Mosquitoes . Both Anopheles gambiae (strain G3) and Aedes aegypti (strain LVP) mosquitoes were internally-sourced from laboratory colonies maintained at the Liverpool School of Tropical Medicine. Mosquitoes were reared from eggs to adults and housed in BugDorm-1 insect rearing cages (Megaview Science, Taiwan; Catalogue #DP1000) at 26–27 °C with 70–80% relative humidity. Experimental exposures were performed as previously described ⁴⁵ . Briefly, adult female mosquitoes, aged 3–7 days, were sugar-starved for 18 hours prior to blood exposure in order to facilitate blood feeding. For experiments involving exposures to Brugia malayi or P. falciparum, mosquitoes were provided with either a standard human bloodmeal (obtained from the local blood bank), or a human bloodmeal spiked with a known concentration of parasites. Exposures to B. malayi were conducted using a Hemotek feeding system (Hemotek Ltd, Blackburn, UK; Catalogue #SP6W1-3), while P. falciparum exposures were performed using a glass feeder (Chemglass Life Sciences, Vineland, NJ; Catalogue #CG-1836). For experiments involving mosquito exposures to T. b. brucei, mosquitoes were provided with a Hemotek feeding system-supplied bloodmeal of defibrinated horse blood (TCS Biosciences, Buckingham, UK; Catalogue #HB030), with or without parasites.

Tsetse flies . Glossina morsitans were reared from larvae and housed in internally-made cages, constructed of lengths of plastic piping covered at each end with netting, at 27 °C ± 2 °C with a relative humidity of 65–75%. Adult flies were fed on defibrinated horse blood, with or without parasites. Feedings occurred by placing blood on an aluminum tray heated to 37 °C. Fly cages were then placed on a silicon membrane positioned directly above the blood, allowing flies to feed through the membrane.

B. malayi . Microfilaria (mf) were generously provided by the anit- Wolbachia Consortium, generated as part of their maintenance of the B. malayi lifecycle ⁴⁶ . Harvested parasites were added to human blood at the appropriate concentrations to generate experimentally desired parasite densities as described below for individual applications.

P. falciparum . Red blood cells containing trophozoites (3D7 strain) were combined with uninfected human serum to produce experimentally desired parasite concentrations as described below for individual applications.

T. b. brucei . The bloodstream form of T. b. brucei, strain AnTat 1.1 90:13 ⁴⁷ , was used for all experimental feedings. Parasites were cultured in HMI-11 medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO ₂. Parasite densities were determined microscopically using a hemocytometer.

B. malayi experiments . All experiments involving B. malayi were performed in accordance with the previously described superhydrophobic cone collection method ⁴⁵ . Briefly, sheets of A4 printer paper were used to create cone-shaped funnels, which were coated in NeverWet (Rust-Oleum, Durham, UK). Cones were then placed inside of mesh-covered un-waxed paper beverage cups, with mosquitoes housed above the cones, allowing E/F produced by the mosquitoes to travel down the walls of the cones and pool at the base of each funnel. For these collections, GenSaver DNA Cards (GenTegra, Pleasanton, CA; Catalogue #GSD4-100) were used in place of the 1.7 mL microcentrifuge tubes that were employed when this method was previously described ⁴⁵ . For all collections, GenSaver DNA Cards, designed with four circular collection areas, were cut into quarters such that each E/F collection even occurred onto a single collection circle.

P. falciparum experiments . When performing experiments involving P. falciparum, E/F was again collected in accordance with the previously described superhydrophobic cone collection method ⁴⁵ briefly described above. For all experiments involving P. falciparum, E/F samples were collected into 1.7 mL microcentrifuge tubes as previously described ⁴⁵ .

