Stomoxysflies (Diptera, Muscidae) are competent vectors of multiple livestock hemopathogens

13 Stomoxys flies are widely distributed and economically significant vectors of various livestock 14 pathogens of veterinary importance. However, the role of Stomoxys spp. in pathogen 15 transmission is poorly understood. Therefore, we studied the feeding patterns of these blood 16 feeders collected from specific locations in Kenya, to identify various vertebrate hosts they fed 17 on, and the livestock hemopathogens they carried, to elucidate their role in pathogens 18 transmission. Our findings show that field-collected Stomoxys flies carried several pathogens 19 including Trypanosoma spp., Anaplasma spp., and Theileria spp. that were also found in the 20 blood of sampled livestock, namely camels and cattle. The findings on blood meal analysis show 21 that Stomoxys flies fed on a variety of domestic and wild vertebrate hosts. We further determined 22 whether Stomoxys spp. are vectors of hemopathogens they harbored by studying the vector 23 competence of S. calcitrans, S. niger niger, and S. boueti species complex, through laboratory 24 and natural experimental in vivo studies. We show that in the process of blood feeding Stomoxys 25 spp. complexes can transmit T. evansi (8.3%) and T. vivax (30%) to Swiss white mice. In 26 addition, field-collected Stomoxy spp. were exposed to healthy mice for blood meal acquisition, 27 and in the process of feeding, they transmitted Theileria mutans and Anaplasma spp. to Swiss 28 white mice (100% infection in the test mice group). All mice infected with both trypanosomes 29 via stomoxys bite died while those infected with Theileria and Anaplasma species did not, 30 demonstrating virulence difference between pathogens. The key finding of this study showing 31 broad feeding host range, cosmopolitan, plethora of pathogens harboured, and efficient vector 32 competence in spreading multiple pathogens suggests profound role of Stomoxys on pathogen 33 transmission and infection prevalence in livestock. 34 Author summary 35 Stomoxys flies are highly adaptable to several ecological settings, including metropolitan areas. 36 In contrast, tsetse flies (genus Glossina), the main biological vectors of African trypanosomes, 37 have a limited distribution to parks and other conservation areas. Stomoxys flies could play a 38 significant role in the spread of animal African trypanosomes, among other hemopathogens, 39 particularly in areas with or without tsetse infestation. Although there have been speculations 40 about the potential role of Stomoxys flies in the transmission of various pathogens, there is lack 41 of data to link hemopathogens occurring in both bloodmeal hosts of Stomoxys and in the flies, 42 and further in vivo experimental studies to confirm the vector competence of Stomoxyine flies. 43 Here, we explored a host and pathogens network, and investigated species diversity at various 44 ecologies, and demonstrated that Stomoxys flies feed on diverse vertebrate hosts and are infected 45 with a plethora of pathogens. We also showed experimentally that they could transmit some of 46 these hemopathogens to mice, for instance, T. vivax, T. evansi, Theileria mutans, and Anaplasma 47 spp. with varying infection success rates. Stomoxys flies could play a significant role in 48 transmitting and spreading various hemopathogens of veterinary importance and possibly 49 maintaining their circulation in livestock, which could explain the occurrence of animal African 50 trypanosomes in the regions outside the tsetse belts. 51 52 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 53 1. Introduction 54 Stomoxys flies are widely distributed globally [1]. They feed on both blood [2], [3] and nectar 55 [4]. These blood feeders pose a significant threat to livestock production worldwide, especially 56 because of their occurrence in wider ecological zones [5]. The economic losses due to livestock 57 infestation by Stomoxys flies are substantial, leading to a significant reduction in meat and milk 58 production [6], due to pathogen infestation. These pathogens cause diseases including; animal 59 trypanosomiasis, Rift Valley fever, African swine fever, lumpy skin disease, and anaplasmosis 60 [3], [7-12]. The annual economic losses in the United States of America (USA) alone due to 61 infestation by a single Stomoxys species, Stomoxys calcitrans – a cosmopolitan species 62 commonly known as stable fly, have been estimated to be around US$ 2.2 billion [8]. The 63 combined economic losses caused by Stomoxys flies infection, nuisance, and treatment costs 64 outside the USA are not well documented at present. 65 Stomoxys feed on their vertebrate hosts' blood once or twice a day [9]. Since the host animals 66 respond to protect themselves against the painful blood-feeding fly bites, they ( Stomoxys) 67 typically do not complete blood-feeding on a single animal [5], [9]. In the event of an interrupted 68 blood meal, the fly can restart feeding on another host by injecting infected saliva before feeding 69 [5]. However, the pathogen transmission mechanism is not clear but could vary from biological, 70 as is the case in the transmission of filariasis [10], [11], to the mechanical transmission of, for 71 instance, animal trypanosomiasis [5], [12],[13]. 72 Animal African trypanosomiasis caused by T. vivax and T. evansi is endemic in most countries in 73 sub-Saharan Africa outside the tsetse belts [3], [12], [14], [15], and also in other regions outside 74 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint Africa, including Asia [16], Latin America [17], and Europe [18]. T. evansi is the causative agent 75 of ‘surra’, and T. vivax, that of nagana – an animal disease that is endemic in large swathes of 76 Africa, Asia, and Latin America, and also present in the Canary Islands (Spain) [22] [23]. 77 However, the establishment, widespread occurrence, maintenance, and circulation of T. vivax 78 and T. evansi particularly in areas outside the tsetse belts is still unclear. 79 Despite the wide geographic distribution and cosmopolitan nature of Stomoxys flies, our 80 knowledge about their blood meal hosts, and the hemopathogens they carry, which will provide 81 insight into ‘vector-host-pathogen’ interactions, and disease transmission dynamics is limited and 82 necessitates compressive research. Thus, in this study, we studied the diversity of Stomoxys spp. 83 collected from various National Reserve to zero grazing ecologies (Fig 1). We conducted a study 84 to assess the vector competence of Stomoxys spp., which refers to their host feeding dynamics, 85 and capacity to become infected and transmit hemopathogens. We show that Stomoxys flies are 86 competent vectors to multiple pathogens and may play a role in the transmission cycles of the 87 pathogens they harbor. 