{"paper_id":"2cfb8ac7-d33a-4d77-ae60-af5855f1f776","body_text":"Full title: Stomoxys flies (Diptera, Muscidae) are competent 1 \nvectors of multiple livestock hemopathogens 2 \nShort title: Disease transmission dynamics by Stomoxys flies 3 \nJulia W. Muita1,2, Joel L. Bargul2, JohnMark O. Makwatta1, Ernest M. Ngatia1,2, Simon K. 4 \nTawich1, Daniel K. Masiga1, Merid N. Getahun1* 5 \n1International Centre of Insect Physiology and Ecology ( icipe), P.O. Box 30772-00100, Nairobi, 6 \nKenya 7 \n2Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology 8 \n(JKUAT), P.O. Box 62000-00200, Nairobi, Kenya 9 \n 10 \n*Corresponding author: Merid N. Getahun,  mgetahun@icipe.org 11 \n12 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nAbstract 13 \nStomoxys flies are widely distributed and economically significant vectors of various livestock 14 \npathogens of veterinary importance. However, the role of Stomoxys spp. in pathogen 15 \ntransmission is poorly understood. Therefore, we studied the feeding patterns of these blood 16 \nfeeders collected from specific locations in Kenya, to identify various vertebrate hosts they fed 17 \non, and the livestock hemopathogens they carried, to elucidate their role in pathogens 18 \ntransmission. Our findings show that field-collected Stomoxys flies carried several pathogens 19 \nincluding Trypanosoma spp., Anaplasma spp., and Theileria spp. that were also found in the 20 \nblood of sampled livestock, namely camels and cattle. The findings on blood meal analysis show 21 \nthat Stomoxys flies fed on a variety of domestic and wild vertebrate hosts. We further determined 22 \nwhether Stomoxys spp. are vectors of hemopathogens they harbored by studying the vector 23 \ncompetence of S. calcitrans, S. niger niger, and S. boueti species complex, through laboratory 24 \nand natural experimental in vivo studies. We show that in the process of blood feeding Stomoxys 25 \nspp. complexes can transmit T. evansi  (8.3%) and T. vivax (30%) to Swiss white mice. In 26 \naddition, field-collected Stomoxy spp. were exposed to healthy mice for blood meal acquisition, 27 \nand in the process of feeding, they transmitted Theileria mutans and Anaplasma spp. to Swiss 28 \nwhite mice (100% infection in the test mice group). All mice infected with both trypanosomes 29 \nvia stomoxys bite died while those infected with Theileria and Anaplasma  species did not, 30 \ndemonstrating virulence difference between pathogens. The key finding of this study showing 31 \nbroad feeding host range, cosmopolitan, plethora of pathogens harboured, and efficient vector 32 \ncompetence in spreading multiple pathogens suggests profound role of Stomoxys on pathogen 33 \ntransmission and infection prevalence in livestock.  34 \nAuthor summary 35 \nStomoxys flies are highly adaptable to several ecological settings, including metropolitan areas. 36 \nIn contrast, tsetse flies (genus Glossina), the main biological vectors of African trypanosomes, 37 \nhave a limited distribution to parks and other conservation areas. Stomoxys flies could play a 38 \nsignificant role in the spread of animal African trypanosomes, among other hemopathogens, 39 \nparticularly in areas with or without tsetse infestation. Although there have been speculations 40 \nabout the potential role of Stomoxys flies in the transmission of various pathogens, there is lack 41 \nof data to link hemopathogens occurring in both bloodmeal hosts of Stomoxys and in the flies, 42 \nand further in vivo experimental studies to confirm the vector competence of Stomoxyine flies. 43 \nHere, we explored a host and pathogens network, and investigated species diversity at various 44 \necologies, and demonstrated that Stomoxys flies feed on diverse vertebrate hosts and are infected 45 \nwith a plethora of pathogens. We also showed experimentally that they could transmit some of 46 \nthese hemopathogens to mice, for instance, T. vivax, T. evansi, Theileria mutans, and Anaplasma 47 \nspp. with varying infection success rates. Stomoxys  flies could play a significant role in 48 \ntransmitting and spreading various hemopathogens of veterinary importance and possibly 49 \nmaintaining their circulation in livestock, which could explain the occurrence of animal African 50 \ntrypanosomes in the regions outside the tsetse belts.  51 \n 52 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n 53 \n1. Introduction 54 \nStomoxys flies are widely distributed globally [1]. They feed on both blood [2], [3] and nectar 55 \n[4]. These blood feeders pose a significant threat to livestock production worldwide, especially 56 \nbecause of their occurrence in wider ecological zones [5]. The economic losses due to livestock 57 \ninfestation by Stomoxys flies are substantial, leading to a significant reduction in meat and milk 58 \nproduction [6], due to pathogen infestation. These pathogens cause diseases including; animal 59 \ntrypanosomiasis, Rift Valley fever, African swine fever, lumpy skin disease, and anaplasmosis 60 \n[3], [7-12]. The annual economic losses in the United States of America (USA) alone due to 61 \ninfestation by a single Stomoxys  species, Stomoxys calcitrans – a cosmopolitan species 62 \ncommonly known as stable fly, have been estimated to be around US$ 2.2 billion [8]. The 63 \ncombined economic losses caused by Stomoxys flies infection, nuisance, and treatment costs 64 \noutside the USA are not well documented at present.  65 \nStomoxys feed on their vertebrate hosts' blood once or twice a day [9]. Since the host animals 66 \nrespond to protect themselves against the painful blood-feeding fly bites, they ( Stomoxys) 67 \ntypically do not complete blood-feeding on a single animal [5], [9]. In the event of an interrupted 68 \nblood meal, the fly can restart feeding on another host by injecting infected saliva before feeding 69 \n[5]. However, the pathogen transmission mechanism is not clear but could vary from biological, 70 \nas is the case in the transmission of filariasis [10], [11], to the mechanical transmission of, for 71 \ninstance, animal trypanosomiasis [5], [12],[13]. 72 \nAnimal African trypanosomiasis caused by T. vivax and T. evansi is endemic in most countries in 73 \nsub-Saharan Africa outside the tsetse belts [3], [12], [14], [15], and also in other regions outside 74 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nAfrica, including Asia [16], Latin America [17], and Europe [18]. T. evansi is the causative agent 75 \nof ‘surra’, and T. vivax, that of nagana – an animal disease that is endemic in large swathes of 76 \nAfrica, Asia, and Latin America, and also present in the Canary Islands (Spain) [22] [23]. 77 \nHowever, the establishment, widespread occurrence, maintenance, and circulation of T. vivax 78 \nand T. evansi particularly in areas outside the tsetse belts is still unclear.  