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Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission

['Angelika Sturm', 'Tropiq Health Sciences', 'Nijmegen', 'The Netherlands', 'Martijn W. Vos', 'Rob Henderson', 'Maarten Eldering', 'Karin M. J. Koolen', 'Avinash Sheshachalam', 'Guido Favia']

Date: 2022-01 

Abstract This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

Citation: Sturm A, Vos MW, Henderson R, Eldering M, Koolen KMJ, Sheshachalam A, et al. (2021) Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission. PLoS Biol 19(12): e3001426. https://doi.org/10.1371/journal.pbio.3001426 Academic Editor: Luis Teixeira, Instituto Gulbenkian de Ciencia, PORTUGAL Received: September 2, 2021; Accepted: December 3, 2021; Published: December 20, 2021 Copyright: © 2021 Sturm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was financially supported by the Bill and Melinda Gates Foundation (https://www.gatesfoundation.org) through grants OPP1067662 and OPP1118462 to KJD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: KJD holds stock in TropIQ Health Sciences. Abbreviations: DMSO, dimethylsulfoxide; MFI, median fluorescence intensity; MMV, Medicines for Malaria Venture; RLU, relative light units; R&D, research and development; SMFA, standard membrane feeding assay; TCP, target candidate profile

Introduction Parasites and viruses that are carried by mosquitoes cause diseases such as malaria, dengue, or yellow fever. Malaria resulted in 229 million cases, causing 409,000 deaths in 2019. The use of insecticides has had large impact on control of malaria [1]. Since World War II, the range of chemical scaffolds with insecticide activity has slowly expanded, resulting in 55 chemically distinct classes of marketed insecticides available in 2019 [2]. Concurrently, resistance to these molecules has developed at a similar rate as a result of widespread use in crop protection, community and household spraying, and impregnation of bed nets [3]. As a more targeted approach, the use of oral insecticides in drug-based vector control is considered [4]. The endectocide ivermectin is used as an oral helminticide but also shows systemic adulticide activity against Anopheles mosquitoes [5]. It has shown promise as a drug that, following repeat mass drug administration to a human population at risk, reduces malaria burden by directly blocking onward pathogen transmission through reduction of the life span of blood-feeding mosquitoes [6]. Ivermectin is relatively rapidly eliminated from the blood circulation in humans, whereas modelling suggest that the duration of the mosquitocidal activity strongly drives impact of drug-based vector control [7]. Therefore, long-acting drug substances and formulations are being pursued [8,9]. As an alternative to use of insecticides for control of vector-borne disease, strategies aimed at biological control of the pathogen stages that underlie spread of the disease are emerging. These approaches have the advantage of a low risk on development of resistance. Arboviruses like Zika and dengue and protozoa such as Leishmania, Plasmodium, and Trypanosoma face a population bottleneck in the insect vector [10,11], with a low number of replication cycles and, hence, a low rate of accumulation of resistance mutations. In the context of malaria elimination, drug interventions targeting the transmission stages of the malaria parasite are explored [12]. Such compounds may kill or sterilise sexual stage parasites that infect mosquitoes [13,14]. Historically, antimalarial compounds have been selected on their ability to clear asexual blood stage parasitaemia that underlies clinical disease, and many of these compounds do not block transmission. More recently, compounds have emerged with a transmission-blocking component in their activity spectrum, although in many cases, this activity is not as potent as their activity against asexual blood stages [15,16]. Therefore, there is a need for novel chemical starting points for the development of malaria transmission-blocking drugs. The requirements for drug candidates that block malaria transmission by killing the mosquito vector or by targeting the sexual stage parasites are outlined in target candidate profiles (TCP) 5 and 6, as put forward by the Medicines for Malaria Venture (MMV) [17]. These TCPs are stimulating and guiding global drug discovery efforts [18]. In the absence of a large array of validated molecular targets, these efforts rely on phenotypic screens that have a relative low throughput and, hence, generate low numbers of chemically diverse starting points [2,19,20]. In pesticide discovery, miniaturised assays in 96-well assays containing larvae are used to predict systemic activity against adult insects [21,22]. Discovery of molecules that block transmission of malaria ultimately relies on laborious membrane feeder experiments that use one container of mosquitoes for each test condition [23]. An increase in throughput of these technologies would accelerate the development of novel malaria interventions. Previous work has demonstrated the use of phage display technology to enable large-scale screens for peptides that block Plasmodium development in the mosquito vector [24]. Killeen and colleagues showed the feasibility of feeding mosquitoes on arrays of blood meals encoded with scFv-displaying phages that could be recovered, identified, propagated, and retested from individual mosquitoes [25]. This introduced the concept of large-scale screens, where active substances could be identified by enrichment of their cognate phage barcode in mosquitoes with the phenotype of interest. Here, we build on this concept and present a technique that significantly improves the throughput of compound testing in phenotypic assays using adult mosquitoes. It allows screening of multiple molecules using barcoded blood meals in multisample arrays. We used a genetically engineered prokaryotic symbiont, α-Proteobacteria of the genus Asaia, which stably associate with a number of sugar feeding insects [26]. Upon ingestion with a glucose or a blood meal, Asaia actively colonises the insect midgut within 1 or 2 days and spreads from there to most other organs [27,28]. We transformed Asaia strains with plasmids that carry individual short DNA barcodes. Following feeding of mosquitoes on arrays of blood meals with test compounds, these DNA barcodes were recovered from the mosquito in order to deconvolute the feeding pattern and identify active compounds. We used this technique to identify systemic insecticides and malaria transmission-blocking compounds from libraries of small molecules.