T. b. brucei experiments . For all experiments involving T. b. brucei, flies/mosquitoes were housed in 50 mL conical tubes allowing for direct deposition of E/F onto the walls of the holding vessel. During the experimental housing of vectors, tubes were covered with mesh netting, and flies/mosquitoes were transferred to new vessels at experimentally specified time intervals. While in tubes, tsetse flies were removed from tubes for feeding on uninfected defibrinated horse blood every second day as described above.

Following superhydrophobic cone collections onto GenSaver DNA Cards . All samples were excised from GenSaver DNA Cards using a standard paper punch (0.64 cm round). For each sample, three punches were placed into a 2.0 mL microcentrifuge tube and the sample was recovered using the GenSolve DNA Recovery Kit (GenTegra; Catalogue #GVR-113) in accordance with the manufacturer’s suggested protocol. Following recovery, each sample was added to a MinElute column (Qiagen, Germantown, MD) for sample binding. Sample washes and DNA recovery procedures occurred utilizing the manufacturer’s recommendations. After recovering the eluate, the total volume of eluate was re-loaded onto the column a second time and again spun through the matrix to maximize sample recovery.

Following superhydrophobic cone collections into microcentrifuge tubes . DNA was extracted from all samples utilizing the QIAamp DNA Micro Kit (Qiagen; Catalogue #56304) following a modified version of the manufacturer’s suggested protocol. Briefly, 180 μL of Buffer AL was added to each E/F sample and tubes were vortexed on a shaking platform for 1 hr. 20 μL of Proteinase K was then added, and samples were incubated at 56 °C for 1 hr with shaking at 1,400 RPM. Following incubation, 200 μL of Buffer AL (containing 5mM carrier RNA) was added to each sample, and samples were incubated at 70 °C for 10 min. Column binding and washing steps were then performed in accordance with the manufacturer’s specifications. Following washes, elution of DNA occurred in 50 μL of Buffer AE. As described above, following the elution of DNA in 50 μL of Buffer AE, eluate was re-loaded onto the column to maximize recovery.

Following collection into 50 mL conical tubes . E/F was eluted from tubes through the direct addition of 7.5 mL of nuclease free water. Following the addition of water, samples underwent agitation on a vortexing platform for 30 min at 56 °C to facilitate the complete resuspension of material. Tubes were then spun at 5,000 RPM for 5 min, and the supernatant was removed from each sample. Pelleted material was resuspended in the residual volume of liquid. Following recovery, each sample underwent DNA isolation in the same manner as described above for superhydrophobic cone-based collections into microcentrifuge tubes.

Tsetse fly midguts were prepared for DNA extraction following the protocol previously described by Cunningham, et al. ⁴⁸ . Briefly, following dissection, midguts were placed in 60 μL of 100% ethanol. 70 μL of nuclease free water was then added to each sample and samples were centrifuged at 13,000 RPM for 15 sec. Following centrifugation, 100 μL of supernatant was aspirated from each sample, and samples underwent three sequential washes with 100 μL of nuclease free water to remove residual ethanol.

In preparation for DNA isolation, 20 μL of Proteinase K, 180 μL of Buffer ATL and a 4.5 mm ball bearing were added to all carcass and midgut samples. Samples were then mechanically homogenized at a setting of 30.0 1/S for 5 min using a TissueLyser II (Qiagen). All DNA extractions were then performed using the DNeasy Blood and Tissue Kit (Qiagen; Catalogue #69581) following the extraction plate procedure. All extractions were conducted in accordance with the manufacturer’s suggested protocol.