79 \nDespite the  wide geographic distribution and cosmopolitan nature of Stomoxys flies, our 80 \nknowledge about their blood meal hosts, and the hemopathogens they carry, which will provide 81 \ninsight into ‘vector-host-pathogen’ interactions, and disease transmission dynamics is limited and 82 \nnecessitates compressive research. Thus, in this study, we studied the diversity of Stomoxys  spp. 83 \ncollected from various National Reserve to zero grazing ecologies (Fig 1).  We conducted a study 84 \nto assess the vector competence of Stomoxys spp., which refers to their host feeding dynamics, 85 \nand capacity to become infected and transmit hemopathogens. We show that Stomoxys flies are 86 \ncompetent vectors to multiple pathogens and may play a role in the transmission cycles of the 87 \npathogens they harbor.  88 \n2. Materials and methods 89 \n2.1 Study sites  90 \nField sampling took place in nine selected counties in Kenya at various times from October 91 \n2021 to November 2023. The sampled ecosystems in each County were inhabited by a variety 92 \nof livestock species, wildlife hosts, and humans. These counties included: Kwale County 93 \n(4.2572° S, 39.3856° E), Isiolo County (0.3257° N, 38.1961° E), Samburu County (1.7299° N, 94 \n37.3079° E), Kiambu County (1.0131° S, 36.9051° E), Kajiado County (1.7617° S, 36.0255° 95 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nE), Marsabit County (2.4426° N, 37.9785° E) Homabay County (0.4368° S, 34.2060° E), 96 \nLaikipia (0.2924° N, 36.8985° E), and Meru County (0.0515° N, 37.6456° E) (Fig 1).  97 \n98 \nFigure 1. A map of Kenya showing the sampling sites across the nine selected counties. Th is99 \nmap was created using the open-source software, QGIS v.3. 100 \n2.2 Ethical approval and animal welfare 101 \nThis study was conducted in strictly adherence to the approved experimental guidelines and102 \nprocedures set forth by the Animal Care and Use Committee (IACUC) of the International103 \nCentre of Insect Physiology and Ecology, icipe (REF No.: icipeACUC2018-003-2023) and the104 \nEthics Review Committee of Pwani University (REF No.: ERC/EXT/002/2020E). F armers and105 \npastoralists were sensitized about the research study and how the findings could benefit the106 \n \nis \nnd \nal \nhe \nnd \nhe \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nfarmers. Sample collection from livestock was done after obtaining verbal consent from farmers, 107 \nas most of them (camel herders) were unable to read or write. Animal experiments complied with 108 \nthe approved guidelines and mice were handled carefully to ensure minimum distress. 109 \n2.3 Blood collection and fly trapping 110 \n2.3.1 Blood collection 111 \nBlood collection from a total of 452 camels and 124 cattle was done from October 2021 to 112 \nNovember 2023. The sample size was determined using our initial data, which indicated an 113 \ninfection rate of 4% in camels and 5.7% in cattle caused by Rickettsiae spp. the lowest 114 \nprevalence among pathogens. We utilized this information as a basis for calculating the sample 115 \nsize, following the formula; /g1866/g3404\n/g2922/g2924/g4666/g2961/g4667\n/g2922/g2924/g4666 /g2869/g2879/g2977/g4667   as outlined by [20]. At least 5 mL of blood was drawn 116 \nfrom the jugular vein of each animal and collected in vacutainer tubes containing disodium salt 117 \nof ethylene diamine tetra123 acetate (EDTA) (Plymouth PLG, UK). The blood samples were 118 \ninitially stored in vacutainers at 4°C until the collection was completed which were transferred to 119 \ncryovials and stored in liquid nitrogen before being transported back to the Nairobi Duduville 120 \ncampus-icipe for molecular identification of pathogens. 121 \n2.3.2 Trapping of flies 122 \nFlies were trapped using five red monoconical traps [21]. The traps were placed 150 meters apart 123 \nand the flies were trapped from nine counties as indicated in (Fig 1). The traps were emptied 124 \ntwice per 24-hour period to avoid flies drying for clear identification and further intended 125 \nanalysis. The flies were later immobilized and preserved in absolute ethanol for further analysis 126 \nand fresh flies were pinned for morphological identification. Trapping lasted for 5 days. Except 127 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nfor Kiambu County, all trapping sites were located in wide woodland savannah ecologies that 128 \nwere not in close proximity to villages. 129 \n2.4 Morphological identification of Stomoxys spp. 130 \nField-collected Stomoxys flies were taxonomically identified to the species level using 131 \nestablished taxonomic keys as outlined by [22] . Briefly, the flies were staged under a dissecting 132 \nStemi 2000-C microscope (Zeiss, Oberkochen, Germany) to identify key morphological 133 \ndifferences. The head frontal index and the distinctive dorso-abdominal patterns on the second 134 \nand third segments  were key distinguishing features in identifying fly sex and species, 135 \nrespectively. Images were captured using a digital microscope connected to an Axio-cam ERc 5s 136 \ncamera (Zeiss). The flies were grouped according to their species and preserved at −20°C for 137 \nmolecular analysis. 138 \n2.5 Molecular identification of Stomoxys spp. and livestock 139 \nhemopathogens 140 \n2.5.1 DNA extraction from blood samples and flies 141 \nIn the initial stages of DNA pre-extraction, the flies were subjected to a 1% sodium hydroxide 142 \nimmersion for 1 minute to eliminate any exogenous material on their bodies. Subsequently, they 143 \nunderwent a 1-minute rinsing procedure with 1 × phosphate-buffered saline (pH = 7.4). Each fly 144 \nwas mechanically homogenized in sterilized 1.5-ml microfuge tubes containing 750 mg of 2.0-145 \nmm yttria-stabilised zirconium (YSZ) oxide beads (Glen Mills, Clifton, NJ, USA) and 80 µL of 146 \n1× PBS using a Mini-Beadbeater-16 (BioSpec, Bartlesville, OK, USA) for one minute. Genomic 147 \nDNA extraction was done using DNeasy blood and tissue kit (Qiagen, Hilden, Germany) 148 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nfollowing the manufacturer’s protocol to obtain DNA from flies, cattle, and camel blood for 149 \npathogen screening. The obtained DNA was quantified using a Nanodrop spectrophotometer 150 \n(Thermo Scientific, Wilmington, DE, United States) by comparing absorbance at 260 and 280 151 \nnm and later stored at −20°C for molecular work. 152 \n2.5.2 Molecular identification of Stomoxys spp. and livestock hemopathogens 153 \nThe extracted DNA was used in the molecular characterization of Stomoxys s p p .  