Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and employ Mrc1a for cargo uptake

Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells within the meningeal layer of the zebrafish brain. We previously reported that BLECs efficiently endocytose extracellular cargo molecules (van Lessen et al., 2017), but how this is accomplished and controlled on the molecular level remains unclear. We here compare BLECs to scavenging endothelial cells (SECs) in the embryonic cardinal vein and find them to accept an identical set of substrate molecules. While there is redundancy in the type of scavenger receptors being used, the two cell populations rely for specific substrate molecules on different cell surface receptors to mediate their physiological role: Stab2 appears more critical within SECs in the cardinal vein, while BLECs depend more on the Mrc1a receptor for internalization of cargo. Given the striking similarities to the substrate specificity of cardinal vein SECs, we postulate that BLECs qualify functionally as SECs of the brain.


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The lymphatic vascular system constitutes a blind-ended network that drains interstitial fluid and 35 macromolecules from tissues and organs and eventually returns its contents back into the blood 36 circulation. Furthermore, lymphatics are essential for immune cell trafficking and fat absorption in the 37 intestine (Schulte-Merker et al., 2011) . For a long time, the brain was considered to be devoid of 38 lymphatic vessels, but recently a lymphatic vascular network in the dura mater of the mouse meninges 39 was (re-)discovered (Mascagni and Bellini, 1816, Aspelund et al., 2015, Louveau et al., 2015. Lymphatic 40 vessels are located in the immediate proximity of the meningeal blood vasculature where they drain 41 cerebral interstitial fluid (ISF), macromolecules and cerebrospinal fluid (CSF) into deep cervical lymph 42 nodes (Aspelund et al., 2015, Louveau et al., 2015. They develop postnatally, originating around the 43 foramina that form the entry and exit sites for the blood vessels (BVs) and nerves, and migrate along 44 blood vessels and the cranial and spinal nerves, eventually resulting in a fully developed lymphatic 45 system at P28 (Antila et al., 2017). The meningeal lymphatic system appears to be conserved across 46 mammals and has been described in humans and non-human primates (Absinta et al., 2017). Whether 47 and how meningeal lymphatics might contribute to the waste removal of the brain has not been 48 experimentally addressed but is a topic of significant interest, since accumulation of protein aggregates 49 is one of the hallmark features of neurodegenerative diseases (Metcalf et al., 2012). 50 Recently we and others have demonstrated the presence of lymphatic endothelial cells in the meningeal 51 layer of the zebrafish brain (Bower et al., 2017, Venero Galanternik et al., 2017, van Lessen et al., 2017. 52 Due to the simultaneous but independent discovery of these cells, they were termed either brain referring to the same cell type. These cells express lymphatic marker genes such as prox1a, lyve-1 and 56 flt4 (vegfr3), develop in a Vegfc, Flt4 and Ccbe1-dependent manner and populate the menigeal layer of 57 the brain. Even though BLECs are in close proximity to the meningeal blood vasculature, they do not 58 share -in contrast to pericytes -a common basement membrane with the endothelial cells (van Lessen 59 et al., 2017). Whole transcriptome profiling of sorted BLECs confirmed that BLECs are a distinct 60 endothelial cell population, which show expression profiles different from macrophages and pericytes, 61 while expressing lymphatic markers such as flt4, lyve-1, prox1a (Bower et al., 2017, van Lessen et al., 62 2017. In addition, inhibition of myelopoesis by administration of a pu.1 (spi1b) morpholino does not 63 affect BLEC development, demonstrating that these cells do not constitute a macrophage lineage. 64 Remarkably these cells do not form any vascular structure, but give rise to a network consisting of 65 individual lymphatic endothelial cells expanding over the whole brain surface (van Lessen et al., 2017, 66 Venero Galanternik et al., 2017, Bower et al., 2017. 67 BLECs originate from the venous choroidal vascular plexus behind the eye and sprout around 56hpf, at 68 which point they downregulate blood vascular specific genes and upregulate lymphatic markers such as 69 flt4. Sprouting occurs bilaterally and cells migrate along the mesencephalic vein (MsV), resulting in 70 symetric loops of single cells that cover the optic tectum (TeO) of the zebrafish embryo. This network 71 of individual cells subsequently expands throughout the development of the fish and covers the whole 72 surface of the brain at around 3 weeks of age. BLECs have been shown to play a role in regenerative 73 processes within the brain (Bower et al., 2017, Chen et al., 2019, and have an enormous ability to take 74 up extracellular substances into subcellular vesicles in a process depending on receptor-mediated 75 endocytosis (RME) (van Lessen et al., 2017). Previously we have shown that the uptake of avidin coupled 76 to pHrhodo which is a pH-sensitive tag that only fluoresces upon internalization into the acidic 77 compartment of the lysosome, can be blocked by mannan. This suggests that the mannose receptor is 78 involved in the uptake of avidin. In line with the high endocytotic capacity of these cells, which becomes 79 evident immediately upon sprouting from the choroidal vascular plexus, BLECs typically have large 80 spherical vacuoles which are interpreted as lysosomal compartments in adult brains (van Lessen et al., 81 2017, Venero Galanternik et al., 2017. 82 Another endothelial subpopulation, that has been reported to posses a high endocytotic capacity, are 83 scavenger endothelial cells (SECs). In all terrestrial vertebrates this specialized cell population is located 84 in the liver sinusoids and termed liver sinusoidal endothelial cells. In teleost fish, sharks and lampreys it 85 was identified in various other organs (Seternes et al., 2002). In embryonic zebrafish, SECs were recently 86 shown to be present in several large veins, including the posterior and common cardinal vein (PCV, CCV), 87 and the caudal vein (CV) where they clear substances, colloidal waste and viral particles from the blood 88 circulation as early as 28hpf. The uptake of cargo molecules by SECs in the CV is mainly dependent on 89 the transmembraine receptor stabilin-2 (Campbell et al., 2018). The questions arise whether BLECs 90 possibly represent a functional equivalent of this cell population serving scavenger functions of the brain 91 and whether they might substitute for the absence of lymphatic vessels in teleost meninges. 92 Here we explore this hypothesis and compare BLECs with SECs in the CV to study the physiological role 93 of BLECs. We have generated and analyzed mutants for some of the classical cargo receptor molecules 94 and tested whether they are required for the internalization of cargo into BLECs. We showed that BLECs 95 are highly endocytic cells, which are are considerably more efficient in macromolecular uptake for the 96 tested substrate than microglia. BLECs and SECs in the CV have the same substrate specificity and take 97 up a range of macromolecules including proteins, liposomes, lipoproteins, polysaccharides and 98 glycoaminglycans. Due to the efficiency of cargo internalization and the similarities of the type of cargo 99 they are able to endocytose, we conclude that BLECs qualify as a novel population of scavenger 100 endothelial cells residing in the brain area. Surprisingly, we find that both cell populations depend on 101 different receptors mediating endocytosis. 102