88 2. Materials and methods 89 2.1 Study sites 90 Field sampling took place in nine selected counties in Kenya at various times from October 91 2021 to November 2023. The sampled ecosystems in each County were inhabited by a variety 92 of livestock species, wildlife hosts, and humans. These counties included: Kwale County 93 (4.2572° S, 39.3856° E), Isiolo County (0.3257° N, 38.1961° E), Samburu County (1.7299° N, 94 37.3079° E), Kiambu County (1.0131° S, 36.9051° E), Kajiado County (1.7617° S, 36.0255° 95 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint E), Marsabit County (2.4426° N, 37.9785° E) Homabay County (0.4368° S, 34.2060° E), 96 Laikipia (0.2924° N, 36.8985° E), and Meru County (0.0515° N, 37.6456° E) (Fig 1). 97 98 Figure 1. A map of Kenya showing the sampling sites across the nine selected counties. Th is99 map was created using the open-source software, QGIS v.3. 100 2.2 Ethical approval and animal welfare 101 This study was conducted in strictly adherence to the approved experimental guidelines and102 procedures set forth by the Animal Care and Use Committee (IACUC) of the International103 Centre of Insect Physiology and Ecology, icipe (REF No.: icipeACUC2018-003-2023) and the104 Ethics Review Committee of Pwani University (REF No.: ERC/EXT/002/2020E). F armers and105 pastoralists were sensitized about the research study and how the findings could benefit the106 is nd al he nd he .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint farmers. Sample collection from livestock was done after obtaining verbal consent from farmers, 107 as most of them (camel herders) were unable to read or write. Animal experiments complied with 108 the approved guidelines and mice were handled carefully to ensure minimum distress. 109 2.3 Blood collection and fly trapping 110 2.3.1 Blood collection 111 Blood collection from a total of 452 camels and 124 cattle was done from October 2021 to 112 November 2023. The sample size was determined using our initial data, which indicated an 113 infection rate of 4% in camels and 5.7% in cattle caused by Rickettsiae spp. the lowest 114 prevalence among pathogens. We utilized this information as a basis for calculating the sample 115 size, following the formula; /g1866/g3404 /g2922/g2924/g4666/g2961/g4667 /g2922/g2924/g4666 /g2869/g2879/g2977/g4667 as outlined by [20]. At least 5 mL of blood was drawn 116 from the jugular vein of each animal and collected in vacutainer tubes containing disodium salt 117 of ethylene diamine tetra123 acetate (EDTA) (Plymouth PLG, UK). The blood samples were 118 initially stored in vacutainers at 4°C until the collection was completed which were transferred to 119 cryovials and stored in liquid nitrogen before being transported back to the Nairobi Duduville 120 campus-icipe for molecular identification of pathogens. 121 2.3.2 Trapping of flies 122 Flies were trapped using five red monoconical traps [21]. The traps were placed 150 meters apart 123 and the flies were trapped from nine counties as indicated in (Fig 1). The traps were emptied 124 twice per 24-hour period to avoid flies drying for clear identification and further intended 125 analysis. The flies were later immobilized and preserved in absolute ethanol for further analysis 126 and fresh flies were pinned for morphological identification. Trapping lasted for 5 days. Except 127 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint for Kiambu County, all trapping sites were located in wide woodland savannah ecologies that 128 were not in close proximity to villages. 129 2.4 Morphological identification of Stomoxys spp. 130 Field-collected Stomoxys flies were taxonomically identified to the species level using 131 established taxonomic keys as outlined by [22] . Briefly, the flies were staged under a dissecting 132 Stemi 2000-C microscope (Zeiss, Oberkochen, Germany) to identify key morphological 133 differences. The head frontal index and the distinctive dorso-abdominal patterns on the second 134 and third segments were key distinguishing features in identifying fly sex and species, 135 respectively. Images were captured using a digital microscope connected to an Axio-cam ERc 5s 136 camera (Zeiss). The flies were grouped according to their species and preserved at −20°C for 137 molecular analysis. 138 2.5 Molecular identification of Stomoxys spp. and livestock 139 hemopathogens 140 2.5.1 DNA extraction from blood samples and flies 141 In the initial stages of DNA pre-extraction, the flies were subjected to a 1% sodium hydroxide 142 immersion for 1 minute to eliminate any exogenous material on their bodies. Subsequently, they 143 underwent a 1-minute rinsing procedure with 1 × phosphate-buffered saline (pH = 7.4). Each fly 144 was mechanically homogenized in sterilized 1.5-ml microfuge tubes containing 750 mg of 2.0-145 mm yttria-stabilised zirconium (YSZ) oxide beads (Glen Mills, Clifton, NJ, USA) and 80 µL of 146 1× PBS using a Mini-Beadbeater-16 (BioSpec, Bartlesville, OK, USA) for one minute. Genomic 147 DNA extraction was done using DNeasy blood and tissue kit (Qiagen, Hilden, Germany) 148 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint following the manufacturer’s protocol to obtain DNA from flies, cattle, and camel blood for 149 pathogen screening. The obtained DNA was quantified using a Nanodrop spectrophotometer 150 (Thermo Scientific, Wilmington, DE, United States) by comparing absorbance at 260 and 280 151 nm and later stored at −20°C for molecular work. 152 2.5.2 Molecular identification of Stomoxys spp. and livestock hemopathogens 153 The extracted DNA was used in the molecular characterization of Stomoxys s p p . a s w e l l a s 154 screening livestock hemopathogens. We conducted amplification using genus-specific primers as 155 outlined in (Supplementary Table 1), using the conventional ProFlex PCR systems thermocycler 156 (Applied Biosystems, Foster City, CA, USA). The PCRs were conducted in 10- μ L reaction 157 volumes, consisting of 5 µL nuclease-free water, 2 μ L 5× HOT FIREPol® Blend Master Mix 158 (Solis BioDyne, Tartu, Estonia), 0.5 μ L of 10 µM forward and reverse primers (Supplementary 159 Table 1), and of 2 μ L DNA template. For negative controls, 2 μ L nuclease-free water was used 160 in place of the DNA template. The amplification conditions were set as described by [23]. PCR 161 amplicons were resolved by electrophoresis under 2% ethidium-bromide-stained agarose gel 162 (100 V for 1 hour), followed by DNA visualization by UV-transillumination (Kodak Gel Logic 163 200 Imaging System, CA, USA). PCR amplicons were purified using ExoSAP-IT (Affymetrix, 164 Santa Clara, CA, USA) as per the manufacturer's protocol, and outsourced for Sanger-165 sequencing at Macrogen Inc. (Amsterdam Netherlands). 