All quantitative real-time PCR (qPCR) testing for the presence/absence of B. malayi occurred using the StepOnePlus Real-Time PCR System (ThermoFisher Scientific, Waltham, MA) and was performed using primers and probe previously described for use with the Bm HhaI real-time PCR assay ⁴⁹ . Cycling conditions consisted of an initial hold at 50 °C for 2 min, followed by a 95 °C incubation for 10 min. These incubations were followed by 45 cycles of sequential denaturation and annealing/extension steps at 95 °C for 15 sec, and 60 °C for 1 min respectively. All qPCR testing for the presence of P. falciparum also occurred using the StepOnePlus Real-Time PCR System and employed the recently described Pf TR1 assay in accordance with suggested reagent concentrations ⁵⁰ . Cycling conditions for P. falciparum detection were identical to those described above for B. malayi detection. All reactions for B. malayi and P. falciparum detection were performed in 25 μL total volumes with 5 μL of template. Genomic DNA positive PCR controls (200 pg/well) and no template control (NTC) wells were run on each reaction plate. Each reaction was conducted using 12.5 μL of TaqPath ProAmp Master Mix (ThermoFisher Scientific; Catalogue #A30867) and a Cq cut-off value of 45 was employed. Depending upon the experiment, samples were tested in duplicate or triplicate reactions and mean Cq values were reported as was the number of positive replicates.

All qPCR testing for the presence/absence of T. b. brucei was performed using the Rotor-Gene Q Instrument (Qiagen) and made use of the previously described Tb117 assay primers at concentrations of 400 nM ⁴⁸ . All reactions for the detection of T. b. brucei were performed in 10 μL volumes, using 5 μL of Type-it HRM PCR Master Mix (Qiagen; Catalogue #206542) and 4 μL of DNA template. Cycling conditions consisted of an initial hold at 96 °C for 5 min, followed by 35 cycles of 95 °C for 15 sec, 60 °C for 30 sec, and 72 °C for 10 sec. As this assay makes use of a saturating fluorescent dye (similar to SYBR Green assay chemistry) a dissociation step was then performed utilizing a temperature gradient gradually increasing from 55 °C to 95 °C. Genomic DNA positive PCR controls (5 genome equivalents/well) and NTC wells were run on each reaction plate. All T. b. brucei testing occurred in duplicate and both mean Cq values and the number of positive replicates were reported.

All digital PCR (dPCR) reactions were performed on the QuantStudio 3D Digital PCR instrument using V2 chips (ThermoFisher Scientific; Catalogue #A26359). Reactions were conducted using the same P. falciparum primer-probe pairings selected for qPCR with identical working concentrations. All reactions were prepared in 15 μL volumes, with 14.5 μL of this prepared reaction mix loaded onto each chip for analysis. Individual reaction mixes contained 7.5 μL of QuantStudio 3D Digital PCR Master Mix v2 (ThermoFisher Scientific; Catalogue #A26316), the appropriate concentrations of primers and probe, and 5 μL of template. Cycling conditions consisted of two initial holds at 96 °C for 10 min and 50 °C for 30 sec. These holds were followed by 39 cycles of 60 °C for 2 min, 98 °C for 30 sec, and 60 °C for 2 min. Two replicate chips were analyzed when testing each sample. For each iteration of samples tested, two NTC chips containing nuclease-free water in place of template were analyzed alongside experimental samples. For a given iteration, NTC results were used to determine positivity by setting the fluorescence threshold for the entire sample set at 125% of the fluorescence reading generated by the NTC well producing the greatest level of background. When visualizing QuantStudio 3D output graphically, signal-producing wells containing true positives should be located in positions along the x-axis directly above the population of wells that failed to amplify. For this reason, as well as for consistency, and for the maintenance of a conservative approach to positivity determination, only wells with a fluorescence unit values of -240 to 240 along the x-axis were analyzed.

B. malayi . Utilizing previously published temporal collection windows ⁴⁵ , infected blood exposures were conducted in order to evaluate the capacity to detect B. malayi signal in the E/F of individual competent ( Ae. aegypti) and incompetent ( An. gambiae) vectors. To evaluate limits of detection, mosquitoes were exposed to either 2,000 B. malayi mf/mL, or 5,000 B. malayi mf/mL. For each species of mosquito, either 10 or 11 replicate exposures were performed and the accumulated E/F was collected at the 48- and 72-hour time points post-exposure. An additional 5 mosquitoes were provided with naïve bloodmeals to serve as uninfected controls, and collections from naïve mosquitoes occurred at the same post-exposure time points. All collections were performed using superhydrophobic cones and E/F was collected onto GenSaver DNA Cards. Following collection, DNA was isolated from all E/F samples and the resulting extracts were analyzed using qPCR.

