Accepted Manuscript

Hyaluronidase activity in the salivary glands of tabanid

Vera Volfova, Viktorie Tothova, Petr Volf

PII: S0965-1748(16)30035-2 DOI: 10.1016/j.ibmb.2016.03.007 Reference: IB 2825

To appear in: Biochemistry and Molecular Biology

Received Date: 13 February 2016 Revised Date: 23 March 2016 Accepted Date: 23 March 2016

Please cite this article as: Volfova, V., Tothova, V., Volf, P., Hyaluronidase activity in the salivary glands of tabanid flies, Insect Biochemistry and Molecular Biology (2016), doi: 10.1016/j.ibmb.2016.03.007.

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1 Hyaluronidase activity in the salivary glands of tabanid flies

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3 Vera Volfova, Viktorie Tothova, Petr Volf *

4 5 Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 6 Prague 2, 128 44 Czech republic 7 8 * Corresponding author. 9 E-mail address: [email protected] (P. Volf ). 10

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11 ABSTRACT 12 Tabanids are haematophagous that act as biological and mechanical vectors of various 13 diseases, including viruses, bacteria and parasites. The saliva of these insects contains strong 14 anticoagulant and vasodilatory activities as well as immunoregulatory peptides. Here we 15 demonstrate pronounced hyaluronidase (hyase) activity in ten tabanid species of the genera 16 Chrysops, , Hybomitra and . Compared to other haematophagous 17 insects, the ability of tabanid hyases to hydrolyze hyaluronic acid (HA) is extremely high, for 18 example the enzyme activity of Hybomitra muehlfeldi was found to be 32-fold higher than the 19 salivary hyase activity of the sand Phlebotomus papatasi . Hyases of all ten tabanid species 20 tested also cleaved chondroitin sulfate A, another glycosaminoglycan present in the 21 extracellular matrix of vertebrates. The pH optimum of the enzyme activity was measured in 22 eight tabanid species; the hyase of Haemopota pluvialis was the only one with optimum at pH 23 4.0, while in the other seven species the activity optimum was at 5.0. SDS PAGE zymography 24 showed the monomeric character of the enzymes in all tabanid species tested. Under non- 25 reducing conditions the activities were visible as single bands with estimated MW between 35 26 and 52 kDa. The very high hyaluronidase activity in tabanid saliva might be related to their 27 aggressive biting behavior as well as to their high efficiency as mechanical vectors. As they 28 are supposedly involved in the enlargement of feediMANUSCRIPTng hematomas, hyases might contribute to 29 the mechanical transmission of pathogens. Pathogens present in vector mouthparts are co- 30 inoculated into the vertebrate host together with saliva and may benefit from increased tissue 31 permeability and the immunomodulatory activity of the salivary hyase. 32 33 Key words : hyaluronidase; saliva; tabanids; bloodfeeding; pathogen transmission

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34 1. Introduction 35 36 Tabanids (Diptera: Tabanidae) are a cosmopolitan group of haematohagous insects 37 comprising more than 4.400 species (Roskov et al., 2015). These pests are considered a 38 serious nuisance, and are characterized by their persistent biting behavior and painful bites 39 that may induce allergic reactions in sensitive individuals (Bircher, 2005). In comparison with 40 other blood-feeding insects, however, tabanids have remained relatively neglected, even 41 though they are efficient biological and mechanical vectors of various human and 42 disease agents including viruses, bacteria and parasites (reviewed in Baldacchino et al., 2014). 43 Nevertheless, over the past few decades the epidemiological relevancy of tabanids as 44 mechanical vectors has been focused on due to the increasing incidence of some veterinary 45 important diseases, above all equine infectious anemia (Herholz et al., 2008), bovine 46 besnoitiosis (Alvarez-Garcia et al., 2013), and livestock trypanosomoses (Desquesnes, 2004; 47 Desquesnes et al., 2013). Of the biologically transmitted pathogens, the filarial nematode Loa 48 loa has recently re-emerged as a disease agent of public health importance, because of its 49 negative impact on the control of onchocerciasis and lymphatic filariasis in areas of co- 50 endemicity (Zoure et al., 2011). 51 The saliva of bloodsucking insects modulatesMANUSCRIPT host hemostasis and immunity in order 52 to enable successful blood-meal acquisition (Fontai ne et al., 2011). Salivary components 53 found in tabanids correspond entirely with their feeding strategy of engorging a large 54 bloodmeal within a few minutes; their saliva exhibits strong anticoagulant and vasodilatory 55 activities (Kazimirova et al., 2002; Rajska et al., 2003), together with a broad repertoire of 56 platelet aggregation inhibitors (Reddy et al., 2000; Xu et al., 2008; Zhang 2014) and 57 immunoregulatory peptides (Yan et al., 2008; Zhao et al., 2009). An extreme diversity of 58 more than 30 anti-thrombosis components has been described in the saliva of Tabanus yao 59 (Ma et al., 2009). Hyaluronidase activity has been detected in the saliva of T. yao and 60 Chrysops viduatus , and transcripts coding for hyaluronidase have been found in 61 sialotranscriptomes of the horseflies T. yao and T. bromius (Ribeiro et al., 2015; Volfova et 62 al., 2008; Xu ACCEPTEDet al., 2008). 63 Hyaluronidases (hyases) are widely distributed enzymes in nature, being found in 64 mammalian somatic tissues, multiple animal venoms and microbial toxins (El-Safory et al., 65 2010). Hyase activity has also been demonstrated in various bloodsucking parasites, e.g. ticks 66 (Neitz and Vermeulen, 1987), leeches (Hoving and Linker, 1999), and some invasive 67 nematodes (Hotez et al., 1994). Hyases preferentially degrade hyaluronic acid (HA), but also