Discussion Conventional testing of the effectiveness of substances on longevity or vector capacity of live insects is labour intense and mostly allows only for a small number of molecules to be tested simultaneously. We have developed a technique that improves the throughput of compound testing in order to fuel pipelines for discovery of pesticides and disease transmission-blocking drugs. To do this, we had to overcome 3 distinct technical challenges: feeding mosquitoes on multiwell plates, tagging blood-fed mosquitoes with a unique well identification code, and multiplex detection of these identification codes. We used a custom designed parafilm membrane stretcher in combination with a hydraulic press to firmly seal 96-well plates filled with blood meals. The plate feeding method proved just as effective as conventional glass feeders. In order to tag mosquitoes stably throughout the course of the experiment, we used the insect midgut symbiont Asaia strain SF2.1, transformed with DNA barcoded plasmids. In line with published data [26,35], we observed efficient colonisation of Anopheles and Aedes mosquitoes when Asaia bacteria were included with the blood meal. Previously, Killeen and colleagues introduced a phenotypic screening concept based on phagemid encoded multisample arrays [25]. This approach led to 95% of successfully tagged mosquitoes with the marker lasting for 3 days only. In capture and release experiments, ectopic DNA oligonucleotides have been used to stably tag mosquitoes during their entire life span [36]. Our data indicate that upon ingestion with the blood meal, DNA oligonucleotides are rapidly eliminated from the mosquito in spite of their nuclease-resistant phosphorothioate backbone. By contrast, ingested Asaia symbionts stay with the mosquito for life and thereby makes long-term applications possible [27]. Sampling intensity of Asaia encoded blood meals differed between wells, and some wells were sampled by more than 1 mosquito. This is in line with the biting behaviour observed with the Vectorchip that contains arrayed glucose meals to sample saliva from individual mosquito bites [37]. By using a surplus of mosquitoes relative to the number of blood meals, we ensured that every well was sampled by multiple mosquitoes. Small contaminations with barcodes from cross-feeding mosquitoes did not impact overall barcode enrichment in pooled analyses. In these analyses, the number of PCR reactions needed to detect individual barcodes was significantly reduced by using common amplification regions and multiplex detection of barcode sequences. Our screen of a collection of pesticides exemplifies the application of the barcoding technology for discovery of novel systemic insecticides. Compounds like fluralaner and nitempyram are used as oral drugs for tick and flea control in veterinary medicine [38,39] and led to enrichment of barcodes in the dead population of mosquitoes. In addition, a number of phenylpyrazoles emerged as hits with blood-borne mosquitocidal activity against Anopheles. Fipronil shows a very long half-life in mammalian circulation [40] and was shown to have potent and long-lasting mosquitocidal effects when administered to cattle [41]. For other compounds from the phenylpyrazole class the systemic insecticide activity in a blood meal is less well documented, but our data show that these molecules show promise for drug-based vector control, provided they show an excellent safety profile in human. Based on the reported mammalian long in vivo half-life of fluralaner [42], this compound was selected as a promising candidate for drug-based vector control and analysed in further detail. The results, which are described elsewhere [9], showed potent killing activity against a wide range of vector species at concentrations that are in line with drug levels predicted to circulate for several months following a single human oral dose. In order to exemplify a screen for vector-borne pathogen transmission, we used the barcoding technology to identify compounds that block Plasmodium development in Anopheles mosquitoes. Using the Pathogen Box collection and a selection criterion of ≥80% barcode enrichment in uninfected mosquitoes, we observed an overall hit rate of 12%. This relatively high hit rate may be explained by a biased composition of the pathogen box towards pharmacologically