P. falciparum . As was done to evaluate limits of detection for B. malayi, the capacity to detect P. falciparum signal in the E/F of mosquitoes exposed to varying blood concentrations of parasite was examined. Exposures of individually housed An. gambiae mosquitoes occurred at 5,000 trophozoites/μL (0.1% parasitemia), 500 trophozoites/μL (0.01% parasitemia), and 50 trophozoites/μL (0.001% parasitemia), with between nine and 14 mosquitoes successfully undergoing exposure at each experimental concentration. An additional five mosquitoes were provided with a parasite-naïve bloodmeal for control purposes. Following exposure, all mosquitoes were individually housed in paper cups facilitating superhydrophobic cone-based collections of E/F into 1.7 mL microcentrifuge tubes. At the 48-hour time point, and again at 72 hours post-exposure, mosquitoes were transferred to new cups and all deposited E/F was prepared for qPCR analysis. In order to investigate whether dPCR could be used as a means of extending detection windows, E/F samples also underwent analysis by dPCR.

T. b. brucei . Previous work has demonstrated the successful detection of T. b. brucei from the E/F produced by pools of 10 mosquitoes following exposure to parasites ⁴⁵ . However, the capacity for detection of T. b. brucei signal from the E/F of individual mosquitoes has not yet been evaluated. The capacity for tsetse fly E/F to similarly allow for T. b. brucei signal detection has also yet to be appraised. To investigate these possibilities, An. gambiae and G. morsitans were exposed to defibrinated horse blood containing either “high dose” (10 ⁵ trypanosomes/mL) or “low dose” (10 ³ trypanosomes/mL) concentrations of parasites. Following exposure for 24 hours, individual flies and mosquitoes were transferred into 50 mL conical tubes for the collection of E/F. In total, E/F samples from 25 flies and 25 mosquitoes exposed to each dose of parasite were evaluated. An additional five flies and five mosquitoes provided with a bloodmeal that was naïve for parasite were included for control purposes. Following an initial 48-hour housing, flies/mosquitoes were transferred to new tubes and soiled tubes were collected for molecular analysis. This collection process was repeated at 96 hours post-exposure, again at 144 hours post-exposure, and finally at 192 hours post-exposure. DNA was then extracted from all collected samples and real-time PCR analysis was performed. Following the 192-hour time point, flies and mosquitoes were sacrificed, and both fly midguts and mosquito carcasses underwent DNA extraction and qPCR analysis.

Prior experimentation has revealed the improved throughput of detection for B. malayi using E/F ⁴⁴ . To investigate if throughput would also improve when detecting P. falciparum, pools of 49 An. gambiae mosquitoes were provided with a parasite-naïve bloodmeal and E/F from each pool was allowed to collect into a single microcentrifuge tube for 72 hours using a hydrophobic cone. Following 72 hours, this tube was transferred to the collecting position beneath a new cone, allowing for the collection of E/F from a single mosquito exposed to P. falciparum at a parasitemia of 0.1%. Accumulation of E/F from this single exposed mosquito continued until the 72-hour post-exposure time point, after which the tube was removed for downstream DNA extraction and qPCR analysis. All samples were tested in triplicate, and positivity was defined as the occurrence of a positive result in two or more reactions with a Cq value ≤ 40. Ten replicate pools were prepared. Additionally, E/F from 10 individual mosquitoes, also exposed to P. falciparum at the same 0.1% parasitemia, were collected for comparative purposes.