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BLECs and SECs within the caudal vein share the same substrate specificity 105 In order to investigate the function of BLECs in the zebrafish embryo, we directly compared SECs in the 106 CV with BLECs. To this end we injected the same macromolecules either into the optic tectum of 107 embryos (Figure 1 A-J) or into the blood circulation ( Figure 1 K-U) at 5dpf to compare the substrate 108 specificity of both cell populations. In most cases at least two dyes were co-injected and the intracellular 109 uptake was monitored. We used different classes of dye-conjugated substrate molecules, including 110 liposomes (DOPG liposomes - Figure  substrate specificity ( Figure 1V), even though the two cell populations serve two completely different 115 anatomical and physiological compartments: BLECs clear the ventricles and the brain extracellular space 116 from macromolecules, while SECs in the CV filter out substrates from the blood stream. 117 BLECs are more efficient in tracer uptake than macrophages 118 A major open question is which function BLECs fulfill on the surface of the brain. Our initial results 119 suggested a role in clearance of extracellular waste products. However, microglia have always been 120 considered as the major force to remove cellular or sub-cellular components from the brain (Platt et al., 1998). We therefore asked how the endocytic capacity of microglia compares to that of BLECs. We 122 injected IgG-Alexa647 into lyve-1:dsRed;mpeg:GFP double transgenic embryos that allow the 123 discrimination of BLECs and microglia ( Figure 2A). We confirmed that IgG-Alexa647 was taken up by 124 nearly all of the mpeg:GFP + microglia ( Figure 2B). Surprisingly, the fluorescence of IgG-Alexa647 that 125 accumulated in the vacuoles of the microglia was significantly lower in intensity than that of IgG-126 Alexa647 accumulating in the BLECs ( Figure 2C red/white arrow heads). In order to quantify this 127 phenomenon and to analyze the dynamics of the dye uptake, IgG-Alexa647 was injected into 5dpf 128 embryos and the fluorescence intensity of IgG-Alexa647 signal in BLECs was quantified (Region 1 and 2 129 depicted in Figure  3hpi and 6hpi. At all three time points, BLECs were found to take up significantly more IgG-Alexa647 132 than mpeg positive microglia ( Figure 2H, n=6 1hpi** p< 0,005; 3hpi **p<0,005; 6hpi **p<0,005). Hence, 133 while microglia are in closer proximity to the ventricles in which the dye has been injected , BLECs were 134 still an order of magnitude more efficient in the internalization of IgG-Alexa647 . 135 mrc1a mutant zebrafish show an increase in BLEC numbers