active compounds. A subset of 125 compounds from this collection is annotated as malaria hit compounds, as they showed IC 50 s of 2.1 μM or better against P. falciparum Dd2 asexual bloodstage parasites (https://www.mmv.org/mmv-open/pathogen-box). Out of these 125, 23 (18%) appear to block transmission in the barcoded screen, which is a higher number than the one predicted on basis of gametocyte viability assays [43]. This is conceivable, as the in vivo transmission assay captures a wide range of potential mode of actions, including ones that incapacitate gametocytes by nonlethal ways, e.g., by prevention of gamete formation or sterilisation of resulting gametes [44]. Hit rates were 9% and 10% for compounds originating from tuberculosis and kinetoplastid hit collections that were well represented in the Pathogen Box with 116 and 70 compounds, respectively. This illustrates the strength of cross-screening bioactive molecules against a large panel of pathogen species. This notion is in line with previous observations that libraries of small molecules preselected for activity against one protozoan parasite showed high hit rates against a wider variety of pathogens [45–47]. MMV675968 identified here as a P. falciparum transmission-blocking molecule belongs to a class of dihydrofolate reductase inhibitors with activity against a range of protozoa and was recently shown to block growth of Acinetobacter baumannii [48,49]. In theory, such cross-reactivity may affect the Asaia bacteria used in our barcoded screening strategy. As our method comprehensively monitors barcode presence in all blood-fed mosquitoes, this would lead to a total absence of the barcode in either phenotype. For the 483 compounds in the combined screens presented here, we observed successful retrieval of barcode in 482 instances, indicating a relatively low hit rate against the barcode-bearing Asaia bacteria. The transmission-blocking hits described here are attractive starting points for further optimisation as they obey to rule of five principles, i.e., have no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, a molecular mass <500 g/mol and a logP < 5 [50]. In addition, all compounds have in vitro and in vivo pharmacokinetic data available (https://www.mmv.org/mmv-open/pathogen-box). For example, in rat pharmacokinetic studies, hit compound MMV687248 showed 38% absorption and clearance of 12.4 ml/min/kg, which is a reasonable starting point for further pharmacological evaluation. Ultimately, these should address the ability of a drug to reduce the parasite load in a mosquito to zero, as a single oocyst that develops in the mosquito midgut can give rise to sufficient salivary gland sporozoites to transmit the disease [51]. Historically, phenotypic screening has driven drug research and development (R&D) pipelines for infectious diseases, and it has been to a larger or lesser extent been in vogue in other therapeutic areas [52]. It is attractive as it captures complex biology in the absence of a priori knowledge of molecular mechanisms of disease. Recent advances in cell biological, imaging, and data analyses techniques have brought it back in the spotlight [53]. The methods described here expand the possibilities for phenotypic live insect screens. In line with published data, we observed stable colonisation of A. stephensi and A. aegypti mosquitoes by Asaia bacteria [26,54]. Applications beyond the examples provided in this paper are conceivable. For example, it should be possible to identify gustatory modulators through barcodes not sampled in arrayed screens. The incubation time in the screen for systemic insecticides presented here could be extended to screen for compounds with a slow mode of action, possibly selecting compounds less prone to development of resistance. Since Asaia is transmitted vertically, screening for barcodes that are absent on eggs or in progeny may identify mosquito contraceptives that reduce fecundity [55]. Asaia has been found to associate with other sugar-feeding, phylogenetically distant genera of insects, for example, the leafhopper Scaphoideus titanus, the vector for Flavescence Dorée, a grapevine disease [56]. This host flexibility makes Asaia an attractive tool for tagging a large variety of pest insects, for the purpose of the discovery of novel molecules for pest and disease intervention.