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68 exhibit a diverse ability to digest chondroitin (Ch) and chondroitin sulphates (CS) (Honda et 69 al., 2012; Stern and Jedrzejas, 2006). Their predominant substrate HA is a multifunctional 70 high molecular weigh glycosaminoglycan ubiquitous in a vast majority of mammalian tissues 71 (Fraser et al., 1997). Being a prominent component of both the pericellular coat and the 72 extracellular matrix (ECM) of the skin, HA contributes to the skin integrity and resistance 73 against penetration or invasion due to its unique physiochemical and biological 74 characteristics. Moreover, HA also participates in numerous physiological and pathological 75 processes including inflammation and wound healing, and the biological functions of HA 76 polymers are dependent on the molecular weight of the molecule (for review see Anderegg et 77 al., 2014; Stern and Maibach, 2008). 78 Among insects, hyases from Hymenoptera venoms are the best characterized. 79 Similarly to other venom hyases, they contribute to local tissue damage via HA 80 degradation and serve as a spreading factor for other venomous components (reviewed by 81 Bordon et al., 2015). Furthermore, these enzymes are also responsible for the cross-reactivity 82 between Hymenoptera venoms, although their relevance differs among species (Jin et al., 83 2010; King et al., 1996). The very first hyase activity in the saliva of a haematophagous insect 84 was documented by Charlab et al. (1999) in the sand fly Lutzomyia longipalpis , with its hyase 85 cDNA showing high similarity to honey bee and verteMANUSCRIPTbrate hyases. Subsequently, hyase 86 activity has been identified in the saliva of all other sand fly species studied, and according to 87 their physicochemical characterization and sequence analyses they have been classified in the 88 endo-β- N acetyl-hexosdiaminidase (E.C.3.2.1.35) family (Cerna et al., 2002; Rohousova et 89 al., 2012; Vlkova et al., 2014). Additionally, hyase has been confirmed as a common 90 constituent in the saliva of other Diptera with pool-feeding modes, namely in black flies 91 (Ribeiro et al., 2000; Volfova et al., 2008) and biting midges (Radrova et al., 2015; Russel et 92 al., 2009). Degrading ECM, salivary hyases may (i) help the diffusion of other bioactive 93 salivary substances, and (ii) promote pathogen invasion due to both increased skin 94 permeability and the immunoregulative activities of generated HA fragments (Ribeiro et al., 95 2000). Moreover, hyases have also been suggested to contribute to pathogen transmission 96 (Charlab et al.ACCEPTED 1999). The enhancing effect of co-inoculated hyase to Leishmania major 97 infection in murine skin has been documented by Volfova et al. (2008). However, the precise 98 role of salivary hyases in pathogen trasmission still needs to be elucidated. 99 In this study, we demonstrated the presence of extremely high levels of hyase activity 100 in the saliva of ten species from four tabanid genera Chrysops , Haematopota , Hybomitra and 101 Tabanus. We described the basic characteristics of the enzymes, including their molecular

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102 weight under reducing and non-reducing conditions, and quantified and compared their 103 activities to the hyases of sand flies, which are the best known among bloodsucking insects. 104 105 2. Material and methods 106 107 2.1. Collection and determination of insects: 108 109 Female tabanids were collected with a Malaise trap or by manual capture on human 110 and cow bait at four trapping sites in Bohemia, the Czech Republic (Table 1). Females were 111 transported to the laboratory and immediately processed as described below. The species used 112 in the study are summarized in Table 2. The species identification was done according to the 113 key by Chvala et al. (1980). 114 115 2.2. Salivary gland extract: 116 117 Female tabanids were immobilized on ice. Salivary glands (SG) were dissected under 118 a stereomicroscope in 20 mM Tris buffered saline (TBS: 150 mM NaCl, pH 7.6). Dissected 119 SGs were transferred to Eppendorf tubes with TBSMANUSCRIPT and stored at -80°C in batches of 5 pairs 120 of SGs in 20 l (small and medium size species of Chrysops spp, Haematopota spp) or 2 pairs 121 of SGs in 50 l (large size species of Hybomitra spp , Tabanus spp). SGs of 5-day-old females 122 of two sand fly species with known hyase activity, Phlebotomus arabicus and P. papatasi 123 originating from laboratory colonies at Charles University in Prague, were used as controls. 124 Salivary gland extracts (SGEs) were obtained by the disruption of tissue by three freeze-thaw 125 cycles in liquid nitrogen, mechanical homogenization and centrifugation at 12,000 g for 5 126 min. Protein concentrations in the resulting SGEs were measured by Quibit Fluorometr 127 (Invitrogen) following the manufacturer’s instructions. 128 129 2.3. Hyaluronidase assay on substrate gels: 130 ACCEPTED 131 The detection of enzyme activity and determination of substrate specificity were done 132 using the dot method on 10% polyacrylamide gels with copolymerized substrate. Gels were 133 prepared using 0.1M acetate buffer, containing 0.1M NaCl, 0.05% Tween-20 (AA buffer) and 134 0.002% substrate. Based on our previous study (Volfova et al. 2008), pH 5.0 was chosen as 135 optimal for the salivary hyase of tabanids. Hyaluronic acid (HA) potassium salt from human