a s  w e l l  a s  154 \nscreening livestock hemopathogens. We conducted amplification using genus-specific primers as 155 \noutlined in (Supplementary Table 1), using the conventional ProFlex PCR systems thermocycler 156 \n(Applied Biosystems, Foster City, CA, USA). The PCRs were conducted in 10- μ L reaction 157 \nvolumes, consisting of 5 µL nuclease-free water, 2 μ L 5× HOT FIREPol® Blend Master Mix 158 \n(Solis BioDyne, Tartu, Estonia), 0.5 μ L of 10 µM forward and reverse primers (Supplementary 159 \nTable 1), and of 2 μ L DNA template. For negative controls, 2 μ L nuclease-free water was used 160 \nin place of the DNA template. The amplification conditions were set as described by [23]. PCR 161 \namplicons were resolved by electrophoresis under 2% ethidium-bromide-stained agarose gel 162 \n(100 V for 1 hour), followed by DNA visualization by UV-transillumination (Kodak Gel Logic 163 \n200 Imaging System, CA, USA). PCR amplicons were purified using ExoSAP-IT (Affymetrix, 164 \nSanta Clara, CA, USA) as per the manufacturer's protocol, and outsourced for Sanger-165 \nsequencing at Macrogen Inc. (Amsterdam Netherlands). 166 \n2.5.3 Phylogenetic analysis 167 \nTo get a better understanding of how the various Stomoxys species and livestock hemopathogens 168 \nare similar and different at the genomic level we performed a phylogenetic tree analysis. The 169 \nnucleotide sequences acquired in this study were cross-referenced against the known sequences 170 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nin the GenBank of NCBI nr database ( https://www.ncbi.nlm.nih.gov/genbank/). BLAST was 171 \nused to validate their identity and establish connections with existing deposited sequences [24]. 172 \nTo show the evolutionary relationships, maximum-likelihood phylogenetic trees were 173 \nconstructed using PhyML v. 3.0, employing automatic model selection based on the Akaike 174 \ninformation criterion. The tree topologies were estimated through 1000 bootstrap replicates, 175 \nincorporating nearest-neighbor interchange improvements [25]. The resulting phylogenetic trees 176 \nwere then visualized using FigTree v. 1.4.4[26]. 177 \n2.5.4 PCR-HRM vertebrate bloodmeal source identification in Stomoxys flies 178 \nThe extracted DNA was used in the analysis of the blood meal source of the fed flies collected in 179 \nthe field. A 10- μ L PCR reaction was carried out, consisting of 1 μ L of DNA template, 6 μ L of 180 \nnuclease-free water, 2 μ L of 5× HOT FIREpol EvaGreen HRM Mix from Solis BioDyne in 181 \nTartu, Estonia, and 0.5 μ M of both forward and reverse primers (Supplementary Table 1). 182 \nPositive control vertebrate host samples used as reference include; cow ( Bos taurus ), camel 183 \n(Camelus dromedarius ), donkey ( Equus asinus), warthog (Phacochoerus africanus ), African 184 \nbuffalo (Syncerus caffer ), goat ( Capra aegagrus  hircus), waterbuck ( Kobus ellipsiprymnus ) 185 \nelephant (Loxodonta africana), sheep (Ovis aries), reticulated giraffe (Giraffa reticulata ), lesser 186 \nkudu ( Tragelaphus imberbis ), cheetah ( Acinonyx jubatus ), zebra (Equus quagga ), baboon 187 \n(Papio), gerenuk ( Litocranius walleri), hartebeest ( Alcelaphus buselaphus), reedbuck ( Redunca 188 \nredunca), hyena (Crocuta crocuta ), gazelle ( Gazella gazella ), impala ( Aepyceros melampus ), 189 \nlion ( Panthera leo), bongo ( Tragelaphus eurycerus ) and human ( Homo sapiens ). The PCR 190 \nthermal cycling conditions for primer were set as described by [3]. After PCR amplification, 191 \nHigh-Resolution Melting (HRM) analysis was conducted within normalized temperature ranges, 192 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nfrom 65°C to 78°C and 88°C to 95°C. The distinct melt curve profiles of the samples were 193 \ncompared against reference standards.  194 \n2.6 Experimental infection assays to determine the vector 195 \ncompetence of Stomoxys to transmit T. evansi and T.vivax 196 \n2.6.1 Experimental animals 197 \nSwiss White Mice ( Mus musculus) obtained from icipe’s Animal Rearing and Quarantine Unit 198 \n(ARQU) were used for the infection experiment. Both male and female mice used for 199 \nexperiments were about 6 – 8 weeks old. Each mouse weighed about 24 – 29 g live body weight. 200 \nThe mice were housed under normal conditions in standard mouse cages and their diet primarily 201 \ncomprised of commercial pellets (Unga® Kenya Ltd) and water, which was provided ad libitum. 202 \nThe mice were kept in a mice experimental room that was free from biting flies. The 203 \nexperimental mice used in pathogen transmission studies were not immune suppressed.  204 \n2.6.2 Establishment of laboratory colonies of Stomoxys spp.  205 \nStomoxys spp. of both sexes were trapped from both Gatundu (1.0131° S, 36.9051° E) and 206 \naround icipe-Duduville campus (1.2921° S, 36.8219° E) and taken to icipe’s insects rearing 207 \nunits. The mixed species of Stomoxys  flies were maintained in 75 cm × 60 cm × 45 cm perspex 208 \ncages (Astariglas ®, Indonesia) and fed once daily between 9 am – 11 am warm defibrinated 209 \nbovine blood obtained from a local slaughterhouse (Choice meats, Nairobi) and supplemented 210 \nwith 10% glucose and Parthenium hysterophorus flowers [27]. The temperature and humidity in 211 \nthe rearing room were kept at 25 ± 1 °C and RH 50 ± 5%, respectively with a 12:12 light/dark 212 \nphotoperiod. Sheep dung was used as an oviposition substrate [28] and developed pupae were 213 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\npicked and transferred to another cage for emergence. The newly emerged or teneral flies were 214 \nused for experimental infection assays. 215 \n2.6. 3 Multiplication of T. evansi and T. vivax isolates in donor mice 216 \nThe strain of Trypanosoma used in this study was T. evansi, which was isolated from a naturally 217 \ninfected camel (Camelus dromedarius) from Marsabit County, and T. vivax IL 2136 which was 218 \ntaken from icipe’s trypanosomes bio-bank. They were let to thaw after which parasitemia was 219 \nchecked using microscopy (Zeiss Primo Star Binocular Microscope, Zeiss, Oberkochen, 220 \nGermany) with a ×40 magnification to ensure the viability of the stabilates before each 221 \ninoculation. 200 µL of each stabilate was then inoculated to the mice through the intraperitoneal 222 \nroute 223 \n2.6.4 Monitoring parasitemia levels in donor mice 224 \nThe mice were monitored daily, three days after pathogen inoculation. Briefly, a drop of blood 225 \nfrom the snipping of the mouse tail using a blood lancet was placed on a clean slide and covered 226 \nusing a coverslip as a wet blood smear and examined under a microscope (Seamer et al., 1993). 227 \nThe parasitemia score was estimated which correlated to a score sheet, as described by [29]. The 228 \nperiod taken from the day post inoculation (dpi) to the first appearance of trypanosomes in blood 229 \nwas recorded for all mice. This was done until the required parasitemia was achieved (1 × 10 8 230 \ntrypanosome/mL blood). 