166 2.5.3 Phylogenetic analysis 167 To get a better understanding of how the various Stomoxys species and livestock hemopathogens 168 are similar and different at the genomic level we performed a phylogenetic tree analysis. The 169 nucleotide sequences acquired in this study were cross-referenced against the known sequences 170 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint in the GenBank of NCBI nr database ( https://www.ncbi.nlm.nih.gov/genbank/). BLAST was 171 used to validate their identity and establish connections with existing deposited sequences [24]. 172 To show the evolutionary relationships, maximum-likelihood phylogenetic trees were 173 constructed using PhyML v. 3.0, employing automatic model selection based on the Akaike 174 information criterion. The tree topologies were estimated through 1000 bootstrap replicates, 175 incorporating nearest-neighbor interchange improvements [25]. The resulting phylogenetic trees 176 were then visualized using FigTree v. 1.4.4[26]. 177 2.5.4 PCR-HRM vertebrate bloodmeal source identification in Stomoxys flies 178 The extracted DNA was used in the analysis of the blood meal source of the fed flies collected in 179 the field. A 10- μ L PCR reaction was carried out, consisting of 1 μ L of DNA template, 6 μ L of 180 nuclease-free water, 2 μ L of 5× HOT FIREpol EvaGreen HRM Mix from Solis BioDyne in 181 Tartu, Estonia, and 0.5 μ M of both forward and reverse primers (Supplementary Table 1). 182 Positive control vertebrate host samples used as reference include; cow ( Bos taurus ), camel 183 (Camelus dromedarius ), donkey ( Equus asinus), warthog (Phacochoerus africanus ), African 184 buffalo (Syncerus caffer ), goat ( Capra aegagrus hircus), waterbuck ( Kobus ellipsiprymnus ) 185 elephant (Loxodonta africana), sheep (Ovis aries), reticulated giraffe (Giraffa reticulata ), lesser 186 kudu ( Tragelaphus imberbis ), cheetah ( Acinonyx jubatus ), zebra (Equus quagga ), baboon 187 (Papio), gerenuk ( Litocranius walleri), hartebeest ( Alcelaphus buselaphus), reedbuck ( Redunca 188 redunca), hyena (Crocuta crocuta ), gazelle ( Gazella gazella ), impala ( Aepyceros melampus ), 189 lion ( Panthera leo), bongo ( Tragelaphus eurycerus ) and human ( Homo sapiens ). The PCR 190 thermal cycling conditions for primer were set as described by [3]. After PCR amplification, 191 High-Resolution Melting (HRM) analysis was conducted within normalized temperature ranges, 192 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint from 65°C to 78°C and 88°C to 95°C. The distinct melt curve profiles of the samples were 193 compared against reference standards. 194 2.6 Experimental infection assays to determine the vector 195 competence of Stomoxys to transmit T. evansi and T.vivax 196 2.6.1 Experimental animals 197 Swiss White Mice ( Mus musculus) obtained from icipe’s Animal Rearing and Quarantine Unit 198 (ARQU) were used for the infection experiment. Both male and female mice used for 199 experiments were about 6 – 8 weeks old. Each mouse weighed about 24 – 29 g live body weight. 200 The mice were housed under normal conditions in standard mouse cages and their diet primarily 201 comprised of commercial pellets (Unga® Kenya Ltd) and water, which was provided ad libitum. 202 The mice were kept in a mice experimental room that was free from biting flies. The 203 experimental mice used in pathogen transmission studies were not immune suppressed. 204 2.6.2 Establishment of laboratory colonies of Stomoxys spp. 205 Stomoxys spp. of both sexes were trapped from both Gatundu (1.0131° S, 36.9051° E) and 206 around icipe-Duduville campus (1.2921° S, 36.8219° E) and taken to icipe’s insects rearing 207 units. The mixed species of Stomoxys flies were maintained in 75 cm × 60 cm × 45 cm perspex 208 cages (Astariglas ®, Indonesia) and fed once daily between 9 am – 11 am warm defibrinated 209 bovine blood obtained from a local slaughterhouse (Choice meats, Nairobi) and supplemented 210 with 10% glucose and Parthenium hysterophorus flowers [27]. The temperature and humidity in 211 the rearing room were kept at 25 ± 1 °C and RH 50 ± 5%, respectively with a 12:12 light/dark 212 photoperiod. Sheep dung was used as an oviposition substrate [28] and developed pupae were 213 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint picked and transferred to another cage for emergence. The newly emerged or teneral flies were 214 used for experimental infection assays. 215 2.6. 3 Multiplication of T. evansi and T. vivax isolates in donor mice 216 The strain of Trypanosoma used in this study was T. evansi, which was isolated from a naturally 217 infected camel (Camelus dromedarius) from Marsabit County, and T. vivax IL 2136 which was 218 taken from icipe’s trypanosomes bio-bank. They were let to thaw after which parasitemia was 219 checked using microscopy (Zeiss Primo Star Binocular Microscope, Zeiss, Oberkochen, 220 Germany) with a ×40 magnification to ensure the viability of the stabilates before each 221 inoculation. 200 µL of each stabilate was then inoculated to the mice through the intraperitoneal 222 route 223 2.6.4 Monitoring parasitemia levels in donor mice 224 The mice were monitored daily, three days after pathogen inoculation. Briefly, a drop of blood 225 from the snipping of the mouse tail using a blood lancet was placed on a clean slide and covered 226 using a coverslip as a wet blood smear and examined under a microscope (Seamer et al., 1993). 227 The parasitemia score was estimated which correlated to a score sheet, as described by [29]. The 228 period taken from the day post inoculation (dpi) to the first appearance of trypanosomes in blood 229 was recorded for all mice. This was done until the required parasitemia was achieved (1 × 10 8 230 trypanosome/mL blood). 