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136 umbilical cord (SIGMA, # H1504) or chondroitin sulfate A (CS-A) sodium salt from bovine 137 trachea (Sigma, # C9819) were incorporated as substrates. SGE samples adjusted to the same 138 protein concentration were dotted on the gel (1.0 g of total salivary protein in a 1l dot) in 139 three independent trials. TBS served as a negative control, and bovine testicular hyaluronidase 140 (SIGMA, # H3884, 1g in 1 l) and Phlebotomus papatasi SGE (0.4 g in 1 l) were used as 141 positive controls. Incubation was carried out for 24 hrs at 37° C in a moist chamber. The gels 142 were then washed in water, soaked in 50% formamide for 30 min and stained in Stains-all 143 (Sigma) solution (100 g/ml in 50% formamide) for 24 hrs in the dark. After a rinse in 144 distilled water the gels were scanned and photographed. Hyase activity was visible as a pink 145 spot on a dark blue background. 146 147 2.4. Hyaluronidase assay on microtitration plates: 148 149 The hyase activity in SGEs of eight tabanid species (Table 2) was quantified using a 150 sensitive method by Frost and Stern (1997) modified by Rohousova et al. (2012). Biotinylated 151 HA (bHA), prepared as described in Cerna et al. (2002), was immobilized onto Covalink NH 152 microtiter plates (NUNC). Fifty l of bHA in 1.62mM N-hydroxysuccinimide was mixed in 153 wells with 50 l of 0.64mM 1-ethyl-3-(3-dimethylaminopropyl)MANUSCRIPT carbodiimide at a final 154 concentration of 1 g of bHA per well, and incubated overnight at 4° C. Following the 155 incubation, the plates were washed three times in PBS, pH 7.2, containing 2M NaCl and

156 50mM MgSO 4. The plates with immobilized bHA were coated for 45 min with 1% BSA in 157 PBS, then washed and equilibrated with the appropriate assay buffer (100 l/well). SGE 158 samples were incubated for 45 min at 37°C in quadruplicates at a final concentration of 159 0.005 g of total salivary protein per well. To obtain a standard curve ranging from 0.003 to 160 0.2 turbidity reducing units (TRU), bovine testicular hyase (SIGMA; 851 units/mg solid) was 161 serially diluted in 0.1M acetate buffer, pH 4.5, 0.1M NaCl, 0.1% Triton X-100. Wells without 162 bHA or without SGE were used as negative controls. The reaction was terminated by 6M

163 guanidine (200 l/well). Plates were washed in PBS containing 2M NaCl, 50 mM MgSO 4, 164 0.05% Tween-20,ACCEPTED pH 7.2. Then, avidin-peroxidase (SIGMA) was added at a final 165 concentration of 0.2 g/well and incubated for 30 min at room temperature. The color reaction 166 was developed with o-phenylenediamine substrate in 0.1M citrate-phosphate buffer, pH 5.5, 167 and absorbance was measured at 492nm (Tecan-Infinite M 200 Fluorometer; Schoeller 168 Instruments). Raw data were evaluated by Measurement Parameters Editor Magelan 6

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169 (Tecan). The obtained results were expressed as relative TRU (rTRU). Three independent 170 experiments were performed with different set of SGE samples in each trial. 171 The pH dependence of enzyme activities in SGE of C. relictus , Ha. pluvialis and Hy. 172 muehlfeldi hyases was tested within the pH range of 3.5 – 8.5. Four different buffers were 173 used: 0.1M citrate, pH 3.5; 0.1M acetate, pH 4.0, 4.5, 5.0 and 5.5; 0.1M bis-Tris, pH 6.0, 6,5 174 and 7.0; 0.1M Tris, pH 7.5, 8.0 and 8.5; all buffers contained 0.1M NaCl and 0.1% Triton X- 175 100. Controls were performed for each pH separately. Two pH values, pH 4.0 and 5.0 (both 176 0.1M acetate buffer) were chosen for comparisons of enzyme activities among various tabanid 177 species. 178 The hyase activities of the three most common tabanid species studied, C. viduatus , 179 Ha. pluvialis and Hy. muehlfeldi , were compared with the enzymes from the saliva of two 180 sand fly species P. papatasi and P. arabicus , as the salivary hyases of sand flies are the best 181 characterized of the haematophagous insects (Cerna et al, 2002; Rohousova et al., 2012). 182 Since the enzyme activity was much stronger in tabanid SGE, different protein loads were 183 used to obtain appropriate levels of absorbance in wells: 0.03 g in sand flies and 0.001 g in 184 tabanids. Three independent experiments were carried out on microtitration plates at pH 5.0 185 as described above. 186 MANUSCRIPT 187 2.5. Zymographic analysis on HA substrate gels: 188 189 The molecular weights (MW) of hyases were determined by SDS PAGE 190 electrophoresis carried out on 10% polyacrylamide gels (0.75mm thick) with 0.002% HA 191 incorporated as described previously (Volfova et al.2008). Considering that the hyase 192 activities varied among tabanid species (see results from microplate assays), different protein 193 contents were loaded per lane in order to obtain bands of approximately equal intensity (Table 194 2). Samples run under reducing conditions were pre-incubated with sample buffer containing 195 2% 2-mercaptoethanol for 40 min at 45° C. Following electrophoresis, gels were rinsed 196 2x20min in 0.1M Tris, pH 7.8, with 1% Triton X-100, 20min in AA buffer to wash out SDS, 197 and then incubatedACCEPTED in AA buffer for 2hrs at room temperature. After rinsing in distilled water, 198 the gels were stained in Stains-all as described above. Hyase activity was visualized as a pink 199 band on a dark background. Invitrogen BenchMark TM Protein Ladder (Invitrogen # 10747- 200 012) stained in Coomassie Brilliant Blue R-250 was used as a standard for the MW 201 determination. 202