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The mannose receptor Mrc1a has previously been shown to be highly expressed in BLECs and is 137 suggested to mediate endocytosis of at least one substrate -avidin -via receptor-mediated endocytosis 138 (van Lessen et al., 2017). In mice, the mannose receptor is maintaining homeostasis of macromolecules 139 in the blood and is responsible for the uptake of Lutrophin ( We have previously shown that mannan, which is a bacterial polysaccharide binding very efficiently to 142 the mannose receptor (Sallusto et al., 1995), could block the uptake of pHr coupled Avidin by BLECs (van 143 Lessen et al., 2017). However, other mannan-binding proteins such as mannan-binding lectin exist. In 144 order to investigate whether zebrafish Mrc1a is mediating the removal of macromolecules from the 145 brain and acts as a clearance receptor, we generated a mutant for mrc1a harbouring a frameshift 146 mutation within exon4 ( Figure 3A). Homozygous mutants did not show any obvious lymphatic or blood 147 vessel phenotype at 5dpf (Figure 3 B,C), and were identified in a normal Mendelian ratio as viable and 148 fertile adults. 149 Interestingly, upon quantification of the number of BLECs in both loops of the optic tectum of the 5dpf 150 zebrafish brain in double transgenic embryos lyve-1:dsRed;fli1a:nucGFP, we found a highly significant 151 increase (56%) in BLEC numbers in mrc1a mutants compared to sibling controls ( Figure 3E). The 152 difference in cell numbers between heterozygous and homozygous mrc1a embryos was already visible 153 at 4dpf (data not shown). This increase in BLEC cell number was also evident in whole brains of adult 154 mrc1a mutant fish. In addition, we found that BLECs display an altered morphology in adult mrc1a 155 mutants ( Figure 3F). 156 mrc1a mutants are deficient in pHr-Avidin uptake 157 When IgG-Alexa647 and pHr-Avidin was co-injected into an mrc1a +/incross, we found that siblings show 158 an uptake of both dyes into BLECs, whereas mrc1a mutant embryos only endocytosed IgG-Alexa647 but 159 did not take up pHR-Avidin ( Figure 3D). We repeated the experiments co-injection of mannan with IgG-160 Alexa647 (van Lessen et al., 2017) and found that IgG-Alexa647 was not only retained at the plasma 161 membrane level, but was completely endocytosed into BLECs whereas pHr-Avidin uptake was blocked 162 completely after mannan administration. When we injected pHr-Avidin first, followed by mannan and 163 IgG-Alexa647, the two dyes were indeed found in identical lysosomes (Supplement S1). We conclude 164 that competitive inhibition using mannan phenocopies the mrc1a mutant phenotype. Analysis of mrc1a 165 mutants demonstrated that the endocytotic uptake of pHr-Avidin, but not of IgG-Alexa647 by BLECs 166 depends on the Mrc1a receptor. 167 168 Stabilin-2 (Stab2) has recently been found to represent an essential receptor for SEC function in the 171 zebrafish caudal vein, clearing the circulation from nanoparticles such as fluorescently labelled 172 hyaluronic acid (fluoHA) and liposomes (Campbell et al., 2018) ( Figure 4A). We therefore asked whether 173 Mrc1a also mediates macromolecule uptake within the caudal vein and whether there is redundancy 174 between Mrc1a and Stab2. 175 To answer these questions, we generated mrc1a -/-;stab2 -/double mutants and compared substrate 176 specificity in wt, mrc1a -/-, stab2 -/and double mutants. We found that the internalization of two known 177 substrates for SEC clearance in the caudal vein -hyaluronic acid and DOPG-liposomes -were affected 178 only in stab2 single and mrc1a -/-;stab2 -/double mutants. Single mrc1a mutants, however, did not exhibit 179 any defects, indicating that Mrc1a is not essential for clearance of these ligands (Figure 4 A). Next we 180 wanted to assess the importance of mrc1a and stab2 for the uptake of hyaluronic acid and DOPG 181 liposomes in BLECs. Interestingly, neither stab2 -/or mrc1a -/single, nor mrc1a -/-;stab2 -/double mutants 182 showed a defect in endocytosis of DOPG liposomes or hyaluronic acid in BLECs ( Figure 4B) suggesting 183 that other receptors must be involved in the endocytosis of these substrates. These results indicate, 184 that even though SECs in the CV and BLECs have identical substrate specificity, there are important 185 differences in the clearance mechanisms between the two cell types. 186 Since zebrafish Mrc1a is essential for the internalization of pHr-Avidin in BLECs, we wondered whether 187 the Mrc1a receptor is mediating preferentially protein uptake in either BLECs or caudal vein SECs. We 188 therefore tested the uptake of additional proteins such as pHr-Avidin, IgG-Alexa647, transferrin and 189 amyloid-β in both cell types in the different mutants. Interestingly, neither Mrc1a nor Stab2 are essential 190 for protein uptake by the SECs of the caudal vein from the blood plasma ( Figure 4C). Similarly, when we 191 analyzed the BLECs, we found that nearly all proteins were still cleared from the brain in mrc1a, stab2 192 and double mutant embryos. Also the endocytosis of modified lipoproteins (acetylated and oxidized 193 LDL) and dextran by BLECs and SECs was unaffected (Supplemental Figure 2). The only exception was 194 pHr-Avidin, whose uptake by BLECs is completely blocked in the mrc1a -/single and mrc1a -/-;stab2 -/-195 double mutants ( Figure 4D, E). 196 mrc1a -/-BLECs are less efficient in the uptake of dextran and IgG-Alexa647