Materials and methods Pilot experiments using modified DNA oligonucleotides In pilot experiments, mosquito blood meals were tagged with a DNA nucleotide with phosphorothioate backbone modifications to increase nuclease resistance (S1 Table). DNA was isolated from individual fed mosquitoes using phenol/chloroform extraction directly after feeding and after 24, 72, and 144 hours postfeeding. Presence of the modified oligo in the extracted DNA samples was assessed by semiquantitative real time PCR using primers MWV 303 and MWV 304 (S1 Table) and a fluorescent TaqMan MGB probe (ThermoFisher, Breda, the Netherlands). Barcode construction and transformation of Asaia SF2.1 Plasmid pMV170 for transformation of Asaia was derived by amplification of a multiple cloning site from pMV-FLPe [57] with primer pair MWV 371 and MWV 374 (S1 Table) and introducing it into the NcoI/AatII sites of vector pBBR122 (Mobitec, Goettingen, Germany). Barcode sequences, compatible with detection using MAGPlex-TAG microspheres (Luminex, ‘s Hertogenbosch, the Netherlands) were generated by hybridisation of complementary primer pairs (S2 Table) and cloned into pMV170 using SpeI/AflII restriction digestion and ligation. Resulting plasmids were introduced into Escherichia coli DH5α competent cells (Thermo Fisher) by heat shock transformation, yielding a collection of 50 barcoded plasmids (S3 Table). Barcoded plasmids were next extracted from E. coli using the PureYield Plasmid Miniprep System (Promega, Leiden, the Netherlands) and subsequently introduced into Asaia sp. SF2.1 described previously [27]. For transformation, Asaia cells were cultured in GLY medium (25 g/litre glycerol, 10 g/litre yeast extract, pH 5), and competent cells were prepared as previously described [27]. Next, 65 μl of the competent cells were mixed with 1 μl (approximately 50 ng/μl) plasmid and electroporated using a BTX electroporation system at 2.0 kV and 186 ohm in a prechilled 1 mm cuvette. Moreover, 935-μl prechilled GLY medium was added, and bacteria were incubated at 30°C for 4 hours without antibiotic before plating on GLY agarose plates containing 100 μg/ml kanamycin. Plates were incubated at 30°C for 48 hours, and single colonies were picked and sequence verified. Preparation of barcoded blood meals and plate feeding Barcoded Asaia bacteria were grown overnight at 30°C to early log phase (OD 600 0.5 to 0.8) in a deep-well plate (Sarstedt, Nümbrecht, Germany) in 300-μl GLY medium supplemented with 100 μg/ml kanamycin per well. Bacteria were next diluted in heat inactivated human serum (type A) and combined with human red blood cells (type O) to achieve a final density of 106 cfu/ml and a haematocrit of 50%. Microtiter plates were filled with 160 μl of blood meal per well and sealed with a membrane (Parafilm M, PM999, VWR, Amsterdam, the Netherlands) that was stretched to about 250% its original dimensions in both directions using a custom build device (S1A Fig) and applied using a lever press (S1B Fig). The plates were kept warm (37°C) and placed upside down on top of a mosquito container sealed with mosquito netting. An aluminium block routed to fit the base of the microtiter plate and preheated to 45°C was put on top to warm the plate (S1C Fig). Experiments were performed with 3- to 5-day-old females of A. stephensi mosquitoes (Sind-Kasur Nijmegen strain) reared at the insectary of the Radboud University Medical Center [58] or A. aegypti (Rockefeller strain, obtained from Bayer, Monheim, Germany) reared at Wageningen University [59]. For a plate containing 48 barcoded blood meals, we used approximately 300 mosquitoes per container, and for experiments with other sample sizes, the number of mosquitoes was adjusted proportionally. Mosquitoes were allowed to feed for 20 minutes after which the mosquitoes were maintained at 26°C and 70% to 80% humidity. Recovery and detection of barcode sequences Mosquitoes were washed in 70% ethanol followed by 3 washes in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 8.0). Individual mosquitoes were transferred to wells in shallow 96-well plates, combined with Zirconium beads and homogenised in 60-μl PBS using a Mini-Beadbeater-96 (Biospec, Bartlesville, Oklahoma, United States). A total of 15 μl of each of the mosquito homogenates was subsequently transferred to a deep-well plate containing 300-μl GLY medium supplemented with 100 μg/ml kanamycin and 2 μg/ml amphotericin B. The plates were sealed with a gas permeable breathing seal (Greiner Bio-One, Alphen aan de Rijn, the Netherlands), and Asaia bacteria were grown to the