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203 2.6. Effect of SDS on hyaluronidase activity in Tabanus bovinus SGE: 204 205 Additional experiments on HA substrate gels were performed to analyze the sensitivity 206 of T. bovinus salivary hyase to the denaturing effect of sodium dodecyl sulfate (SDS). The 207 enzyme activity was studied in the presence and the absence of SDS on two different 10% 208 polyacrylamide gels (0.75 mm slab) with 0.002% HA incorporated. Gel A was mixed using 209 AA assay buffer, pH 5.0, as described above in the dot method. In gel B, the assay buffer was 210 replaced by 1.5M TRIS-HCl, 0.4% SDS, pH 8.8, which is routinely used for electrophoretic 211 gels (with a final concentration of 0.1% SDS in the gel). T. bovinus SGEs diluted either in 212 TBS without SDS (TAB1) or in a sample buffer containing 1% SDS (TAB2), were dotted on 213 the gel at a concentration of 1.5g of the total salivary protein per 1l dot. Bovine testicular 214 hyase (1 g in 1 l) with and without SDS served as a positive control. Incubation was carried 215 out for 24 hrs at 37° C in a moist chamber. Afterwards, gel A was washed and stained as 216 described in the dot method. Gel B was processed as described above for the gels after the 217 zymographic procedure. Then the gels were scanned and photographed. 218 219 3. Results 220 MANUSCRIPT 221 3.1. Protein content in salivary gland extracts: 222 223 The protein concentrations in SGEs of the ten tabanid species studied are summarized 224 in Table 2. The protein content mostly reflected the body and salivary gland size of the 225 species and differed more than fifteen-fold between the most and the least concentrated SGE 226 samples (T. autumnalis versus C. relictus ). The protein concentration of SGE in controls was 227 0.34 g, and 0.28 g per gland in Phlebotomus arabicus , and P. papatasi , respectively. 228 229 3.2. Hyaluronidase assays on substrate gels: 230 231 The dotACCEPTED method on substrate gels was performed to examine the hyase activity in SGE 232 and to study the substrate specificity of the enzyme. Pronounced activity was observed in the 233 saliva of all species, and HA was found to be the preferential substrate for tabanid salivary 234 hyases (Fig. 1A), whereas CS-A showed solely moderate hydrolysis (Fig. 1B). The highest 235 degradation of HA was detected in T. maculicornis and both Hybomitra species, while the

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236 activities of the other large-size tabanids T. autumnalis and T. bovinus were lower. Among 237 small species, the hyase of C. relictus acted most intensively. 238 The results obtained from the CS-A substrate gel were consistent with those from the 239 HA gel, although digestion of the substrate was much slower. The enzymes from Hy. 240 muehlfeldi , Hy. ciureai and T. maculicornis SGEs were the most active, followed by C. 241 caecutiens , while the lowest activity was observed in SGE samples of T. autumnalis and T. 242 bovinus (Fig 1B). 243 244 3.3. Measuring hyaluronidase activities on microtitration plates: 245 246 The pH dependence of hyase activity in SGEs of C. relictus , Ha. pluvialis and Hy. 247 muehlfeldi was characterized using an assay on microtitration plates with incorporated bHA. 248 In Ha. pluvialis and Hy. muehlfeldi considerable activities were observed within a pH range 249 of 4.0 – 5.5, with pH optimums at 4.0 and 5.0, respectively. Nevertheless, in both of the 250 species the activity was well detectable over a broad pH range from 3.5 to 7.5 (Fig. 2A, 2B). 251 In C. relictus substantial activity occurred at pH 4.0 with a sharp peak at pH 5.0; at higher pH 252 it went down rapidly, being reduced to 10% at pH 5.5 and almost undetectable under alkaline 253 conditions (Fig. 2C). MANUSCRIPT 254 A sensitive assay on microtitration plates was also used to quantify the hyase activity 255 in SGSs of eight tabanid species (T. bovinus and Ha. italica SGEs were not included due to 256 the lack of material for repeated experiments). Since two different values of pH optima were 257 detected in the previous tests (see above), the quantitative assays were performed at pH 4.0 258 and at pH 5.0. Average activities related to the total protein content in SGEs are summarized 259 in Table 2, and the relative activities at both pH 4.0 and pH 5.0 are compared in Fig. 2D. The 260 activity levels detected corresponded mostly to the results on HA substrate gels. The highest 261 activity was seen in Hy. muehlfeldi , followed by other large and medium size species Hy. 262 ciureai , T. maculicornis and T. autumnalis (Fig 2D). In C. relictus SGE, much higher activity 263 was found at pH 5.0 than at pH 4.0, which corresponded well with the sharp peak at the pH 264 optima in thisACCEPTED species (Fig. 2C). By contrast, insignificant differences between the activity at 265 pH 4.0 and pH 5.0 were found in T. autumnalis. The lowest hydrolysis of HA was detected in 266 C. caecutiens SGE at both pH values tested. Hyaluronidase from Ha. pluvialis SGE was the 267 only one among the tested enzymes with an optimum at pH 4.0. 268 As the hyase activity in all tabanid species appeared to be high, an additional 269 experiment (repeated three times) was carried out on microtitration plates at pH 5.0 to