231 2.6.5 Determination of T. evansi and T. vivax survival rates in Stomoxys fly 232 Given that mechanical transmission of trypanosome parasitemia is known to be dose-dependent 233 [30], our experimental design included the testing of two doses that are typically encountered in 234 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint natural infection [3] approximately 1 × 10 8 trypanosomes/ml blood and 5 × 10 8 235 trypanosomes/mL blood. Following the successful induction of high parasitemia, the next step 236 involved the extraction of whole blood from the donor mouse. This was done by sacrificing the 237 mouse in accordance to the standard protocol defined by the Institutional Animal Care and Use 238 Committee (IACUC). Fino-Ject disposable syringe 5 mL/cc with needle was used for blood 239 collection by cardiac puncture [31] which resulted to 200 μ L of blood. The parasitemia was 240 checked microscopically, to ensure the blood still had enough parasite concentration. The 241 infected blood was then carefully diluted in clean pre-warmed defibrinated bovine blood 242 collected from the slaughterhouse (Choice meats, Nairobi) at a 1:1 ratio. The resulting blood 243 mixture, approximately 400 µL, was applied onto clean cotton placed in a petri dish. A total of 244 60 teneral Stomoxys flies which were starved for 24 hours were placed in a clear acrylic plastic 245 cage with dimensions of 10 × 10 × 15 cm, which was made of a 6-mm-thick perspex sheet from 246 Astariglas® in Indonesia. The flies were fed on the blood-soaked cotton wool that was provided 247 on a petri dish. Following a feeding period of five minutes, until all flies were fully engorged, the 248 infection and spread of trypanosomes within the Stomoxys flies were monitored at various time 249 points post-feeding, beginning one hour after the feeding event and continuing at each 250 subsequent hour. To analyze the distribution and prevalence of the parasite within the bodies of 251 the Stomoxys flies, we conducted dissections of various body parts, including the mouthparts, 252 crop, and gut. At least five insects per exposure time were examined after immediate interrupted 253 feeding. 254 2.6.6. Experimental trials of in vivo transmission of T. evansi and T. vivax 255 Various protocols were tested in the in vivo transmission trials for optimization. At first, after the 256 successful induction of high parasitemia within the donor mouse, the donor mouse and recipient 257 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint mouse were restrained using a restrainer which was made of stainless-steel woven wire mesh 258 with measurements of 0.9 mm per hole and a 400 µm wire diameter, and placed in a 10 ×10 ×15-259 cm cage made of 6-mm (thick) perspex clear acrylic plastic sheet (Astariglas®, Indonesia). The 260 teneral flies n = 20 were introduced and the flies were disturbed by the observer to allow them to 261 move from donor to recipient mice. Unfortunately, after more than 20 trials using this 262 experimental method, we did not get any results. We optimized our experiment whereby, once a 263 donor mouse with high parasitemia was achieved, the donor mouse and recipient mouse were 264 restrained using a restrainer which was made of stainless-steel woven wire mesh with 265 measurements of 0.9 mm per hole and a 400 µm wire diameter, and both were placed in separate 266 10 ×10 ×15-cm cage made of 6-mm (thick) perspex clear acrylic plastic sheet (Astariglas®, 267 Indonesia). One-day-old teneral flies which were not fed on any blood meal where n=20 per 268 experiment, were released in the cage of the donor mouse and allowed to feed for ≤ 1 minute. The 269 timing was done once the proboscis had pierced the mouse’s skin to ensure feeding had started 270 and the flies ingested blood from the infected mouse. This was followed by disrupted feeding 271 where only the fed flies were individually picked using a respirator and transferred to the next 272 cage containing restrained recipient healthy mice and flies were allowed to complete their blood 273 meal until fully engorged (Fig 2). The flies were subsequently dissected to confirm the presence 274 of parasites in their gut to assess the parasite-feeding success rate. The restrained recipient mouse 275 was then released into their standard mouse cages and monitored daily after three days post-276 infection. 277 278 279 280 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 281 282 Figure 2: Laboratory in vivo transmission of T. evansi and T. vivax experimental design. 283 2.6.7 Screening for T. evansi and T. vivax in the recipient mice by PCR 284 After three days post-infected fly bites a combination of microscopy and molecular methods 285 were used to confirm the presence of parasites in the blood of infected animals for up to 30 days 286 post-infection (dpi). For microscopy, it was done as described above, daily. Molecular screening 287 was done by collecting blood samples from snipping the mice tails and collecting them in 1.5 ml 288 eppendorf tubes which contained 80 μ L 1× PBS buffer, pH = 7.4. Blood collection was done 289 after every two days. This was followed by total DNA extraction using a DNeasy blood and 290 tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. PCR, gel 291 electrophoresis, and gene sequencing were performed as described above 292 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 2.7 Determination of vector competence through field bioassay 293 Fly trapping was done as described above in various study sites. The traps were emptied after 6 294 hours and the flies were put in 10 × 10 × 15-cm cage made of 6-mm (thick) perspex clear acrylic 295 plastic sheet (Astariglas®, Indonesia). The recipient mice were restrained using the restrainer that 296 was made of stainless-steel woven wire mesh with measurements of 0.9 mm per hole and a 400 297 µm wire diameter and released into the cage. The flies were left to feed for 30 minutes before 298 releasing the mice. This was followed by daily evaluation of pathogens in the recipient mice 299 through microscopy and molecular screening as described above. 300 2.8 Data analysis 301 The Shannon diversity index (H) was utilized to define the diversity index of biting flies among 302 study counties and was calculated using R statistical software (R version 4.4.1.). Estimated 303 minimum infection rates (MIRs) of pathogens obtained for the flies were calculated as the 304 number of positive per total number of flies tested ×100. Graphs were visualized using GraphPad 305 software (GraphPad Software, Inc, USA). The bipartite R package's interaction network [32] 306 visualized the vectors blood-feeding behavior and pathogen interactions between hosts and 307 vectors which was generated by R statistical software (R version 4.4.1.). An Upset plot displayed 308 the number of flies feeding on specific animal species and those containing bloodmeals from one 309 or more host species and was plotted using R statistical software (R version 4.4.1.). Transmission 310 rates of the experimental infection assays were performed by calculating the number of infected 311 mice per total number of transmission trials done ×100. 