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Although all tested molecules (except for pHr-Avidin) and particles were still endocytosed by BLECs in 198 mrc1a mutants, we noticed differences in the amount of endocytosed material between the different 199 genotypes in some instances. To quantify this effect, we co-injected acLDL-488, pHr-Dextran and IgG-200 Alexa647 into mrc1a mutants and siblings and subsequently measured the fluorescence intensity of the 201 dye accumulating in the vesicles of the BLECs as depicted in Figure 5A and B. Importantly, this analysis 202 demonstrated that the amount of pHr-Dextran and IgG-Alexa647 accumulating in the vesicles of mrc1a 203 mutant BLECs was significantly reduced compared to wild type sibling controls (t-test, **** p<0.0001).

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The amount of endocytosed acLDL was unaltered in mrc1a mutants. This shows that even though the 205 mrc1a mutant fish can still endocytose pHr-Dextran and IgG-Alexa647 in the absence of a functional 206 Mrc1a receptor, dye internalization is much less efficient suggesting that the uptake of dextran and IgG-207 Alexa647 are partially mediated by mrc1a. 208 Taken together, our results demonstrate that BLECs are highly endocytic cells that accept the same 209 substrate molecules as SECs in the CV and qualify as scavenger endothelial cells of the brain. However, 210 the two cell populations rely at least in parts on different receptor molecules for cargo internalization.