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270 compare hyase activity of three tabanid and two sand fly species. Significant differences 271 between tabanids and sand flies were found: the hyase activity (expressed in rTRU per 1 g of 272 total salivary protein) reached 1.15±0.15 in P. arabicus and 1.64±0.14 in P. papatasi , whereas 273 activities in tabanid flies were an order of magnitude higher, 11.04±2.7 in C. viduatus , 274 15.19±2.1 in Ha. pluvialis and 52.90±4.6 in Hy. muehlfeldi . These data confirmed the 275 extremely high hyase activity in the saliva of tabanids (Fig. 3). 276 277 3.4. Zymographic analysis on HA substrate gels: 278 279 Ten tabanid species were analyzed by SDS PAGE zymography on HA substrate gels 280 to estimate the molecular weight of their salivary hyases. In all but one species, marked 281 activities were detected under both non-reducing and reducing conditions. In non-reduced 282 samples enzyme activities were visualized as monomers providing a single diffuse band. The 283 estimated MW of the enzymes ranged between 35 – 52 kDa (Fig. 4A). In Haematopota spp ., 284 the bands had a MW around 40 kDa, in Hy. muehlfeldi and Hy. ciureai 35 and 37 kDa, 285 respectively. In Tabanus species, the activity bands were detected at around 38 kDa in T. 286 autumnalis, and 48 kDa in T. maculicornis . The highest variability was found in Chrysops 287 species, with MWs of 52, 48 and 40 kDa in C. viduatusMANUSCRIPT, C. caecutiens and C. relictus , 288 respectively. 289 Under reducing conditions the enzymatic activity gave sharper bands that allowed 290 more accurate estimations of MW, which ranged from 43 to 52 kDa. In most species the 291 activity bands had higher MW than in non-reducing conditions, suggesting that the hyases of 292 tabanids are monomers that run slower in the reducing environment due to conformation 293 changes after the breakage of disulfide bonds by 2-mercaptoethanol (Fig. 4B). This difference 294 was most pronounced in Hy. muehlfeldi with MWs around 35 and 46 kDa under non-reducing 295 and reducing conditions, respectively. On the other hand, the hyaluronidase activity of C. 296 viduatus gave a 52 kDa band in both conditions tested. In T. bovinus, no activity was detected 297 when the protein content loaded per lane was similar to the other tabanids tested. Weak bands 298 appeared afterACCEPTED the protein concentration was increased tenfold, and their MW occurred in the 299 same range as those in T. autumnalis, 38kDa under non-reducing and 48 kDa under reducing 300 conditions (Fig. 3A, B). For the protein loads applied see Table 2. 301 302

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303 3.5. The effect of SDS on hyaluronidase activity: 304 305 The pronounced hydrolysis of HA found in T. bovinus SGE on substrate gels 306 contrasted with the scarcely detectable activity found by zymographic analysis. To clarify this 307 unique discrepancy, hyase of T. bovinus was additionally tested for sensitivity to SDS by the 308 dot method on HA substrate gels in the presence and the absence of SDS. This experiment 309 revealed an irreversible sensitivity of the enzyme to the denaturing effect of SDS. On 310 substrate gels without any incorporated SDS, the hyase activity remained visible only in 311 samples diluted in TBS. The samples prepared using a sample buffer with 1% SDS did not 312 show any degradation of HA (Fig. 5A). Furthermore, on gels copolymerized with 0.1% SDS 313 the activity was completely inhibited in both of samples, the one with added SDS and the 314 second with TBS alone (Fig. 5B). In the control, the activity of bovine testicular hyase was 315 not affected by the presence of SDS in either the substrate gels or in the sample buffer. 316 317 4. Discussion 318 319 We demonstrated pronounced hyase activity in ten tabanid species of the genera 320 Chrysops , Haematopota , Hybomitra and Tabanus .MANUSCRIPT Hyase has been previously shown to be a 321 ubiquitous component in the saliva of bloodsucking insects with pool-feeding mode (Ribeiro 322 et al., 2000; Volfova et al., 2008). However, compared to other pool-feeders, the ability of 323 tabanid hyases to hydrolyze HA is unusually high. Here we showed that the enzyme activity 324 of Hybomytra muehlfeldi is 32-fold higher than hyase of Phlebotomus papatasi , which had 325 previously been found to be one of the most active among sand flies (Cerna et al., 2002). The 326 very high levels of the enzyme activity in tabanids might be related to their aggressive biting 327 behavior. Tabanids are able to ingest up to 200 mg of blood within a few minutes (Chvala et 328 al., 1980), and the presence of strong hyase activity in their saliva supports the hypothesis 329 about the essential role of this enzyme in the formation of the feeding hematoma in blood- 330 sucking insects with pool-feeding mode (Ribeiro et al., 2000). Depolymerization of HA by 331 tabanid salivaryACCEPTED hyases might facilitate more the efficient spread of other bioactive salivary 332 substances essential for a prompt blood-meal acquisition. 333 We found significant differences in hyase activity among the tabanid species studied; 334 the substrate degradation was several-fold lower in the small species than in medium- and 335 large-sized ones. This finding might be explained by dissimilarities in the feeding behavior of 336 various tabanid species. Small species like Chrysops are more frequently able to complete

