Fig 1.
The genomes of filarial worms contain a reduced complement of divergent chemoreceptors.
Chemoreceptors were mined from 39 nematode genomes, and the phylogeny of chemoreceptors from a down-sampled species set was constructed with ML inference. (A) General life cycle of mosquito-transmitted filarial worms. (B) Filarial worm (clade IIIc) genomes contain far fewer chemoreceptors than other nematodes, and they are enriched for srsx, srab, srbc, and srx receptors. Each box in the heatmap is normalized to the total number of chemoreceptors per species. (C) Family and superfamily categorizations from C. elegans were used to annotate the final phylogeny. Clade IIIc chemoreceptors are diverged from C. elegans and other nematodes, without any one-to-one homologs. Filarial worm chemoreceptors are notably diverged in srsx, srab, srbc, and srx. Nodal values represent percent bootstrap support of 1,000 separate replicates. Branches consisting of only C. elegans receptors were collapsed to aid visualization. (D) A decrease in chemoreceptor count is correlated with an increase in extrahost (e.g., terrestrial) stages within nematode life cycles. Completely free-living nematodes such as C. elegans, C. briggsae, Pristionchus pacificus, and Panagrellus redivivus have many more chemoreceptors than parasitic nematodes that are vector transmitted or host contained such as the filarial worms and Trichinella spiralis. Note that the x-axis is categorical, and slight jitter has been added to the points to decrease point/label overlap. ρ was calculated with Spearman’s rank correlation with the null hypothesis that ρ = 0. Raw data for (B) and (D) can be found at https://github.com/zamanianlab/BrugiaChemo-ms. Raw tree data for (C) can be found in Newick format in S1 Data. L1, first stage larvae; L2, second stage larvae; L3, third stage larvae; L4 fourth stage larvae; mf, microfilaria; ML, maximum-likelihood.
Fig 2.
Chemoreceptors are clustered in the B. malayi genome and are enriched in specific life stages and adult tissues.
The chromosomal location of annotated B. malayi and C. elegans chemoreceptor genes are shown with chromosomes in white, and chemoreceptor loci are depicted as black lines. C. elegans chemoreceptors are found throughout the genome but are heavily clustered on chromosome V, and these clusters can be enriched for specific families and superfamilies [40]. Likewise, B. malayi chemoreceptors are clustered on chromosomes II and IV. RNA expression data reveal distinct patterns of chemoreceptor expression across the life cycle and in discrete adult male and female tissues. Raw data can be found at https://github.com/zamanianlab/BrugiaChemo-ms. DPI, days postinfection; HPI, hours postinfection; L1, first stage larvae; L2, second stage larvae; L3, third stage larvae; L4, fourth stage larvae; mf, microfilaria; TPM, transcripts per million.
Fig 3.
Filarial worms possess unique complements of broadly conserved nematode TRP and CNG channels.
The phylogenies of (A) TRP and (B) CNG channels were constructed with Bayesian inference. Nodal values represent the posterior probability. (C) osm-9 and (E) ocr-1/2 subtrees were drawn from (A), and (D) tax-4 and (F) tax-2 subtrees were drawn from (B). Filarial worms have one-to-one orthologs of C. elegans osm-9, tax-4, and tax-2. In contrast, the two ocr-1/2–like genes from filarial worms are more closely related to each other than with the homologous Cel-ocr-1 and Cel-ocr-2 and belong to a diverged clade IIIc grouping of OCR-1/2–like channel subunits. Raw tree data for (A) and (B) can be found in S2 Data and S3 Data, respectively. CNG, cyclic nucleotide–gated; TRP, transient receptor potential.
Fig 4.
Treatment with a TRPV agonist dysregulates chemotaxis of B. pahangi infective larvae.
(A) L3 parasites were extracted from mosquitoes and subjected to chemotaxis assays with or without 250 μM NAM treatment. Chemotaxis assays were performed by adding L3s to the middle of a 0.8% agarose plate (M), with either test cue (T) or water (C) added to the opposite sides of the plate. The plate was placed at 37°C for 30 minutes, scored after incubation, and the CI was calculated. (B) NAM dysregulates attraction of freshly extracted L3s to serum and NaCl but has no effect on aversion to 3-methyl-1-butanol. (C) NAM has no effect on translational movement of freshly extracted L3s. (D) Bpa-osm-9 expression is unchanged by in vitro culture at physiological or room temperature 4 HPE. (E) L3s cultured for 1 DPE do not show chemotaxis toward serum and have reduced motility on the chemotaxis plate when compared with untreated freshly extracted parasites (p = 0.028, t test). Data for (A–C) represent the combined results of three independent biological replicates, except for the experiments with 3-methyl-1-butanol, which included two replicates (cohorts of mosquito infections). Data for (E) represent the results of two biological replicates. Each point represents a single chemotaxis plate with 8–10 L3s. Red diamonds and bars indicate the mean and standard error of the mean. Comparisons of means were performed using t tests (**p ≤ 0.01). Raw data for (B) through (E) can be found at https://github.com/zamanianlab/BrugiaChemo-ms. CI, chemotaxis index; DPE, days postextraction; FBS, fetal bovine serum; HPE, hours postextraction; L3, third stage larvae; NAM, nicotinamide; ns, not significant; TRP, transient receptor potential.
Fig 5.
Treatment with a TRPV agonist impairs the coiling response in cooled B. pahangi infective larvae.