To evaluate whether the provision of a second bloodmeal following an initial infected blood exposure would facilitate an extended window of parasite detection, three pools of 10 An. gambiae mosquitoes were exposed to P. falciparum-containing blood at a parasitemia of 0.01%, and an additional control pool, also containing 10 An. gambiae mosquitoes, was provided with a parasite-naïve bloodmeal. Using a superhydrophobic cone, E/F from each pool of mosquitoes was collected into a microcentrifuge tube for a 72-hour period following exposure. Mosquito pools were then transferred to new cones, and E/F was allowed to accumulate for an additional 72 hours into a new microcentrifuge tube. At 144 hours post-feeding, mosquitoes were again transferred to new cones/tubes and a naïve bloodmeal was provided. Following this second blood exposure, an additional 72-hour collection was performed. All collected samples then underwent DNA extraction and triplicate testing by qPCR ( Figure 1).

Statistical differences between groups were determined by means of a Student’s two-tailed t-test performed using GraphPad’s “ t test calculator” freely available from graphpad.com. A p value < 0.05 was considered to be statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001). Where appropriate, confidence intervals were calculated using the previously described E. B. Wilson method ^{51, 52} utilizing software freely available at http://vassarstats.net/prop1.html.

B. malayi . Individual competent vector ( Ae. aegypti) and incompetent vector ( An. gambiae) mosquitoes were exposed to B. malayi at blood concentrations of 2,000 mf/mL or 5,000 mf/mL. E/F collection occurred at the 48- and 72-hour post-exposure time points. Irrespective of time point, qPCR analysis resulted in the detection of parasite signal from the E/F of 10 of 11 Ae. aegypti mosquitoes exposed at a parasitemia of 5,000 mf/mL, and from 9 of 11 Ae. aegypti mosquitoes exposed at a parasitemia of 2,000 mf/mL ( Figure 2A). Results for An. gambiae exposures were similar, with positive detection occurring in 8 of 10 samples produced from mosquitoes exposed at a parasitemia of 5,000 mf/mL and in 10 of 11 samples produced following exposure at a parasite density of 2,000 mf/mL ( Figure 2B). Consistency of detection across time points was greater when testing E/F produced by Ae. aegypti, occurring for six mosquitoes following exposure at 5,000 mf/mL, and six mosquitoes following exposure at 2,000 mf/mL. In E/F produced by An. gambiae, detection across multiple time points occurred from only three mosquitoes and one mosquito following exposures to B. malayi at 5,000 mf/mL and 2,000 mf/mL respectively. Unsurprisingly, for both species, mean Cq values were lower, suggesting greater concentrations of target DNA, in the E/F produced by mosquitoes exposed to higher blood concentrations of B. malayi ( Figure 2C). Of note, a single negative control mosquito, not exposed to B. malayi, did give a positive signal. Contamination, resulting in amplification, likely occurred either during mosquito rearing or during DNA extraction. The use of no-template negative controls during PCR suggests that the contamination was unlikely to have occurred during the PCR.

P. falciparum . Following exposure of individual An. gambiae mosquitoes to P. falciparum at parasitemias of 0.1% (5000/μL), 0.01% (500/μL), and 0.001% (50/μL), E/F was collected at the 48-hour and 72-hour time points. As measured by qPCR, all nine mosquitoes exposed at 0.1% produced E/F that gave positive results: four samples were positive at the 48-hour time point, while six were positive at the 72-hour time point. Following exposure at 0.01% parasitemia, 13 of 14 mosquitoes produced E/F that gave positive qPCR results: 12 were positive at the 48-hour time point, while only one sample was positive at the 72-hour time point. Exposures at 0.001% parasitemia resulted in positive detection from the E/F produced by seven of 11 mosquitoes: four were positive at the 48-hour point, while three were positive at the 72-hour point ( Figure 3A). Interestingly, regardless of concentration, only one mosquito produced sample that was detectable at both collection time points (0.1%, sample 2). This result was in sharp contrast with findings for B. malayi ( Figure 2A, B). Taken together, these results may mean that deposition of parasite material occurs largely as the result of a solitary excretion event. When signal detection occurs across time points, it may be that this excretion event spans collection intervals, resulting in multiple positive time points from an isolated excretion occurrence. Whether the duration of this excretion event is longer following a B. malayi exposure, or these findings are chance results, remains an open question.