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337 their blood-meal during one continuous feeding, while larger Hybomitra and Tabanus are 338 usually interrupted by host defensive reactions and require several partial attempts before 339 reaching engorgement (Desquesnes and Dia, 2003; Magnarelli and Anderson, 1980; Muzari et 340 al., 2010; for review see Baldacchino et al., 2014). Hypothetically, small species may not 341 need such potent salivary hyase for blood-meal acquisition. Nevertheless, the hyase activity 342 quantified here did not entirely correlate with total salivary protein content within the studied 343 genera, and might also be affected by adaptations to a different spectrum of hosts. Among 344 Chrysops spp. , the smallest species, C. relictus , displayed the highest enzyme activity. 345 Similarly, medium-sized Tabanus maculicornis showed more effective degradation of HA 346 than the large species T. autumnalis and T. bovinus . According to Chvala et al. (1980), C. 347 relictus has the broadest host spectrum of the studied Chrysops species. Analogously, T. 348 maculicornis feed on a wide range of large , in contrast to T. autumnalis and T. 349 bovinus . Therefore, the range of hosts might be another aspect of feeding strategy that affects 350 the pharmacological potency of tabanid saliva. A similar observation has been published by 351 Kazimirova et al. (2002) in a study of the anticoagulant activities in the saliva of 19 tabanid 352 species belonging to six genera: most tabanid species with the strongest anticoagulant 353 activities were those having a broad host spectrum. 354 The DNA sequence encoding for the tabanidMANUSCRIPT hyase obtained from a Tabanus yao 355 Macquart salivary gland cDNA library shared 60% sequence similarity with hyase from wasp 356 venoms, and has been classified to the glycoside hydrolase family 56 (Ma et al., 2011). In 357 addition to the HA hydrolysis, these enzymes may also display a diverse capability to 358 catabolize chondroitin and chondroitin sulfates, glycosaminoglycans that are structurally 359 related to HA (El-Safory et al., 2010). Our study demonstrated the ability of all tabanid 360 species tested to utilize CS as an alternative substrate. The highest activity on CS substrate 361 gels was observed in both Hybomitra spp . and T. maculicornis , the species with the strongest 362 hydrolysis of HA, whereas species with lower activity on HA gels also showed weaker 363 digestion of the alternative substrate. Previously, salivary extracts of various blood feeding 364 insects has been shown to hydrolyze CS, although the intensity of digestion is usually lower 365 compared to HA.ACCEPTED Interestingly, the hydrolysis of CS significantly differs among insect taxons; 366 pronounced degradation of CS has been detected in Culex quinquefasciatus (Volfova et al., 367 2008) while moderate hydrolysis has been documented in all sand flies studied (Cerna et al., 368 2002). A high variability of CS degradation has been observed within the genus Culicoides: 369 C. newsteadi and C. nubeculosus displayed strong activity, whereas the hydrolysis in C. 370 imicola , C. pulicaris and C. punctatus was only moderate, and no activity towards CS was

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371 observed in C. obsoletus (Radrova et al., 2016). The hydrolysis of alternative substrates by 372 hyases might be related to the supposed ability of HA and CS to form complexes in nature 373 and to the adaptation of these enzymes to degrade these two polymers simultaneously, at 374 approximately a speed that reflects their relative abundance in tissues (Stern et al., 2007). In 375 mammalian skin, HA is expressed in both the epidermis and dermis, in contrast to CS which 376 is associated mainly with the upper part of the dermis (Tammi and Tammi, 1998; Coolen et 377 al., 2010). Moreover, the pH optima for the hydrolytic activity of hyases toward different 378 substrates may slightly differ (Honda et al., 2012). Thus, the relatively broad pH range of 379 hyase activity detected in the saliva of some tabanid species might facilitate the efficient ECM 380 degradation required for the successful feeding. 381 A pH of 5.0 was found as the optimum for hyase activity in seven of eight tested 382 tabanid species, with the hyase of Ha. pluvialis the only one tested with an optimum at pH 383 4.0. In other bloodsucking insects, salivary hyase activities have also been reported to have 384 optima at acidic pH, which is probably an adaptation to the slightly acidic environment of the 385 skin. In sand flies, Cerna et al. (2002) demonstrated HA hydrolysis over a broad pH range 386 between 4.0 and 8.0 with an optimum at pH 5.0. The hyase of Simulium vittatum black fly 387 showed substantial activity from pH 5.0 - 7.0 and a peak at pH 6.0 (Ribeiro et al., 2000). 388 SDS PAGE zymography revealed the monomericMANUSCRIPT character of the enzymes in all 389 tested tabanid species. Under non-reducing conditio ns, the activities were visible as single 390 bands with estimated MW ranging between 35 and 52 kDa. Similar MWs have also been 391 demonstrated for two tabanids studied previously, C. viduatus and T. yao (Volfova et al., 392 2008; Xu et al., 2008). In contrast to other the tabanid species studied, however, the salivary 393 hyase of T. bovinus showed irreversible sensitivity to the denaturing effect of SDS. Such an 394 inhibition of hyase activity by SDS has previously been reported in Culex quinquefasciatus 395 mosquito SGE (Volfova et al., 2008) and several Culicoides species (Radrova et al., 2015). 396 Likewise, irreversible denaturation in the presence of SDS was also observed by Hotez et al. 397 (1994) in hyase from invasive stages of the gastrointestinal nematode Anisakis simplex , 398 whereas hyase from Ancylostoma caninum remained active after exposure to SDS. 399 SalivaryACCEPTED hyases of some bloodsucking Diptera are known as medically and veterinary 400 relevant allergens. In the saliva of biting midges Culicoides nubeculosus and C. obsoletus , 401 hyases are primary allergens that induce insect bite hypersensitivity, an economically 402 important allergic dermatitis in horses (Schaffartzik et al., 2011; van der Meide et al., 2013). 403 Three allergens, hyase, antigen 5-related protein and apyrase, have been purified from the 404 salivary glands of the horsefly Tabanus yao (An et al., 2011; Ma et al., 2011), and are the