312 313 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 3. Results 314 3.1 Stomoxys species diversity relative abundance is ecology 315 dependent 316 Diverse Stomoxys species were trapped throughout the year . A total of 11,323 adult Stomoxys 317 flies were collected from various sites, from National Reserve including; Shimba Hills National 318 Reserve and Nguruman Conservation Reserve, to zero grazing ecologies and identified 319 morphologically using specific keys to the species level according to [22] as S. calcitrans, S. 320 sitiens, S. niger niger, S. niger bilineatus, S. boueti, and S. taeniatus ( Fig 3A). The sampling 321 sites varied in species richness with some having only three species, while others had up to six. 322 S. calcitrans was identified in all study sites while S. taeniatus was only found in Kajiado 323 County. S. calcitrans exhibited a body appearance characterized by three dark spots on each of 324 the second and third segments. S. sitiens abdominal segments resemble that of S. calcitrans but 325 the dark spots are more transversely elongated. S. niger niger appears to have grey coloration 326 with well-defined and dark stripes on abdominal segments. The dorsal view of S. boueti appears 327 to have an indistinct dark abdomen and is much smaller in size. S. niger bilineatus has a 328 brownish appearance with the abdominal segment having defined the dark stripes in a dorsal 329 view. S. taeniatus has a brighter golden brown to almost yellowish color and it is larger than the 330 other species (Fig 3A). Our molecular taxonomy using CO1 (Cytochrome Oxidase I gene) DNA 331 sequence confirmed the morphological identification of those samples clustered distinctly and 332 with previously documented DNA sequences demonstrating they are different species (Fig 3B). 333 New CO1 sequences including S. boueti, (GenBank Accession number, PP587243) and S. 334 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint taeniatus (GenBank Accession number PQ203543) were deposited that were not available in the 335 NCBI. 336 Kajiado followed by Kwale County recorded the highest number of Stomoxys species diversity, 337 six and five species, respectively (Fig 3C) The Shannon diversity index shows the varying levels 338 of Stomoxys species diversity (Supplementary Table 2) across Kenyan counties with Kwale 339 County having the highest Shannon diversity index of 1.36 and Meru County having the lowest 340 Shannon diversity index of 0.29. Overall, the species distribution showed that S. calcitrans was 341 the dominating species, except in Kajiado and Homabay counties, which was accounted for by 342 (n= 5,547, 49%), followed by S. niger niger, (n= 2,938, 25.95%), S. boueti (n= 1,471, 12.99%), 343 S. niger bilineatus (n= 778, 6.87%), S. sitiens (n= 495, 4.37%), and finally S. taeniatus (n=94, 344 0.83%). Using one of our sites (Kiambu County) we studied the seasonal dynamics of Stomoxys 345 flies. Stomoxys flies were caught all year round with seasonal variation (Fig 3D). The abundance 346 of Stomoxys increase with the rainfall data, there was an annual rainfall of 674 mm with a 347 monthly average of 56 mm in the study County during the study period. 348 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 349 Figure 3: Stomoxys flies morphological identification, molecular characterization, species 350 diversity, and seasonality (A) Image showing the dorsal and lateral view demonstrating the 351 distinct morphological features of the six Stomoxys species encountered in various study sites (B) 352 Neighbour-joining tree constructed based on aligned sequences of CO1 tree showing the 353 relatedness of the various Stomoxys species. (C) The species diversity and their relative 354 abundance in various ecologies. (D). Seasonality of Stomoxys at Gatundu site from Kiambu 355 County. 356 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 3.2 Stomoxys flies blood meal host network analysis demonstrates 357 Stomoxys flies feed on a wide host range 358 Stomoxys flies feed on diverse wild and domestic animals. In total 225 fed Stomoxys flies were 359 successfully identified to analyze the Stomoxys-host feeding network. Fifteen distinct vertebrate 360 blood-meal hosts were identified, including cattle ( Bos taurus), camel ( Camelus dromedarius), 361 warthog ( Phacochoerus africanus ), African buffalo (Syncerus caffer ), goat (Capra aegagrus 362 hircus), waterbuck ( Kobus ellipsiprymnus) elephant ( Loxodonta africana), sheep ( Ovis aries ), 363 reticulated giraffe (Giraffa reticulata ), zebra (Equus quagga ), baboon ( Papio), reedbuck 364 (Redunca redunca), gazelle (Gazella gazella), impala (Aepyceros melampus), and human (Homo 365 sapiens) (Fig. 4). S. calcitrans had the most diverse blood meal hosts followed by S. boueti and 366 lastly S. niger niger (Fig 4). We observed the diversity of blood meal sources is dependent. 367 Wildlife conservation (Shimba Hills National Reserve) had the most variety of identified blood-368 meal hosts. Kiambu and Meru counties had the lowest host diversity due to zero grazing in 369 regions where Stomoxys were trapped, resulting in a limited number of hosts mostly only cattle. 370 In general, cattle were the most detected and most preferred host across all species S. calcitrans 371 (n=65/265), S. niger niger (11/205), and S. boueti (n=13/205) (Fig.4). Multiple host feeding was 372 also revealed in some flies where HRM melt curves revealed two peaks that matched the 373 standard reference (Fig. 4). This was most commonly found in livestock, including cattle and 374 goats, cattle and sheep, cattle and camels, and detected once in wildlife, including waterbuck and 375 buffalo may be due to interrupted feeding before completion (Supplementary Table 3). 376 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 377 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint Figure 4: Identification of vertebrate hosts from bloodmeal analysis of Stomoxys spp. (A ) A 378 bipartite network graph showing feeding interactions between hosts and blood-fed Stomoxys spp. 379 The top bar indicates hosts while the bottom bar indicates the Stomoxys spp. while the lines 380 illustrate the interaction. The size of a bar reflects the number of blood-fed Stomoxys (if it is a 381 bottom bar) or the number of mammalian hosts that were fed on the vector (if it is a top bar). The 382 thickness of a line corresponds to the number of blood-fed hosts detected in the various Stomoxys 383 spp. (B) An Upset plot showing the total number of hosts fed per species and also multiple host 384 feeding. 