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Whereas Mrc1a is essential for endocytosis in BLECs and thereby likely plays an essential role in waste 212 removal from the brain, it is not crucial for waste removal from the blood by SECs in the CV. On the 213 other hand, Stab2 is essential for internalization of selected substrates by the caudal vein SECs, but 214 appears dispensable for BLECs. 215

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Clearance of macromolecules from the brain parenchyma is a crucial process and has always been 217 considered to be carried out by microglia in vertebrates. Recently, teleost BLECs were discovered, which 218 cover the meningeal layer of the optic tectum and other parts of the brain as an extensive network of 219 loosly connected cells in close proximity to blood vessels. They remain as single cells and never form 220 lumenized structures, even after having expanded significantly during growth of the individuum. The 221 question which function these cells fulfill under normal physiological conditions has not been answered. 222 In response to cerebrovascular damage, however, BLECs are able to popupulate the brain parenchyma 223 and can fulfill a guidance function for regrowing blood vessels (Chen et al., 2019). 224 It is striking that within the same anatomical compartment (meninges) of teleosts and mammals there 225 are lymphatic endothelial cells to be found, but that these cells form different structures while still 226 possibly serving similar physiological functions such as waste removal. Since BLECs cannot, in the 227 absence of lumenized vessels,mechanistically work the same way as mammalian lymphatics, we here 228 investigated whether BLECs represent a scavenger endothelial cell population of the brain. We 229 compared BLECs to a recently discovered SECs in the CV of the zebrafish embryo, which has been 230 demonstrated to clear the blood from macromolecules (Campbell et al., 2018), and in that sense are 231 functionally homologous to mammalian liver sinusoidal endothelial cells. While trying numerous 232 different substrate classes such as proteins, liposomes, lipoproteins, polysaccharides and 233 glycoaminoglycans, we could not find any difference in the substrate specificity of caudal vein SECs and 234 BLECs. We therefore conclude that BLECs function as bona fide scavenger endothelial cells. 235 Since microglia were always considered to constitute a cleaning mechansim which responds efficiently latter would suggest that microglia might be more efficient in clearing the brain from macromolecules, 248 particularly since they reside in the brain parenchyma while BLECs are not in direct contact with neural 249 tissue. By directly comparing microglia and BLECs efficacy, we found that BLECs are significantly more 250 efficient in clearing the brain from the protein we tested, compared to macrophages at 1hpi, 4hpi and 251 6hpi ( Figure 2H) . This is counterintuitive, for the reasons mentioned above. Hence, we conclude here 252 that BLECs fulfill the function of scavenger endothelial cells of the brain that can internalize IgG-253 Alexa647 in a very efficient way. Interestingly, within the mammalian liver, there is a similar division of 254 labour between liver sinusoidal endothelial cells and the liver resident macrophages (Kupffer cells), 255 which together form the reticuloendothelial system (RES) within this organ, clearing the blood plasma 256 from endogenous and exogenous waste. We suggest that BLECs and microglia possibly function in a 257 smilar way in the brain. 258 Concerning the spectrum of possible scavenger receptor molecules, we have focused on Mrc1a and 259 Stab2. We found that Mrc1a mediates the uptake of several substrates ( Figure 2D, Figure 5E,H) in BLECs.