stationary phase by incubation at 30°C with continuous shaking (220 rpm) for at least 72 hours. In initial experiments, barcodes were amplified from individual Asaia cultures by PCR. In later phenotypic screening experiments, Asaia cultures from mosquitoes with the phenotype of interest were pooled. For comparative analyses (e.g., live versus dead mosquitoes), mock cultures with an unrelated barcode were added to make up for differences in sample sizes between the 2 pools. This to prevent differences in amplification efficiencies due to different numbers of PCR templates and, as a result, a bias in the barcode representation. Barcodes were amplified using forward primer MWV 486 and a 5’-biotinylated reverse primer MWV 358 (S1 Table) using standard PCR conditions with Gotaq G2 flexi DNA polymerase (Promega). The biotinylated PCR products were then hybridised to a pool of MagPlex-TAG microspheres (S3 Table) according to the manufacturer’s instructions (Luminex) with some adaptations. Briefly, 33 μl of microsphere mixture was prepared in 1.5 X TMAC hybridisation solution (1× TMAC = 3M Tetramethyl ammonium chloride, 50 mM Tris, 1 mM EDTA, and 0.1% SDS at pH 8.0) with about 1,000 beads per barcode for all 50 barcode sequences. This was then mixed with 2 μl from the barcode amplification reactions and 15 μl TE buffer (10 mM Tris/1 mM EDTA, pH 8) and incubated for 15 at 52°C. Next, 35 μl of reporter mix was added, containing 14.3 ug/ml SAPE (Streptavidin, R-Phycoerythrin Conjugate) and 0.24% bovine serum albumin in TMAC buffer, resulting in a final concentration of 5.9 ug/ml SAPE and 0.1% BSA per reaction. After a second incubation at 52°C for 15 minutes, 50 μl was analysed on a MAGPIX instrument (Luminex). Screening of a collection of pesticides A collection of pesticides was obtained through the Innovative Vector Control Consortium (Liverpool, United Kingdom) and the MMV (Geneva, Switzerland). Compounds were first diluted in DMSO and then in human serum type A to a concentration 4 times above the final test concentration. Blood meals were prepared by mixing 40 μl of diluted compound with 40 μl of 4.106 CFU/ml barcoded Asaia and 80 μl human type O red blood cells. Controls included vehicle (0.1% DMSO) and positive controls fipronil and deltamethrin, both at 10 μM. Blood meals were prepared in duplicate for each compound and transferred to 96-well plates in 2 different layouts (S2 Fig). A. stephensi mosquitoes were allowed to feed for 20 minutes and maintained at 26°C and 70% to 80% humidity. Moreover, 48 hours after feeding, live and dead mosquitoes were processed in separate pools as described above. Screening for malaria transmission-blocking compounds Infectious P. falciparum gametocytes of parasite line NF54-HGL, expressing a GFP-luciferase fusion protein under control of the hsp70 promoter, were cultured in RPMI 1640 medium supplemented with 367 μM hypoxanthine, 25 mM HEPES, 25 mM sodium bicarbonate, and 10% human type A serum in a semiautomated system as previously described [20,60]. Furthermore, 72-μl aliquots of cultures containing mature stage V gametocytes were transferred to 96-well v-bottom plates (Corning Life Sciences, Amsterdam, the Netherlands) in duplicate in 2 different layouts (S2 Fig). Test compounds from the Pathogen Box (MMV, Geneva, Switzerland) were diluted in DMSO and then in RPMI 1640 medium supplemented with 10% human serum type A, and 8 μl of diluted compound was added to the gametocytes in the plate to achieve a final compound concentration of 10 or 20 μM and a final DMSO concentration of 0.2%. Positive and negative controls included 10 μM and 0.2% DMSO, respectively. Plates were incubated at 37°C, 4% CO 2 and 3% O 2 for 24 hours in accordance with established methods for maintenance of infectious gametocytes [31,61]. Subsequently, plates were centrifuged briefly (750xg, 5′), and 70-μl supernatant was removed and replaced with 42.7 μl of heat inactivated human type A serum, 48-μl human type O red blood cells, and 5.3 μl of barcoded Asaia bacteria to a final density of 105 CFU/ml. All procedures were performed at 37°C. Plates were then sealed and used for feeding to A. stephensi mosquitoes as described above. Following feeding, mosquitoes were maintained at 26°C and 70% to 80% humidity and starved for 2 days. From day 3 onwards, the mosquitoes were presented with cotton pads wetted in a 5% glucose solution supplemented with 100 μg/ml kanamycin twice a day for a duration of 2 hours each to minimise barcode cross-contamination through the glucose pads. Eight days after feeding, mosquitoes were