231 \n2.6.5 Determination of T. evansi and T. vivax survival rates in Stomoxys fly 232 \nGiven that mechanical transmission of trypanosome parasitemia is known to be dose-dependent 233 \n[30], our experimental design included the testing of two doses that are typically encountered in 234 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nnatural infection [3] approximately 1 × 10 8 trypanosomes/ml blood and 5 × 10 8 235 \ntrypanosomes/mL blood. Following the successful induction of high parasitemia, the next step 236 \ninvolved the extraction of whole blood from the donor mouse. This was done by sacrificing the 237 \nmouse in accordance to the standard protocol defined by the Institutional Animal Care and Use 238 \nCommittee (IACUC).  Fino-Ject disposable syringe 5 mL/cc with needle was used for blood 239 \ncollection by cardiac puncture [31] which resulted to 200 μ L of blood. The parasitemia was 240 \nchecked microscopically, to ensure the blood still had enough parasite concentration. The 241 \ninfected blood was then carefully diluted in clean pre-warmed defibrinated bovine blood 242 \ncollected from the slaughterhouse (Choice meats, Nairobi) at a 1:1 ratio. The resulting blood 243 \nmixture, approximately 400 µL, was applied onto clean cotton placed in a petri dish. A total of 244 \n60 teneral Stomoxys flies which were starved for 24 hours were placed in a clear acrylic plastic 245 \ncage with dimensions of 10 × 10 × 15 cm, which was made of a 6-mm-thick perspex sheet from 246 \nAstariglas® in Indonesia. The flies were fed on the blood-soaked cotton wool that was provided 247 \non a petri dish. Following a feeding period of five minutes, until all flies were fully engorged, the 248 \ninfection and spread of trypanosomes within the Stomoxys flies were monitored at various time 249 \npoints post-feeding, beginning one hour after the feeding event and continuing at each 250 \nsubsequent hour. To analyze the distribution and prevalence of the parasite within the bodies of 251 \nthe Stomoxys flies, we conducted dissections of various body parts, including the mouthparts, 252 \ncrop, and gut. At least five insects per exposure time were examined after immediate interrupted 253 \nfeeding. 254 \n2.6.6. Experimental trials of in vivo transmission of T. evansi and T. vivax  255 \nVarious protocols were tested in the in vivo transmission trials for optimization. At first, after the 256 \nsuccessful induction of high parasitemia within the donor mouse, the donor mouse and recipient 257 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nmouse were restrained using a restrainer which was made of stainless-steel woven wire mesh 258 \nwith measurements of 0.9 mm per hole and a 400 µm wire diameter, and placed in a 10 ×10 ×15-259 \ncm cage made of 6-mm (thick) perspex clear acrylic plastic sheet (Astariglas®, Indonesia). The 260 \nteneral flies n = 20 were introduced and the flies were disturbed by the observer to allow them to 261 \nmove from donor to recipient mice. Unfortunately, after more than 20 trials using this 262 \nexperimental method, we did not get any results. We optimized our experiment whereby, once a 263 \ndonor mouse with high parasitemia was achieved, the donor mouse and recipient mouse were 264 \nrestrained using a restrainer which was made of stainless-steel woven wire mesh with 265 \nmeasurements of 0.9 mm per hole and a 400 µm wire diameter, and both were placed in separate 266 \n10 ×10 ×15-cm cage made of 6-mm (thick) perspex clear acrylic plastic sheet (Astariglas®, 267 \nIndonesia). One-day-old teneral flies which were not fed on any blood meal where n=20 per 268 \nexperiment, were released in the cage of the donor mouse and allowed to feed for ≤ 1 minute. The 269 \ntiming was done once the proboscis had pierced the mouse’s skin to ensure feeding had started 270 \nand the flies ingested blood from the infected mouse. This was followed by disrupted feeding 271 \nwhere only the fed flies were individually picked using a respirator and transferred to the next 272 \ncage containing restrained recipient healthy mice and flies were allowed to complete their blood 273 \nmeal until fully engorged (Fig 2). The flies were subsequently dissected to confirm the presence 274 \nof parasites in their gut to assess the parasite-feeding success rate. The restrained recipient mouse 275 \nwas then released into their standard mouse cages and monitored daily after three days post-276 \ninfection.  277 \n 278 \n 279 \n 280 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n 281 \n 282 \nFigure 2: Laboratory in vivo transmission of T. evansi and T. vivax experimental design. 283 \n2.6.7 Screening for T. evansi and T. vivax in the recipient mice by PCR 284 \nAfter three days post-infected fly bites a combination of microscopy and molecular methods 285 \nwere used to confirm the presence of parasites in the blood of infected animals for up to 30 days 286 \npost-infection (dpi). For microscopy, it was done as described above, daily. Molecular screening 287 \nwas done by collecting blood samples from snipping the mice tails and collecting them in 1.5 ml 288 \neppendorf tubes which contained 80 μ L 1× PBS buffer, pH = 7.4. Blood collection was done 289 \nafter every two days. This was followed by total DNA extraction using a DNeasy blood and 290 \ntissue kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. PCR, gel 291 \nelectrophoresis, and gene sequencing were performed as described above 292 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n2.7 Determination of vector competence through field bioassay 293 \nFly trapping was done as described above in various study sites. The traps were emptied after 6 294 \nhours and the flies were put in 10 × 10 × 15-cm cage made of 6-mm (thick) perspex clear acrylic 295 \nplastic sheet (Astariglas®, Indonesia). The recipient mice were restrained using the restrainer that 296 \nwas made of stainless-steel woven wire mesh with measurements of 0.9 mm per hole and a 400 297 \nµm wire diameter and released into the cage. The flies were left to feed for 30 minutes before 298 \nreleasing the mice. This was followed by daily evaluation of pathogens in the recipient mice 299 \nthrough microscopy and molecular screening as described above. 300 \n2.8 Data analysis 301 \nThe Shannon diversity index (H) was utilized to define the diversity index of biting flies among 302 \nstudy counties and was calculated using R statistical software (R version 4.4.1.). Estimated 303 \nminimum infection rates (MIRs) of pathogens obtained for the flies were calculated as the 304 \nnumber of positive per total number of flies tested ×100. Graphs were visualized using GraphPad 305 \nsoftware (GraphPad Software, Inc, USA). The bipartite R package's interaction network [32] 306 \nvisualized the vectors blood-feeding behavior and pathogen interactions between hosts and 307 \nvectors which was generated by R statistical software (R version 4.4.1.). An Upset plot displayed 308 \nthe number of flies feeding on specific animal species and those containing bloodmeals from one 309 \nor more host species and was plotted using R statistical software (R version 4.4.1.). Transmission 310 \nrates of the experimental infection assays were performed by calculating the number of infected 311 \nmice per total number of transmission trials done ×100. 