(A) L3s were extracted from mosquitoes and treated with NAM, subjected to a temperature shift, and analyzed for cooling-induced coiling behaviors. (B) Representative images of untreated (control) individuals displaying the coiled phenotype and individuals exposed to 1 mM NAM that are uncoiled and thrashing. (C) Blinded coiling score given to each treatment after 24 hours and 48 hours posttreatment (higher score indicates less coiling). (D) Mean motility calculated by an optical flow algorithm. Red diamonds and bars indicate the mean and standard error of the mean from three biological replicates, each composed of >3 technical replicates scored by three different researchers. Comparisons of means were performed using one-sided t tests (*p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001; ****p ≤ 0.0001). Raw data for (C) and (D) can be found at https://github.com/zamanianlab/BrugiaChemo-ms. L3, third stage larvae; NAM, nicotinamide; ns, not significant; TRP, transient receptor potential.
Fig 6.
Treatment with a TRPV agonist reduces the ability of mf to establish infection in mosquitoes.
(A) NAM added to blood at up to 50 mM increases the proportion of blood-fed mosquitoes when allowed to feed to repletion but reduces mosquito blood feeding at concentrations greater than 50 mM. Black points represent technical replicates, and gray diamonds represent the mean. (B) Replication of blood-feeding experiments with 5 mM and 25 mM showed a significant increase in the proportion of blood-fed mosquitoes when blood was supplemented with 5 or 25 mM NAM. These concentrations were used for subsequent parasite treatment. Points represent the values from three independent biological replicates (cohorts of mosquitoes). (C) Blood supplemented with 5 or 25 mM NAM does not alter the size of distended mosquito abdomens after blood feeding, indicating an unaltered size of blood meal. Points represent the measured abdomens of individual mosquitoes from a single blood-feeding experiment. (D-E) Pretreatment of B. pahangi mf with NAM prior to mosquito infection causes a dose-dependent reduction in the number of L3s recovered per mosquito. (F) Reduction in L3 recovery was due to a decrease in larval parasites in the mosquito thorax (i.e., the flight muscles, the migratory destination for mf and site of development for L1, L2, and early-L3 parasites). Data from (D-F) represent the combined results of three independent biological replicates (cohorts of mosquito infections); each point represents the parasites recovered from an individual mosquito. Red diamonds and bars indicate the mean and standard error of the mean. Tests of significance for (B), (D), and (F) were performed with Tukey’s post hoc tests and adjusted for multiple comparisons (*p ≤ 0.05; **p ≤ 0.01). Raw data can be found at https://github.com/zamanianlab/BrugiaChemo-ms. L1, first stage larvae; L2, second stage larvae; L3, third stage larvae; mf, microfilaria; NAM, nicotinamide; ns, not significant; TRP, transient receptor potential.
Fig 7.
dsRNA treatment of chemosensory pathway receptors causes defective chemotaxis of B. pahangi infective larvae.
(A) Injection of 250 ng dsRNA into B. pahangi-infected Ae. aegypti LVP was performed 9 DPI, and L3 parasites were recovered via dissection at 14 DPI. Recovered parasites were immediately used in chemotaxis experiments. Chemotaxis assays were performed by adding L3s to the middle of a 0.8% agarose plate (M), with either test cue (T) or water (C) added to the opposite sides of the plate. The plate was placed at 37°C for 30 minutes, scored after incubation, and the CI was calculated. Intramosquito developmental dynamics were adapted from [8]. (B) dsRNA treatment of Bpa-osm-9 or Bpa-tax-4 resulted in a reduced ability of L3s to migrate to serum. Control parasites were recovered from mosquitoes injected with lacZ dsRNA. (C) dsRNA exposure does not inhibit general translational motility on the chemotaxis plate. Data represent the combined results of three independent biological replicates (cohorts of mosquito infections); each point represents the CI of an individual plate. Red diamonds and bars indicate the mean and standard error of the mean. Comparisons of means were performed using t tests (*p ≤ 0.05; ****p ≤ 0.0001). Raw data can be found at https://github.com/zamanianlab/BrugiaChemo-ms. CI, chemotaxis index; DPI, days postinfection; dsRNA, double-stranded RNA; L1, first stage larvae; L2, second stage larvae; L3, third stage larvae; LVP, Liverpool strain; mf, microfilaria.
Fig 8.
Heterologous expression of B. malayi osm-9 partially rescues loss-of-function sensory defects in C. elegans.
Bma-osm-9 was cloned and expressed under the control of the endogenous Cel-osm-9 promoter and 3′ UTR [30,32]. The Cel-osm-9 open reading frame was used as a positive control. Avoidance defects (OSM-9 functioning in ASH) to (A) concentrated benzaldehyde or (B) mechanical nose touch were partially rescued by the positive control and by Bma-osm-9. Data represent the combined results of at least three independent biological replicates, each consisting of five technical replicates. Each point represents the recorded value of an individual worm. Comparisons to the loss-of-function strain were performed using t tests (*p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001). Raw data can be found at https://github.com/zamanianlab/BrugiaChemo-ms.
Fig 9.
Heterologous expression of B. malayi tax-4 partially rescues loss-of-function chemotaxis defects in C. elegans.
Bma-tax-4 was cloned and expressed under the control of the endogenous Cel-tax-4 promoter. The Cel-tax-4 open reading frame was used as a positive control. Both constructs partially rescued the chemotaxis defect to isoamyl alcohol (TAX-4 functioning in AWC). Comparisons to the loss-of-function strain were performed using t tests (*p ≤ 0.05, ****p ≤ 0.0001). Raw data can be found at https://github.com/zamanianlab/BrugiaChemo-ms.