As expected, and as seen following B. malayi exposures, Cq values increased with declining numbers of parasites, suggesting greater amounts of template in the E/F produced by mosquitoes exposed to higher concentrations of pathogen ( Figure 3B). Digital PCR analysis of samples resulted in the improved overall sensitivity of detection, as more positive results were seen at the 48–72-hours time point (61.8%) compared to the qPCR results (29.4%) ( Figure 3A, C). In total, across all parasite concentrations, 17 qPCR negative samples demonstrated positivity when tested by dPCR, while only 1 sample which was qPCR positive produced a negative result by dPCR ( Figure 3D).

T. b. brucei . Following exposure to T. b. brucei, E/F was collected from individually housed G. morsitans and A. gambiae at 48-hour intervals. Overall, following exposure to “high dose” T. b. brucei (10 ⁵ trypanosomes/mL), 23 of 25 individually housed G. morsitans produced at least one E/F sample that was qPCR positive for parasite (92.0%). In contrast, only six of the 25 individual An. gambiae mosquitoes exposed to the "high dose" produced E/F which was qPCR positive for T. b. brucei (24.0%). Following “low dose” exposures (10 ³ trypanosomes/mL), six out of 25 tsetse flies produced at least one parasite-positive E/F sample (24.0%), while two mosquitoes out of the 25 exposed produced a sample that was T. b. brucei positive by qPCR (8.0%) ( Figure 4A). Interestingly, T. b. brucei detection from E/F produced by G. morsitans readily occurred at the 192-hour time point following both “high dose” (70.8%) and “low dose” (20.8%) exposures to parasite ( Table 1). In contrast, only the E/F produced by a single mosquito resulted in positive T. b. brucei detection by qPCR at a time point later than 96 hours post-infection (4.2%), and this sample was derived from an individual of “low dose” exposure ( Table 1).

Following sacrifice at the 196-hour time point, qPCR analysis of DNA extracted from G. morsitans midguts and A. gambiae carcasses was performed. Testing revealed T. b. brucei positivity in 15 of 20 midgut-derived samples from G. morsitans subjected to “high dose” exposures (75.0%), and in 3 of 14 samples collected from “low dose” individuals (21.4%). Neither “high” nor “low dose” mosquitoes produced a single T. b. brucei-positive carcass ( Figure 4B).

To investigate the capacity for high throughput sampling when testing mosquito E/F for the presence of P. falciparum by qPCR, comparative analysis of samples containing the pooled E/F from 50 mosquitoes (49 unexposed and 1 P. falciparum-exposed) and mosquitoes individually exposed to P. falciparum was performed. Eight of 10 samples containing pooled E/F gave positive qPCR results with a mean Cq value of 31.37 for all positive samples. By comparison, nine of 10 control samples containing the E/F from individually exposed mosquitoes resulted in the detection of P. falciparum signal, with a mean Cq value of 28.33 for all positive samples, a difference in means that was statistically significant ( p=0.0067) ( Figure 5).

Following an initial exposure to an infected bloodmeal, detection of P. falciparum signal in the E/F from all three experimental pools of 10 mosquitoes occurred. As expected, at the 144-hour post-exposure time point, signal detection was no longer possible. Following the provision of a second, uninfected bloodmeal, signal remained undetectable, indicating that such an exposure was not capable of extending, or re-initiating the post-parasite exposure collection window ( Table 2).

Open Science Framework: Laboratory evaluation of molecular xenomonitoring using mosquito excreta/feces to amplify Plasmodium, Brugia, and Trypanosoma DNA. https://doi.org/10.17605/OSF.IO/EWRTJ ⁵³

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).