312 \n  313 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n3. Results 314 \n3.1 Stomoxys species diversity relative abundance is ecology 315 \ndependent 316 \nDiverse Stomoxys species were trapped throughout the year . A total of 11,323 adult Stomoxys 317 \nflies were collected from various sites, from National Reserve including; Shimba Hills National 318 \nReserve and Nguruman Conservation Reserve, to zero grazing ecologies and identified 319 \nmorphologically using specific keys to the species level according to [22] as S. calcitrans,  S. 320 \nsitiens, S. niger niger, S. niger bilineatus,  S. boueti, and S. taeniatus  ( Fig 3A). The sampling 321 \nsites varied in species richness with some having only three species, while others had up to six. 322 \nS. calcitrans  was identified in all study sites while S. taeniatus  was only found in Kajiado 323 \nCounty. S. calcitrans exhibited a body appearance characterized by three dark spots on each of 324 \nthe second and third segments. S. sitiens abdominal segments resemble that of S. calcitrans but 325 \nthe dark spots are more transversely elongated. S. niger niger  appears to have grey coloration 326 \nwith well-defined and dark stripes on abdominal segments. The dorsal view of S. boueti appears 327 \nto have an indistinct dark abdomen and is much smaller in size. S. niger  bilineatus has a 328 \nbrownish appearance with the abdominal segment having defined the dark stripes in a dorsal 329 \nview. S. taeniatus has a brighter golden brown to almost yellowish color and it is larger than the 330 \nother species (Fig 3A). Our molecular taxonomy using CO1 (Cytochrome Oxidase I gene) DNA 331 \nsequence confirmed the morphological identification of those samples clustered distinctly and 332 \nwith previously documented DNA sequences demonstrating they are different species (Fig 3B).  333 \nNew CO1 sequences including S. boueti,  (GenBank Accession number, PP587243)  and S. 334 \n    \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\ntaeniatus (GenBank Accession number  PQ203543) were deposited that were not available in the 335 \nNCBI.  336 \nKajiado followed by Kwale County recorded the highest number of Stomoxys species diversity, 337 \nsix and five species, respectively (Fig 3C) The Shannon diversity index shows the varying levels 338 \nof Stomoxys species diversity (Supplementary Table 2) across Kenyan counties with Kwale 339 \nCounty having the highest Shannon diversity index of 1.36  and Meru County having the lowest 340 \nShannon diversity index of 0.29. Overall, the species distribution showed that S. calcitrans was 341 \nthe dominating species, except in Kajiado and Homabay counties, which was accounted for by 342 \n(n= 5,547, 49%), followed by S. niger niger, (n= 2,938, 25.95%), S. boueti (n= 1,471, 12.99%), 343 \nS. niger bilineatus (n= 778, 6.87%), S. sitiens (n= 495, 4.37%), and finally S. taeniatus (n=94, 344 \n0.83%). Using one of our sites (Kiambu County) we studied the seasonal dynamics of Stomoxys 345 \nflies. Stomoxys flies were caught all year round with seasonal variation (Fig 3D). The abundance 346 \nof Stomoxys increase with the rainfall data, there was an annual rainfall of 674 mm with a 347 \nmonthly average of 56 mm in the study County during the study period. 348 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n 349 \nFigure 3: Stomoxys flies morphological identification, molecular characterization, species 350 \ndiversity, and seasonality (A) Image showing the dorsal and lateral view demonstrating the 351 \ndistinct morphological features of the six Stomoxys species encountered in various study sites (B) 352 \nNeighbour-joining tree constructed based on aligned sequences of CO1 tree showing the 353 \nrelatedness of the various Stomoxys species. (C) The species diversity and their relative 354 \nabundance in various ecologies. (D). Seasonality of Stomoxys at Gatundu site from Kiambu 355 \nCounty. 356 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n3.2 Stomoxys flies blood meal host network analysis demonstrates 357 \nStomoxys flies feed on a wide host range 358 \nStomoxys flies feed on diverse wild and domestic animals.  In  total 225 fed Stomoxys flies were 359 \nsuccessfully identified to analyze the Stomoxys-host feeding network. Fifteen distinct vertebrate 360 \nblood-meal hosts were identified, including cattle ( Bos taurus), camel ( Camelus dromedarius), 361 \nwarthog ( Phacochoerus africanus ), African buffalo (Syncerus caffer ), goat (Capra aegagrus  362 \nhircus), waterbuck ( Kobus ellipsiprymnus) elephant ( Loxodonta africana), sheep ( Ovis aries ), 363 \nreticulated giraffe (Giraffa reticulata ), zebra (Equus quagga ), baboon ( Papio), reedbuck 364 \n(Redunca redunca), gazelle (Gazella gazella), impala (Aepyceros melampus), and human (Homo 365 \nsapiens) (Fig. 4). S. calcitrans  had the most diverse blood meal hosts followed by S. boueti and 366 \nlastly S. niger niger (Fig 4). We observed the diversity of blood meal sources is dependent. 367 \nWildlife conservation (Shimba Hills National Reserve) had the most variety of identified blood-368 \nmeal hosts. Kiambu and Meru counties had the lowest host diversity due to zero grazing in 369 \nregions where Stomoxys were trapped, resulting in a limited number of hosts mostly only cattle.  370 \nIn general, cattle were the most detected and most preferred host across all species S. calcitrans 371 \n(n=65/265), S. niger niger (11/205), and S. boueti (n=13/205) (Fig.4). Multiple host feeding was 372 \nalso revealed in some flies where HRM melt curves revealed two peaks that matched the 373 \nstandard reference (Fig. 4). This was most commonly found in livestock, including cattle and 374 \ngoats, cattle and sheep, cattle and camels, and detected once in wildlife, including waterbuck and 375 \nbuffalo may be due to interrupted feeding before completion (Supplementary Table 3). 376 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n 377 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nFigure 4: Identification of vertebrate hosts from bloodmeal analysis of Stomoxys spp. (A ) A 378 \nbipartite network graph showing feeding interactions between hosts and blood-fed Stomoxys spp. 379 \nThe top bar indicates hosts while the bottom bar indicates the Stomoxys spp. while the lines 380 \nillustrate the interaction. The size of a bar reflects the number of blood-fed Stomoxys (if it is a 381 \nbottom bar) or the number of mammalian hosts that were fed on the vector (if it is a top bar). The 382 \nthickness of a line corresponds to the number of blood-fed hosts detected in the various Stomoxys 383 \nspp. (B) An Upset plot showing the total number of hosts fed per species and also multiple host 384 \nfeeding. 