385 3.3 Stomoxys flies and domestic animals harbor various 386 hemopathogens 387 Another data required to elucidate the role of Stomoxys for various pathogen transmission 388 dynamics besides blood meal source is to study the pathogens network between Stomoxys and 389 some of the most preferred host animals they feed on. Various pathogens were detected both in 390 the blood of livestock which were also common in the Stomoxys flies. Anaplasma sp., Theileria 391 sp., and Trypanosoma sp. were shared across all analyzed domestic animal hosts and Stomoxys 392 flies. Ehrlichia sp. and Rickettsiae sp. were detected in both camels and cattle. Coxiella burnetti 393 was only detected in camels. In camels (n= 452), Anaplasma sp., was the most prevalent 394 pathogen affecting 64.7% of the camels. Trypanosoma sp. was detected in 12.3% and Ehrlichia 395 sp. in 12.2% of camels sampled. Coxiella burnetti was found in 6% of the camels, while 396 Rickettsiae sp. was the least detected in 4% of the camels. We did not detect any 397 Theileria/Babesia spp. in camels. Additionally, in cattle out of n=124, we found a high 398 prevalence of Theileria/Babesia sp. with 56.6% and Anaplasma sp. in 54.1%. Trypanosoma sp. 399 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint was detected in 10% of cattle, Rickettsiae sp. in 5.7% of the cattle and Ehrlichia sp. was the least400 prevalent, found in only 1.6% of cattle. Among all hosts, sheep had the least pathogen diversity401 with only Theileria/Babesia sp. being detected in 4% of the sheep. A total of 3,451 Stomoxys402 were screened for pathogen diversity across the study counties. Among these, Anaplasma sp.,403 was the most frequently detected with 49.1%, 19.1% having Theileria/Babesia sp. and 9.1%404 having Trypanosoma sp. For comparison, Glossina pallidipes (n=1000) co- inhabit with405 Stomoxys had pathogen prevalence of Trypanosoma sp., Anaplasma sp., and Theileria/Babesia406 sp., which were detected in 7.5%, 4%, and 11% of the flies, respectively. 407 408 Figure 5: Pathogen diversity in host animals and ve ctors from various sites through molecular409 screening and neighbor-joining tree showing pathogens. (A) A bipartite network graph showing410 pathogen interactions between hosts (camel, cattle, and sheep) and vectors Stomoxys sp. and411 Glossina sp. The top bar indicates pathogens while the bottom bar indicates the hosts and vectors412 while the lines i llustrate the interaction. The thickness of a line corresponds to the number of413 ast ity ys p., % ith sia lar ng nd rs of .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint pathogens detected in either the hosts or vectors. (B) Neighbor-joining tree showing pathogens 414 from host animals and vectors. 415 3.4 Stomoxys flies are competent mechanical vectors of T. evansi and 416 T. vivax 417 Stomoxys feeds about 9.98 ± (5.5) mL blood when fully engorged and needs on average 4.6 ± (2) 418 minutes to fully engorged, the number in parenthesis is the standard deviation of the mean (n=10 419 flies). T. evansi survived in various tissues of Stomoxys after immediate disruption of feeding. 420 About 30% of Stomoxys fed on infected mice showed parasites in the proboscis if feeding was 421 interrupted within one minute. However, more than 80% of the flies fed on infected mice had 422 parasites in their crops and gut when feeding was interrupted within one minute. T. evansi 423 survived up to 5 hours in the gut of Stomoxys which shows a possibility of delayed transmission 424 of trypanosomiasis. In the first three hours, the trypanosomes were very active swimmers, and 425 gradually became inactive after 4 hours and were all dead 6 hours post-feeding by flies. We 426 demonstrate that Stomoxys flies transmit T. evansi through in vivo experiments using laboratory 427 mice, with 8.3% (2/ 24) mice with patent parasitemia detected by microscopy by day 7 after 428 infection assays. The wild T. evansi strain showed moderate virulence as the mice maintained a 429 peak of parasitemia (1 × 10 8 trypanosomes/ml blood) for several days and died between the 10 th 430 and 14th days, respectively with mild clinical symptoms. Concerning T. vivax, we found longer 431 survival times in Stomoxys guts as compared to T. evansi, as we found live T. vivax at 16 hours, 432 as opposed to 6 hours for T. evansi (Fig. 6). Furthermore, we found a higher transmission success 433 rate as compared to T. evansi , as we showed 30% (3/10) transmission was successful. The 434 incubation period varied from 6 days to 11 to 34 days in the three T. vivax-infected mice. The 435 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint T. vivax IL 2136 strain exhibited a moderate level of virulence as the infected mice also 436 sustained a high parasitemia of 1 × 10 8 trypanosomes/ml blood for several days and died on the 437 6th, 8th, and 12 th days. For comparison, we did the same mechanical infection experiment with 438 G.pallidipes with 5 trails and all transmitted T.evansi to five mice, demonstrating variation 439 between Stomoxys spp. and G.pallidipes. 440 441 Figure 6: Vector competence of Stomoxys spp. to transmit trypanosomes. (A). Graph showing the 442 survival of T. evansi and T. vivax in Stomoxys gut (B). The success of infection. 443 444 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 3.5 Natural pathogen transmission assays through feeding bites on 445 experimental mice by field-collected Stomoxys spp. 446 We finally asked if field-collected Stomoxys flies are capable of transmitting pathogens they 447 harbored by allowing field-trapped Stomoxys flies to feed on healthy mice. We demonstrated 448 wild caught Stomoxys flies are capable of delayed transmission of various pathogens they 449 harbored in in vivo experiments . Stomoxys flies transmitted Theileria mutans (GenBank 450 Accession Number, PP918990) into healthy mice after delayed feeding in the field (Fig. 5B) 451 Furthermore, all mice showed Anaplasma spp. infection microscopically only. These pathogens 452 had low virulence as the mice showed no clinical symptoms and no mortality of the mice was 453 recorded for /g3410 120 days. 454 4. Discussion 455 In this study we aim to understand Stomoxys -host-pathogens network interaction to get insight 456 about the role of Stomoxys flies in disease transmission dynamics, and how transmission 457 networks of pathogens-vectors-host are functioning. Out of 18 species of Stomoxys that are found 458 globally 14 of them are found in Africa [1]. We found year-round wide distribution of six species 459 of Stomoxys including Stomoxys calcitrans, S. sitiens, S. niger niger, S. niger bilineatus, S. boueti 460 and S. taeniatus that varies in their abundance and diversity in nine regions including in three 461 tsetse infested ecologies. With our wider geographic coverage, we reported only six species of 462 Stomoxys as compared to Mihok et al., 1996 [33] who did trapping from Nairobi National Park 463 and reported ten species of Stomoxys. Stomoxys species complexity varies between ecologies, 464 national reserve got