of predicting and validating suspected drivers of helminth transmission in this context are considered based on advances in predictive modelling and molecular tools.

Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.