385 \n3.3 Stomoxys flies and domestic animals harbor various 386 \nhemopathogens 387 \nAnother data required to elucidate the role of Stomoxys for various pathogen transmission 388 \ndynamics besides blood meal source is to study the pathogens network between Stomoxys and 389 \nsome of the most preferred host animals they feed on. Various pathogens were detected both in 390 \nthe blood of livestock which were also common in the Stomoxys flies. Anaplasma sp., Theileria 391 \nsp., and Trypanosoma sp. were shared across all analyzed domestic animal hosts and Stomoxys 392 \nflies. Ehrlichia sp. and Rickettsiae sp. were detected in both camels and cattle. Coxiella burnetti 393 \nwas only detected in camels. In camels (n= 452), Anaplasma sp., was the most prevalent 394 \npathogen affecting 64.7% of the camels. Trypanosoma sp. was detected in 12.3% and Ehrlichia 395 \nsp.  in 12.2% of camels sampled. Coxiella burnetti was found in 6% of the camels, while 396 \nRickettsiae sp. was the least detected in 4% of the camels. We did not detect any 397 \nTheileria/Babesia spp. in camels.  Additionally, in cattle out of n=124, we found a high 398 \nprevalence of Theileria/Babesia sp. with 56.6% and Anaplasma sp. in 54.1%. Trypanosoma sp. 399 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nwas detected in 10% of cattle, Rickettsiae sp. in 5.7% of the cattle and Ehrlichia sp. was the least400 \nprevalent, found in only 1.6% of cattle. Among all hosts, sheep had the least pathogen diversity401 \nwith only Theileria/Babesia  sp. being detected in 4% of the sheep. A total of 3,451 Stomoxys402 \nwere screened for pathogen diversity across the study counties. Among these, Anaplasma sp.,403 \nwas the most frequently detected with 49.1%, 19.1% having Theileria/Babesia sp. and 9.1%404 \nhaving Trypanosoma sp. For comparison, Glossina pallidipes (n=1000) co- inhabit with405 \nStomoxys had pathogen prevalence of Trypanosoma sp., Anaplasma sp., and Theileria/Babesia406 \nsp., which were detected in 7.5%, 4%, and 11% of the flies, respectively.  407 \n408 \nFigure 5: Pathogen diversity in host animals and ve ctors from various sites through molecular409 \nscreening and neighbor-joining tree showing pathogens. (A)  A bipartite network graph showing410 \npathogen interactions between hosts (camel, cattle, and sheep) and vectors Stomoxys sp. and411 \nGlossina sp. The top bar indicates pathogens while the bottom bar indicates the hosts and vectors412 \nwhile the lines i llustrate the interaction. The thickness of a line corresponds to the number of413 \nast \nity \nys \np., \n% \nith \nsia \nlar \nng \nnd \nrs \nof \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\npathogens detected in either the hosts or vectors. (B) Neighbor-joining tree showing pathogens 414 \nfrom host animals and vectors.  415 \n3.4 Stomoxys flies are competent mechanical vectors of T. evansi and 416 \nT. vivax   417 \nStomoxys feeds about 9.98 ± (5.5) mL blood when fully engorged and needs on average 4.6 ± (2) 418 \nminutes to fully engorged, the number in parenthesis is the standard deviation of the mean (n=10 419 \nflies). T. evansi survived in various tissues of Stomoxys after immediate disruption of feeding. 420 \nAbout 30% of Stomoxys fed on infected mice showed parasites in the proboscis if feeding was 421 \ninterrupted within one minute. However, more than 80% of the flies fed on infected mice had 422 \nparasites in their crops and gut when feeding was interrupted within one minute.  T. evansi 423 \nsurvived up to 5 hours in the gut of Stomoxys which shows a possibility of delayed transmission 424 \nof trypanosomiasis. In the first three hours, the trypanosomes were very active swimmers, and 425 \ngradually became inactive after 4 hours and were all dead 6 hours post-feeding by flies. We 426 \ndemonstrate that Stomoxys flies transmit T. evansi  through in vivo experiments using laboratory 427 \nmice, with 8.3% (2/ 24) mice with patent parasitemia detected by microscopy by day 7 after 428 \ninfection assays. The wild T. evansi  strain showed moderate virulence as the mice maintained a 429 \npeak of parasitemia (1 × 10 8 trypanosomes/ml blood) for several days and died between the 10 th 430 \nand 14th days, respectively with mild clinical symptoms. Concerning T. vivax, we found longer 431 \nsurvival times in Stomoxys guts as compared to T. evansi, as we found live T. vivax at 16 hours, 432 \nas opposed to 6 hours for T. evansi (Fig. 6). Furthermore, we found a higher transmission success 433 \nrate as compared to T. evansi , as we showed 30% (3/10) transmission was successful. The 434 \nincubation period varied from 6 days to 11 to 34 days in the three T. vivax-infected mice. The 435 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nT. vivax IL 2136 strain exhibited a moderate level of virulence as the infected mice also 436 \nsustained a high parasitemia of 1 × 10 8 trypanosomes/ml blood for several days and died on the 437 \n6th, 8th, and 12 th days. For comparison, we did the same mechanical infection experiment with 438 \nG.pallidipes with 5 trails and all transmitted T.evansi to five mice, demonstrating variation 439 \nbetween Stomoxys spp. and G.pallidipes.   440 \n 441 \nFigure 6: Vector competence of Stomoxys spp. to transmit trypanosomes. (A). Graph showing the 442 \nsurvival of T. evansi and T. vivax in Stomoxys gut (B). The success of infection. 443 \n  444 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n3.5 Natural pathogen transmission assays through feeding bites on 445 \nexperimental mice by field-collected Stomoxys spp. 446 \nWe finally asked if field-collected Stomoxys flies are capable of transmitting pathogens they 447 \nharbored by allowing field-trapped Stomoxys flies to feed on healthy mice. We demonstrated 448 \nwild caught Stomoxys flies are capable of delayed transmission of various pathogens they 449 \nharbored in in vivo  experiments . Stomoxys flies transmitted Theileria mutans (GenBank 450 \nAccession Number, PP918990) into healthy mice  after delayed feeding in the field (Fig. 5B) 451 \nFurthermore, all mice showed Anaplasma spp. infection microscopically only. These pathogens 452 \nhad low virulence as the mice showed no clinical symptoms and no mortality of the mice was 453 \nrecorded for /g3410 120 days. 454 \n4. Discussion 455 \nIn this study we aim to understand Stomoxys -host-pathogens network interaction to get insight 456 \nabout the role of Stomoxys flies in disease transmission dynamics, and how transmission 457 \nnetworks of pathogens-vectors-host are functioning. Out of 18 species of Stomoxys that are found 458 \nglobally 14 of them are found in Africa [1]. We found year-round wide distribution of six species 459 \nof Stomoxys including Stomoxys calcitrans, S. sitiens, S. niger niger, S. niger bilineatus, S. boueti 460 \nand S. taeniatus that varies in their abundance and diversity  in nine regions including in three 461 \ntsetse infested ecologies. With our wider geographic coverage, we reported only six species of 462 \nStomoxys as compared to Mihok et al., 1996 [33] who did trapping from Nairobi National Park 463 \nand reported ten species of Stomoxys. Stomoxys species complexity varies between ecologies, 464 \nnational reserve got more species of Stomoxys as compared to zero grazing ecologies.  