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405 only native allergens purified and characterized from tabanids to date. Subsequently, An et al. 406 (2012) confirmed hyase as one of the cross-reactive allergens between wasp venom and 407 horsefly saliva responsible for the so-called wasp-horsefly syndrome. This coexistent 408 hypersensitivity to Hymenoptera and tabanids has been demonstrated in individuals that are 409 hypersensitive to Tabanus americanus (Freye and Litwin, 1996), Haematopota pluvialis and 410 Chrysops spp (Quercia et al. 2008). At present, the lack of well-characterized allergen extracts 411 and commercially available tools for immunotherapy impedes the diagnosis and treatment of 412 allergies to tabanid bites (Quercia et al., 2008). Thus, our demonstration of pronounced hyase 413 activity in various tabanid species might be useful for further immunological and 414 allergological studies. 415 The hydrolysis of HA as a prominent component of the ECM and the pericellular coat 416 might allow some pathogens to overcome that barrier and enhance their infectivity, due to 417 reduced viscosity of the ECM, increased accessibility of cell membrane components to host 418 cell-infectious agent interactions, and/or the supposed immunomodulatory activities of 419 external hyases (Bookbinder et al., 2006; Cermelli et al., 2011; Huang et al., 2014). HA 420 fragments generated during the degradation of the ECM display a broad spectrum of 421 biological functions, depending upon the HA polymer size, microenvironment, localization, 422 and availability of specific binding partners (for reviewMANUSCRIPT see Petrey and de la Motte, 2014). 423 The co-administration of recombinant human PH20 hyase was shown to significantly increase 424 the dispersion of injected adenoviruses in vivo (Bookbinder et al., 2006), and the enhancing 425 effect of co-inoculated bovine testicular hyaluronidase was also reported in mice infected by 426 Leishmania major (Volfova et al., 2008). However, no experimental data are available for 427 purified salivary hyases from blood-feeding insects, and it is difficult to hypothesize about an 428 immunological role for these enzymes during the invasion of transmitted pathogens. 429 Tabanids are the biological vectors of various filarial nematodes, of which Loa loa is 430 of significant epidemiological importance (Baldacchino et al., 2014; Zoure et al., 2011). In 431 contrast to biological transmission, the range of pathogens transmitted mechanically by 432 tabanids is relatively broad, since these Diptera display all the attributes of an efficient 433 mechanical vector:ACCEPTED (i) high mobility and good vision, (ii) interrupted blood feeding due to 434 host defensive reactions to painful bites, (iii) a tendency to switch hosts when dislodged, (iv) 435 a large amount of residual blood on the mouthparts, and (v) pharmacologically active saliva 436 (for review see Baldacchino et al., 2014; Carn, 1996). The very high hyase activity in tabanid 437 saliva might be related to their high efficiency as mechanical vectors of various pathogens. As 438 they are supposedly involved in the enlargement of feeding hematomas, hyases might

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439 contribute to this mechanical transmission. Pathogens imported on vector mouthparts, along 440 with residual blood from the previous feeding, are co-inoculated with the saliva and may 441 benefit from increased tissue permeability and the immunomodulatory activity of the tabanid 442 hyase. Clearly, further studies on the role of vector salivary hyases in both the biological and 443 mechanical transmission of pathogens are needed. 444 445 Acknowledgments 446 We would like to thank to Dr. J. Votypka, Dr. L. Mikes (Charles University in Prague) and 447 Dr. J. Jezek (National Museum in Prague) for help with laboratory assays, field work and 448 tabanid determination. This study was partially supported by the Ministry of Education of the 449 Czech Republic (MSM0021620828). 450 451

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ACCEPTED

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620 Table 1. Trapping localities details. 621 Locality GPS coordinates Altitude [m] Biotope characteristics Golcuv Jenikov 49°48´38´´N; 15°28´15´´E 395 meadow near a pond Hrncire, Prague 50°00´15´´N; 14°30´20´´E 300 meadow near a pond Kladruby nad Labem 50°03´10´´N; 15°29´00´´E 210 pasture near a river Ruda 49°09´00´´N; 14°41´30´´E 420 edge of a wood and a swampy meadow 622 623 624 Table 2. Protein content in tabanid SGEs, hyase activities per 1 l of total salivary protein and 625 protein loads used for SDS PAGE zymography. 626 Mean of Hyase activity SDS PAGE: SGEs: [rTRU/ g of total salivary protein loads per protein protein]** well [ g] content non- reduced pH 4.0 pH 5.0 Tabanid species Abbr. [g/gland]* reduced Chrysops caecutiens CHC 4.3 ± 0.2 2.60 ± 0.12 4.35 ± 0.22 0.02 0.04 (Linnaeus, 1758) Chrysops relictus CHR 2.3 ± 0.2 13.90 ± 1.21 35.61 ± 3.20 0.03 0.06 Meigen, 1820 Chrysops viduatus CHV 5.7 ± 1.5 7.19MANUSCRIPT ± 1.68 9.10 ± 2.09 0.03 0.06 (Fabricius, 1794) Haematopota italica HAI 3.5 ± 0.5 nd nd 0.04 0.08 Meigen, 1804 Haematopota pluvialis HAP 2.6 ± 0.5 18.26 ± 3.83 12.15 ± 2.14 0.03 0.07 (Linnaeus, 1758) Hybomitra ciureai HYC 24.7 ± 2.7 32.16 ± 3.51 41.77 ± 5.01 0.02 0.04 (Séguy, 1937) Hybomitra muehlfeldi HYM 15.2 ± 1.2 50.42 ± 3.98 56.52 ± 5.54 0.02 0.04 (Brauer, 1880) TAA 36.9 ± 0.8 27.00 ± 1.51 28.91 ± 2.50 0.04 0.08 Linnaeus, 1761 Tabanus bovinus TAB 21.5 ± 0.5 nd nd 0.24 0.61 Linnaeus, 1758 Tabanus maculicornis ACCEPTEDTAM 12.5 ± 0.4 29.56 ± 1.57 40.02 ± 4.81 0.02 0.04 Zetterstedt, 1842 627 628 *Data represent the mean ± SEM of three independent measurements, each containing three 629 replicates of the tested SGEs. 630 ** Data represent the mean ± SEM of three independent measurements, each containing four 631 replicates of the tested SGEs. 632