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Avidin is so far the only substrate that we identified, that completely failed be taken up by the mrc1a 261 mutant cells. Importantly, avidin that is isolated from hen egg white contains abundant high-mannose 262 glycans (Fiete et al., 1997, DeLange, 1970, Green and Toms, 1970, Bruch and White, 1982. 263 Glycoproteins containing these glycans are known ligands for the mannose receptor and of such 264 substances are rapidly cleared from the blood circulation via liver sinusoidal endothelial cells in 265 mammals (Hubbard et al., 1979). In contrast, transferrin normally does not contain abundant high-266 mannose glycans, providing an explanation for the selective requirement for Mrc1a in avidin clearance. 267 Although the other substrates we injected can be internalzed via alternative pathways, we show that 268 also the pHr-Dextran and IgG-Alexa647 uptake is significantly reduced in mrc1a receptor mutants, 269 highlighting the key role of Mrc1a in the removal of different substance classes (Figure 5 E,H). Both of 270 these substrate classes are known ligands for the mammalian mannose receptor as well (Goetze et al., 271 2011, Kato et al., 2000. In mrc1a mutant situation other receptors might take over the clearance of 272 macromolecules, which normally have lower affinity for those particular substrates and might be not as where pHr-Avidin uptake depends on Mrc1a, pHr-Avidin is still efficiently endocytosed in mrc1a mutant 283 SECs in the CV (Figure 4 A,C). This and the notion that for other substrates uptake efficiency is markedly 284 reduced in mrc1a mutants, supports the conclusion that Mrc1a is important for endocytosis of many 285 substrates within the meningeal BLECs, but is of less importance in SECs in the CV. Converseley, the 286 analysis of stab2 mutant fish revealed that Stabilin-2 is completely dispensable for the uptake of the 287 endocytosis of all tested substrates in BLECs. Since Stab2 is the main receptor in liver sinusoidal 288 endothelial cells for the binding of hyaluronic acid in mice (Schledzewski et al., 2011, Adachi and289 Tsujimoto, 2002), it is surprising that in stab2 mutant fish, fluorescent hyaluronic acid can still be 290 internalized by BLECs. This indicates, that at least in BLECs, additional receptors are involved in the 291 endocytosis of different macromolecules. Since it has been reported that Stab1 is considered as a 292 potential endocytosis receptor (Hansen et al., 2005) which is able to mediate the uptake of acetylated 293 low-density protein and advanced glycation end products (Adachi and Tsujimoto, 2002), it is very likely 294 that it plays at least a redundant role in macromolecular internalization, and this can be analyzed once 295 the stab1 mutants are available. Other potential receptors which would be interesting to look at are 296 lyve-1 and cd44, which have been reported to constitute hyaluronic acid receptors and to be important 297 for dendritic cell trafficking in mammals (Johnson et al., 2017). Since lyve-1 is also expressed in BLECs 298 (Bower et al., 2017), it might also mediate endocytosis of hyaluronic acid in the BLECs and might play 299 an essential role for macromolecule internalization. Yet another candidate involved in the 300 internalization of cargo is Toll-like receptor 2 (TLR2), which forms a complex with the mannose receptor 301 in macrophages (Tachado et al., 2007) and could be a promising candidate for taking over the 302 endocytosis of different dyes in the mrc1a mutants. Notably, various TLRs including TLR2 are also 303 expressed in human lymphatic endothelial cells (Garrafa et al., 2011). Future studies therefore need to 304 investigate additional scavenger receptors, and double or even triple mutants might have to be 305 employed in order to shed more light onto the molecular machinery involved in the endocytosis of 306 proteins, liposomes, lipoproteins, polysaccharides and glycoaminoglycans. 307 Since the discovery of the mammalian lymphatic vasculature in the brain has recently received 308 significant attention and has been linked to a clearance system involved in physiological and 309 pathophysiological conditions such as ageing (Ma et al., 2017) and Alzheimer's disease (Da Mesquita et 310 al., 2018), the experimental analysis of BLECs provided here adds further insight into how endothelial 311 cells contribute to the maintenance of brain homeostasis. Since BLECs have the ability to scavenge very 312 efficiently macromolecules from the brain and qualify as scavenger endothelial cells for the brain, it 313 raises the question which impact the absence of those cells would have on physiological and 314 pathological conditions and whether a functionally related cell type might be conserved in mammalian 315 brains. This needs to be addressed in future studies. Genotyping 342 mrc1a -7bp and stab2 -4bp embryos were genotyped by KASP using the primers indicated in Table S1. 343

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Injection regimes 345 Injections were carried out with a Pneumatic PicoPump. Embryos were anesthetized and embedded in 346 1.5% low melting agarose (ThermoFischer, #16520100) dissolved in embryo medium containing MS222 347 (Sigma, #A5040) and injected with a total volume of 0.5 nl -1 nl per injected bolus. For intratectal 348 injection and injection into the cerebrospinal fluid, needles were inserted into the brain in a sloped 349 angle. Care was taken not to penetrate deep into the brain tissue.

Statistical analysis 408
Data sets were tested for normality (Shapiro-Wilk) and equal variance. P-values were determined by 409 Student's t-test. When normality test failed, Mann-Whitney test was performed. 410   The funders had no role in study design, data collection and interpretation, or the decision to 553 submit the work for publication.