For 465 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nexample, species diversity was notably high in Kwale County, most likely due to the availability 466 \nof numerous breeding sites generated by the forested terrain [34]. Kiambu County, where zero 467 \ngrazing is implemented, had a high population density of mainly three species, which is due to 468 \nthe availability of readily available breeding substrate [35]. Isiolo (n = 2,514, 22.20%), Kajiado 469 \n(n= 1,373, 12.12%), and, Homabay (n = 545, 4.81%) counties exhibited a considerably high 470 \npopulation of Stomoxys, which could be attributed to the habitat, which has a semi-arid climate 471 \nthat encourages the growth of the flies (Mavoungou et al., 2017). Marsabit (n = 74, 0.65%) and 472 \nSamburu (n = 26, 0.23%) counties had the least abundance, which could be attributed to the 473 \nenvironment, which is a hot and arid climate that is not friendly to Stomoxys because high 474 \ntemperatures have been reported to cause a drop in the fly population due to reduced survival of 475 \nlarvae and pupae [36].  476 \nTo get insight of the role of Stomoxys  in disease transmission dynamics we need to understand 477 \nthe natural feeding habits of Stomoxys flies from various ecologies. We showed Stomoxys flies 478 \nfeed on a wide range of wild and domestic animals, including humans which is comparable to 479 \ntsetse flies [37], [38] and which also corresponds to prior research findings [2], [3], [39].  From 480 \nour study Stomoxys  need an average of 4 minutes to complete feeding, this may induce host 481 \ndefense and interrupted feeding, which will result in multiple hosts feeding and pathogen 482 \ntransmission [40]. This disruption of Stomoxys-feeding before bloodmeal completion enables the 483 \nvectors to switch to new hosts to continue feeding, which serves as the basis of mechanical 484 \ntransmission of pathogens [5]. In general, the relatively wide variety of feeding suggests that 485 \nStomoxys may take a more opportunistic approach to host selection, potentially responding to the 486 \navailability of susceptible hosts in its environment [39].  We found 225 blood-fed Stomoxys out 487 \nof 3451 showing that most Stomoxys flies caught using traps are often seeking hosts for a blood 488 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nmeal, which is also true for other hematophagous insects [41]. Moreover, blood digestion starts 489 \nmore rapidly in Stomoxys as compared to other hematophagous flies [42]. Thus, the low rate of 490 \nblood meal identifications could be explained by the degradation of host DNA during digestion 491 \nin the fly midgut or furthermore, Stomoxys takes too little blood quantity in the midgut, even in 492 \nstrained animals Stomoxys takes only 10 mg in 4 minutes. Nevertheless, the diversity of hosts we 493 \nsuccessfully identified includes diverse wild, domestic animals and humans (Homo sapiens). The 494 \ndiversity of blood meals can be due to the flies high mobility, their opportunistic feeding 495 \nbehavior, and their frequent feeding habit. Furthermore, trap position and ecologies may 496 \ninfluence the range of host species Stomoxys may feed on. For instance, Mavoungou et al., 2008 497 \ndemonstrated that Stomoxys flies sampled in canopies mainly feed on arboreal species [43]. We 498 \ncan also notice the absence of small mammals (e.g., rodents) within the diversity of host 499 \nvertebrates we identified. This may be explained by the trophic preferences of Stomoxys flies, the 500 \nsame as tsetse for large vertebrates [37], [44], [45].  501 \nSuch a diverse feeding host will expose Stomoxys to diverse pathogens as the host varies in their 502 \npathogen reservoir capacity [46], which is shown in our pathogen network result. The 503 \nepidemiology of African trypanosomiasis includes the biting rate of vectors on infected hosts and 504 \nthe probability of vectors feeding on different hosts as key parameters for understanding the 505 \ntransmission of these infections.  Molecular pathogen screening led to the identification of 506 \nvarious pathogens that showed epidemiological overlap and interactions between hosts and 507 \nvectors in the study area. Concerning the pathogens network, we detected high infection rates of 508 \nAnaplasma spp. both in selected domestic animals and Stomoxys. The detection of the pathogen 509 \nfrom the biting flies confirms the possibility of these flies acquiring and maintaining these 510 \npathogens. Ticks including Rhipicephalus decolaratus, R. microplus , Hyalomma marginatum 511 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nrufipes, R. evertsi , and R. simus  are among the Anaplasma  biological vectors [47]. However, 512 \nScoles et al. (2005) found that stable flies can transmit A. marginale [48]  whereby they 513 \ndemonstrated that the Florida strain of A. marginale which cannot be transmitted by ticks, was 514 \nmore effectively maintained in stable fly mouth parts compared to the tick-transmittable St. 515 \nMaries strain [5]. Similarly, Bargul et al. (2021) demonstrated A. camelii transmission by 516 \nHippobosca camelina , but the same pathogens were not detected in ticks collected from A. 517 \ncamelii-infected camel [49]. According to a report conducted by Oliveira et al., 2011, 518 \nseroprevalence and the presence of tabanids and stable flies are associated with bovine exposure 519 \nto A. marginale, which is widespread in Costa Rican dairy herds [50]. Another pathogen found 520 \nwith high prevalence both in the host and Stomoxys was Theileria spp. and our in vivo  521 \nexperiment demonstrated the successful transmission of Theileria mutans  by Stomoxys flies. 522 \nSimilarly, Theileria DNA was detected in stable flies, in the case of T. orientalis at least for two 523 \nhours after blood-feeding in a study done by [51]. Interestingly, we did not detect anyof 524 \nEhrlichia spp. and Rickettsia spp. in Stomoxys or Glossina but the pathogens were present in 525 \nboth hosts, demonstrating the poor vector competence of Stomoxys for these particular 526 \npathogens.  