harvested and homogenised in 96-well plates as described above. Infection status of individual mosquitoes was analysed by determining luciferase activity in 45 μl of the mosquito homogenate as described previously [62]. Background luminescence was determined by analysing 10 uninfected (unfed) mosquitoes. Mosquitoes were considered infected when the luminescence signal was greater than the mean + 5xσ of the signal in the negative control mosquitoes as described previously [20]. Asaia cultures from uninfected and infected mosquitoes were collected in separate pools for further analysis of barcode signals. Standard membrane feeding assays using glass feeders Results from barcoded experiments were validated through standard membrane feeding assays using traditional glass feeders [20]. For testing for systemic insecticide activity, compounds were serially diluted in DMSO and then in DMEM medium and combined with human type A serum and type O red blood cells to achieve a final DMSO concentration of 0.1% in 40% haematocrit in a volume of 300 μl. Blood meals were placed in glass feeders warmed at 37°C and A. stephensi mosquitoes were allowed to feed for 15 minutes. Following feeding, nonfed mosquitoes were removed and the blood-fed mosquitoes were maintained at 26°C and 70% to 80% humidity for 48 hours. Subsequently, the number of live and dead mosquitoes was determined for each test condition. Testing for compound effects on transmission of P. falciparum gametocytes to A. stephensi mosquitoes was performed as described previously [20]. Replicates and data analyses To obtain sufficient numbers of fed mosquitoes, all test compounds were presented in replicate blood meals (S2 Fig). An average of 6 mosquitoes per blood meal was used in barcoded feeding experiments. With a 90% feeding efficiency, this resulted in approximately 10 fed mosquitoes per test condition. Mosquitoes were processed individually and rescued barcoded Asaia bacteria were pooled according to phenotype. Here, the Asaia from the replicate plates were combined for each phenotype. For each pool, barcode fragments were amplified and analysed in triplicate. Fluorescence intensity was determined by analyses of at least 40 microspheres per barcode and expressed as relative median fluorescence intensity (MFI). MFI values were averaged from the triplicates observations for each pool and corrected for average background signals from negative control (GLY medium without barcoded Asaia) samples. Barcodes were considered as sampled when the signal was above the mean + 3σ of the negative control samples. In comparative phenotypic analyses, data were expressed as the relative proportion of the barcode signal in the phenotype of interest. For example, when comparing uninfected and infected mosquitoes, the percentage of the barcode signal in the uninfected mosquitoes was calculated by where I u and I i are the background corrected median fluorescence intensities in the uninfected and infected mosquitoes, respectively. In standard membrane feeding experiments using glass feeders, all conditions were tested in 2 replicate feeders, and at least 24 mosquitoes were analysed per feeder. Data were analysed and visualised using the Prism software package (GraphPad Software, San Diego, US). IC 50 values for systemic insecticides were determined by fitting a 4 parameter logistic regression model using least squares to find the best fit. IC 50 values in Plasmodium transmission-blocking experiments were determined by assuming a beta binomial distribution and logistic regression using maximum likelihood to find the best fit as described previously [63]. Effects of Pantoea or Asaia on P. falciparum were analysed by ANOVA using a Kruskal–Wallis test and Dunn multiple comparison test.

Acknowledgments We wish to thank Claudia Damiani and Aida Capone for help with the Asaia SF2.1 strain, Marcelo Jacobs-Lorena for his kind gift of Pantoea agglomerans, and Sander Koenraadt for provision of Aedes aegypti mosquitoes. Bernd Engelbrecht, Katharina Schumacher, and colleagues at Irmato Industrial Solutions are gratefully acknowledged for help with the design of the plate sealing process. The authors wish to thank Geert-Jan van Gemert and Laura Pelsen-Posthumus for expert technical assistance in mosquito rearing. Sarah Rees is acknowledged for provision of a collection of pesticides. The authors thank Isaac Sandoval Capuchino for providing artwork. Manuel Llinás and Robert Sauerwein are gratefully acknowledged for critical reading of the manuscript.

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