more species of Stomoxys as compared to zero grazing ecologies. For 465 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint example, species diversity was notably high in Kwale County, most likely due to the availability 466 of numerous breeding sites generated by the forested terrain [34]. Kiambu County, where zero 467 grazing is implemented, had a high population density of mainly three species, which is due to 468 the availability of readily available breeding substrate [35]. Isiolo (n = 2,514, 22.20%), Kajiado 469 (n= 1,373, 12.12%), and, Homabay (n = 545, 4.81%) counties exhibited a considerably high 470 population of Stomoxys, which could be attributed to the habitat, which has a semi-arid climate 471 that encourages the growth of the flies (Mavoungou et al., 2017). Marsabit (n = 74, 0.65%) and 472 Samburu (n = 26, 0.23%) counties had the least abundance, which could be attributed to the 473 environment, which is a hot and arid climate that is not friendly to Stomoxys because high 474 temperatures have been reported to cause a drop in the fly population due to reduced survival of 475 larvae and pupae [36]. 476 To get insight of the role of Stomoxys in disease transmission dynamics we need to understand 477 the natural feeding habits of Stomoxys flies from various ecologies. We showed Stomoxys flies 478 feed on a wide range of wild and domestic animals, including humans which is comparable to 479 tsetse flies [37], [38] and which also corresponds to prior research findings [2], [3], [39]. From 480 our study Stomoxys need an average of 4 minutes to complete feeding, this may induce host 481 defense and interrupted feeding, which will result in multiple hosts feeding and pathogen 482 transmission [40]. This disruption of Stomoxys-feeding before bloodmeal completion enables the 483 vectors to switch to new hosts to continue feeding, which serves as the basis of mechanical 484 transmission of pathogens [5]. In general, the relatively wide variety of feeding suggests that 485 Stomoxys may take a more opportunistic approach to host selection, potentially responding to the 486 availability of susceptible hosts in its environment [39]. We found 225 blood-fed Stomoxys out 487 of 3451 showing that most Stomoxys flies caught using traps are often seeking hosts for a blood 488 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint meal, which is also true for other hematophagous insects [41]. Moreover, blood digestion starts 489 more rapidly in Stomoxys as compared to other hematophagous flies [42]. Thus, the low rate of 490 blood meal identifications could be explained by the degradation of host DNA during digestion 491 in the fly midgut or furthermore, Stomoxys takes too little blood quantity in the midgut, even in 492 strained animals Stomoxys takes only 10 mg in 4 minutes. Nevertheless, the diversity of hosts we 493 successfully identified includes diverse wild, domestic animals and humans (Homo sapiens). The 494 diversity of blood meals can be due to the flies high mobility, their opportunistic feeding 495 behavior, and their frequent feeding habit. Furthermore, trap position and ecologies may 496 influence the range of host species Stomoxys may feed on. For instance, Mavoungou et al., 2008 497 demonstrated that Stomoxys flies sampled in canopies mainly feed on arboreal species [43]. We 498 can also notice the absence of small mammals (e.g., rodents) within the diversity of host 499 vertebrates we identified. This may be explained by the trophic preferences of Stomoxys flies, the 500 same as tsetse for large vertebrates [37], [44], [45]. 501 Such a diverse feeding host will expose Stomoxys to diverse pathogens as the host varies in their 502 pathogen reservoir capacity [46], which is shown in our pathogen network result. The 503 epidemiology of African trypanosomiasis includes the biting rate of vectors on infected hosts and 504 the probability of vectors feeding on different hosts as key parameters for understanding the 505 transmission of these infections. Molecular pathogen screening led to the identification of 506 various pathogens that showed epidemiological overlap and interactions between hosts and 507 vectors in the study area. Concerning the pathogens network, we detected high infection rates of 508 Anaplasma spp. both in selected domestic animals and Stomoxys. The detection of the pathogen 509 from the biting flies confirms the possibility of these flies acquiring and maintaining these 510 pathogens. Ticks including Rhipicephalus decolaratus, R. microplus , Hyalomma marginatum 511 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint rufipes, R. evertsi , and R. simus are among the Anaplasma biological vectors [47]. However, 512 Scoles et al. (2005) found that stable flies can transmit A. marginale [48] whereby they 513 demonstrated that the Florida strain of A. marginale which cannot be transmitted by ticks, was 514 more effectively maintained in stable fly mouth parts compared to the tick-transmittable St. 515 Maries strain [5]. Similarly, Bargul et al. (2021) demonstrated A. camelii transmission by 516 Hippobosca camelina , but the same pathogens were not detected in ticks collected from A. 517 camelii-infected camel [49]. According to a report conducted by Oliveira et al., 2011, 518 seroprevalence and the presence of tabanids and stable flies are associated with bovine exposure 519 to A. marginale, which is widespread in Costa Rican dairy herds [50]. Another pathogen found 520 with high prevalence both in the host and Stomoxys was Theileria spp. and our in vivo 521 experiment demonstrated the successful transmission of Theileria mutans by Stomoxys flies. 522 Similarly, Theileria DNA was detected in stable flies, in the case of T. orientalis at least for two 523 hours after blood-feeding in a study done by [51]. Interestingly, we did not detect anyof 524 Ehrlichia spp. and Rickettsia spp. in Stomoxys or Glossina but the pathogens were present in 525 both hosts, demonstrating the poor vector competence of Stomoxys for these particular 526 pathogens. 