527 \nIn our in vivo experimental studies, we discovered that T. evansi could actively persist in several 528 \ntissues of Stomoxys flies. Stomoxys flies displayed motile T. evansi  in the proboscis after 529 \nimmediate feeding disruption, which could be observed for up to 5 minutes. These findings 530 \nimply that the mouthparts of Stomoxys species do not promote trypanosome survival for long 531 \n[12]. This may be due to the direct transit of blood to the midgut during eating, which leaves 532 \nvery little blood in the proboscis [52]. Our findings are consistent with those of Sumba et al., 533 \n1998, who confirmed that motile and presumably viable trypanosomes remained in or on the 534 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nproboscis for around 5-7 minutes after feeding was terminated [12]. While in the midgut, we 535 \nestablished T. evansi  could survive for up to 5 hours in the gut of Stomoxys. This survival 536 \ncapability allows T. evansi  to allow for a second possible mechanism of transmission, namely 537 \nregurgitation [13]. Reports in other literature summarized different survival times for various 538 \ntrypanosome species in different biting flies. For instance, Sumba et al. 1998 found that T. 539 \ncongolense could live up to 3 and half hours and T. evansi up to 8 hours in the guts of S. niger  540 \nand S. taeniatus. Additionally, Getahun et al. 2022 found that T. congolense  could live for 3 541 \nhours and trypanozoons for 5 hours in the midgut of S. calcitrans [3] , [12], [13]. Additionally, 542 \nStomoxys flies are efficient mechanical vectors of T.vivax. We showed that T.vivax survived in 543 \nthe Stomoxys gut for a longer period as compared to T.evansi for unknown reasons . The 544 \nexperimental assay showed that in vivo transmission of T. evansi  and T.vivax by Stomoxys  flies 545 \nwas successful with variable success rates. Our findings align with that of Mihok et al., 1995 546 \nwhere it was established S. calcitrans transmits multiple trypanosome species with various 547 \ntransmission rates. A contrary finding by [53] using relevant host cattle- T. vivax-and S. 548 \ncalcitrans interaction reported that S. calcitrans could not transmit T. vivax to cattle this could be 549 \ndue to the transmission experiment design. The authors released the flies into a pen with both 550 \nhealthy and infected animals, flies may be more attracted to an infected host than a healthy one 551 \n[54] in our protocol establishment when both the infected mice and healthy mice were kept 552 \ntogether we found no transmission, despite 20 trials. Furthermore, the interrupting feeding was 553 \ndone after 1.5 minutes, in our protocol establishment experiment when flies were allowed to feed 554 \nfor more than 1 minute, and transferred to a new host, they lost motivation to feed immediately 555 \nand even those that fed later did not transmit, it seems mechanical transmission of trypanosomes 556 \nis time sensitive. Furthermore, mechanical transmission is parasitemia dependent [30] we found 557 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nthe best parasitemia range for mechanical transmission was 1 × 10 8 trypanosomes/mL blood 558 \nrequired The other factor could be Stomoxys species may vary in their vector competence, in our 559 \nexperiment we kept the Stomoxys species complex intact mainly composed of three species as a 560 \nmatrix.  561 \n  562 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\n5. Conclusion 563 \nThe wider geographic distribution, fast reproduction, species diversity, year all presence, diverse 564 \nfeeding habits, the plethora of pathogens harbored, and their successful vectorial capacity of 565 \ntransmitting T. evansi, T. vivax, Anaplasma spp., and Theileria mutans as shown by our in vivo  566 \nexperiments demonstrate Stomoxys flies are significant but overlooked vectors of various 567 \npathogens of livestock. Stomoxys flies may play a significant role in the spread and maintenance 568 \nof T. evansi  and T. vivax in the wide geographic regions of the world. In the future, it is 569 \nimportant to do vector competence experiments using a specific Stomoxys spp. with relevant host 570 \nanimals -pathogens interaction. In our experiment we kept the natural species complex, 571 \ncomposed of mainly S. calcitrans S. niger niger, and S. boueti matrix intact, which means we did 572 \nnot try to separate them by species, in the future it is important to do individual species vector 573 \ncompetence.  574 \nData availability 575 \n All relevant data are in the manuscript and supplementary data. All sequences have been 576 \ndeposited in the NCBI database 577 \nAuthor’s contributions 578 \nJ.W.M; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, 579 \nVisualization, Writing – original draft, Writing – review & editing. J.L.B; Conceptualization, 580 \nsupervision, Writing-review & editing. J.M.O.M; Data curation, Methodology, Formal analysis, 581 \nWriting-review & editing. E.M.N; Data curation, Methodology, Formal analysis, Writing-review 582 \n& editing. S.K.T; Data curation, Methodology, Formal analysis, Writing-review & editing. 583 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nD.K.M; Conceptualization, Funding acquisition, Resources, Supervision, and, Writing-review & 584 \nediting. M.N.G; Conceptualization, Funding acquisition, Resources, Supervision, Writing – 585 \noriginal draft, Writing-review & editing, and Investigation. All authors read and commented on 586 \nthe content. 587 \nFunding 588 \nThis project has received funding from the European Union’s Horizon 2020 research and 589 \ninnovation program under grant agreement no101000467, the acronym ‘COMBAT’ (Controlling 590 \nand Progressively Minimizing the Burden of Animal Trypanosomiasis). Additionally, this 591 \nproject was funded by the Max Planck Institute for Chemical Ecol ogy-icipe partner group. The 592 \nauthors gratefully acknowledge the financial support for this research by the following 593 \norganizations and agencies the Swedish International Development Cooperation Agency (Sida); 594 \nthe Swiss Agency for Development and Cooperation (SDC); the Australian Centre for 595 \nInternational Agricultural Research (ACIAR); the Norwegian Agency for Development 596 \nCooperation (Norad); the German Federal Ministry for Economic Cooperation and Development 597 \n(BMZ); and the Government of the Republic of Kenya. The views expressed herein do not 598 \nnecessarily reflect the official opinion of the donors.” 599 \n 600 \nAcknowledgments 601 \nWe would like to thank Dr. Geoffrey Gimonneau and Dr. Marc Desquesnes for useful discussion 602 \nabout infection experiment protocol development. We acknowledge Dr. Steve Mihok for the 603 \nuseful discussion and his support in Stomoxys identification. James Kabii for his technical 604 \nsupport; John Ngiela, Victor Omondi, and Peter Ahuya helped. We are grateful to Shadrack 605 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint \n\nKibet for designing the map of sampling sites. Joseck Esikuri for supplying mice for 606 \nexperimental pathogen transmission assays. 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