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633 Fig. 1. Substrate specificity characterized by the dot method on 10% polyacrylamide 634 substrate gels, pH 5.0. Copolymerized 0.002% HA (A) and 0.002% CS-A (B) were used as 635 substrates. Samples of tabanid SGE were applied at a concentration of 1.0 g of total salivary 636 protein in each 1l dot. 20 mM Tris buffered saline (TBS; 1 l) served as a negative control. 637 Bovine testicular hyaluronidase (BTH; 1 g in 1 l) and Phlebotomus papatasi SGE (PHP; 0.4 638 g in 1 l) were used as positive controls. For abbreviations of the tabanid species see Table 2. 639 640 Fig.2 pH dependence of hyase activity characterized in three tabanid species: Haematopota 641 pluvialis (A), Hybomitra muehlfeldi (B) and Chrysops relictus (C). Two pH values, pH 4.0 642 and 5.0 were chosen for comparisons of enzyme activities among eight tabanid species using 643 the microtitration plate method (D). Wells were loaded with 0.005 g of total salivary protein, 644 with bovine testicular hyaluronidase used as a standard. Data are expressed as the relative 645 activity; the values of hyase activities expressed in rTRU per g of total protein are given in 646 Table 2. 647 648 Fig. 3 Comparison of hyase activity in SGEs of three tabanid and two sand fly species. 649 Wells were loaded with 0.001 g and 0.03 g of total salivary protein in tabanids and sand 650 flies, respectively. The assay was performed at pH 5.0 using the microtitration plate method 651 with bovine testicular hyase as a standard. The mea MANUSCRIPTn ± SEM of three independent 652 measurements are shown, each containing four replicates of the tested SGEs, and are 653 expressed as the relative activity; the values of hyase activities expressed in rTRU per g of 654 total protein are given in the results. Phlebotomus arabicus = PHA, Phlebotomus papatasi = 655 PHP, Chrysops viduatus = CHV, Haematopota pluvialis = HAP, Hybomitra muehlfeldi = 656 HYM. 657 658 Fig. 4. SDS PAGE zymography on 10% polyacrylamide gels with 0.002% HA 659 incorporated. SGEs from females of ten tabanid species under non-reducing conditions (A), 660 and the same species tested in a reducing environment (B). Abbreviations of the tabanid 661 species and protein loads of SGEs applied per lane are given in Table 2. 662 ACCEPTED 663 Fig. 5. Inhibiting effect of SDS to hyase activity in Tabanus bovinus SGE. The enzyme 664 activity was tested by the dot method on polyacrylamide gels with 0.002% HA incorporated 665 in the presence and the absence of SDS. Samples of T. bovinus SGE and bovine testicular 666 hyase were diluted to a final concentration of 1.5 g of the total salivary protein/ l and

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667 1g/ l, respectively, either in TBS (TAB1, BTH1) or in a sample buffer containing 1% SDS 668 (TAB2, BTH2). Gel A was prepared without SDS using the assay buffer described in the dot 669 method. Gel B was copolymerized with SDS in the electrophoretic buffer (with a final 670 concentration of 0.1% SDS).

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ACCEPTED ACCEPTED MANUSCRIPT A B 120 120 100 100 80 80 60 60 40 40 20 20 Relative activity (%) Relative activity (%) 0 0 3,5 4 4,5 5 5,5 6 6,5 7 7,5 3,5 4 4,5 5 5,5 6 6,5 7 7,5 pH pH MANUSCRIPT C D 120 120 pH 4 100 100 pH 5 80 80 60 60 40 ACCEPTED 40 20 20 Relative activity (%) Relative activity (%) 0 0 3,5 4 4,5 5 5,5 6 6,5 7 7,5 CHC CHR CHV HAP HYC HYM TAA TAM pH ACCEPTED MANUSCRIPT 120

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Relative activity (%) 20 ACCEPTED 0 PHA PHP CHV HAP HYM ACCEPTED MANUSCRIPT

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Pronounced hyaluronidase activity was found in saliva of ten tabanid species studied. Tabanid hyaluronidases cleave chondroitin sulfate and have pH optimum at 4.0 or 5.0. The enzymes are monomers with estimated molecular weight between 35 and 52 kDa. The high activity may correspond to the high efficiency of tabanids as mechanical vectors.

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