527 In our in vivo experimental studies, we discovered that T. evansi could actively persist in several 528 tissues of Stomoxys flies. Stomoxys flies displayed motile T. evansi in the proboscis after 529 immediate feeding disruption, which could be observed for up to 5 minutes. These findings 530 imply that the mouthparts of Stomoxys species do not promote trypanosome survival for long 531 [12]. This may be due to the direct transit of blood to the midgut during eating, which leaves 532 very little blood in the proboscis [52]. Our findings are consistent with those of Sumba et al., 533 1998, who confirmed that motile and presumably viable trypanosomes remained in or on the 534 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint proboscis for around 5-7 minutes after feeding was terminated [12]. While in the midgut, we 535 established T. evansi could survive for up to 5 hours in the gut of Stomoxys. This survival 536 capability allows T. evansi to allow for a second possible mechanism of transmission, namely 537 regurgitation [13]. Reports in other literature summarized different survival times for various 538 trypanosome species in different biting flies. For instance, Sumba et al. 1998 found that T. 539 congolense could live up to 3 and half hours and T. evansi up to 8 hours in the guts of S. niger 540 and S. taeniatus. Additionally, Getahun et al. 2022 found that T. congolense could live for 3 541 hours and trypanozoons for 5 hours in the midgut of S. calcitrans [3] , [12], [13]. Additionally, 542 Stomoxys flies are efficient mechanical vectors of T.vivax. We showed that T.vivax survived in 543 the Stomoxys gut for a longer period as compared to T.evansi for unknown reasons . The 544 experimental assay showed that in vivo transmission of T. evansi and T.vivax by Stomoxys flies 545 was successful with variable success rates. Our findings align with that of Mihok et al., 1995 546 where it was established S. calcitrans transmits multiple trypanosome species with various 547 transmission rates. A contrary finding by [53] using relevant host cattle- T. vivax-and S. 548 calcitrans interaction reported that S. calcitrans could not transmit T. vivax to cattle this could be 549 due to the transmission experiment design. The authors released the flies into a pen with both 550 healthy and infected animals, flies may be more attracted to an infected host than a healthy one 551 [54] in our protocol establishment when both the infected mice and healthy mice were kept 552 together we found no transmission, despite 20 trials. Furthermore, the interrupting feeding was 553 done after 1.5 minutes, in our protocol establishment experiment when flies were allowed to feed 554 for more than 1 minute, and transferred to a new host, they lost motivation to feed immediately 555 and even those that fed later did not transmit, it seems mechanical transmission of trypanosomes 556 is time sensitive. Furthermore, mechanical transmission is parasitemia dependent [30] we found 557 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint the best parasitemia range for mechanical transmission was 1 × 10 8 trypanosomes/mL blood 558 required The other factor could be Stomoxys species may vary in their vector competence, in our 559 experiment we kept the Stomoxys species complex intact mainly composed of three species as a 560 matrix. 561 562 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint 5. Conclusion 563 The wider geographic distribution, fast reproduction, species diversity, year all presence, diverse 564 feeding habits, the plethora of pathogens harbored, and their successful vectorial capacity of 565 transmitting T. evansi, T. vivax, Anaplasma spp., and Theileria mutans as shown by our in vivo 566 experiments demonstrate Stomoxys flies are significant but overlooked vectors of various 567 pathogens of livestock. Stomoxys flies may play a significant role in the spread and maintenance 568 of T. evansi and T. vivax in the wide geographic regions of the world. In the future, it is 569 important to do vector competence experiments using a specific Stomoxys spp. with relevant host 570 animals -pathogens interaction. In our experiment we kept the natural species complex, 571 composed of mainly S. calcitrans S. niger niger, and S. boueti matrix intact, which means we did 572 not try to separate them by species, in the future it is important to do individual species vector 573 competence. 574 Data availability 575 All relevant data are in the manuscript and supplementary data. All sequences have been 576 deposited in the NCBI database 577 Author’s contributions 578 J.W.M; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, 579 Visualization, Writing – original draft, Writing – review & editing. J.L.B; Conceptualization, 580 supervision, Writing-review & editing. J.M.O.M; Data curation, Methodology, Formal analysis, 581 Writing-review & editing. E.M.N; Data curation, Methodology, Formal analysis, Writing-review 582 & editing. S.K.T; Data curation, Methodology, Formal analysis, Writing-review & editing. 583 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint D.K.M; Conceptualization, Funding acquisition, Resources, Supervision, and, Writing-review & 584 editing. M.N.G; Conceptualization, Funding acquisition, Resources, Supervision, Writing – 585 original draft, Writing-review & editing, and Investigation. All authors read and commented on 586 the content. 587 Funding 588 This project has received funding from the European Union’s Horizon 2020 research and 589 innovation program under grant agreement no101000467, the acronym ‘COMBAT’ (Controlling 590 and Progressively Minimizing the Burden of Animal Trypanosomiasis). Additionally, this 591 project was funded by the Max Planck Institute for Chemical Ecol ogy-icipe partner group. The 592 authors gratefully acknowledge the financial support for this research by the following 593 organizations and agencies the Swedish International Development Cooperation Agency (Sida); 594 the Swiss Agency for Development and Cooperation (SDC); the Australian Centre for 595 International Agricultural Research (ACIAR); the Norwegian Agency for Development 596 Cooperation (Norad); the German Federal Ministry for Economic Cooperation and Development 597 (BMZ); and the Government of the Republic of Kenya. The views expressed herein do not 598 necessarily reflect the official opinion of the donors.” 599 600 Acknowledgments 601 We would like to thank Dr. Geoffrey Gimonneau and Dr. Marc Desquesnes for useful discussion 602 about infection experiment protocol development. We acknowledge Dr. Steve Mihok for the 603 useful discussion and his support in Stomoxys identification. James Kabii for his technical 604 support; John Ngiela, Victor Omondi, and Peter Ahuya helped. We are grateful to Shadrack 605 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint Kibet for designing the map of sampling sites. Joseck Esikuri for supplying mice for 606 experimental pathogen transmission assays. Caroline Muya helped in handling the administrative 607 aspects relating to this study. 608 Conflict of interest 609 The authors declare no conflict of interest. 610 611 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted October 8, 2024. ; https://doi.org/10.1101/2024.10.07.611962doi: bioRxiv preprint

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