Souphalone Luangsay

Malika Ait-Goughoulte

Maud Michelet

Océane Floriot

Marc Bonnin

Marion Gruffaz

Michel Rivoire

Simon Fletcher

Hassan Javanbakht

Julie Lucifora

! Floriot

Fabien Zoulim
email: fabien.zoulim@inserm.fr

David Durantel
email: david.durantel@inserm.fr

Expression and functionality of Toll-and RIG-like receptors in HepaRG cells

Keywords: pathogen recognition receptor (PRR), toll-like receptor (TLR), hepatocyte, 51

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

101!

The liver is targeted by several important human pathogens, including plasmodium

Material and Methods

153!

Cell culture and HBV infection

154! HepaRG cells were cultured and differentiated in presence of DMSO (cell culture 155! grade, Sigma) as previously described [START_REF] Gripon | ! Infection! of! a! human! 491! hepatoma!cell!line!by!hepatitis!B!virus.![END_REF]. Primary human hepatocytes (PHH) were 156! freshly prepared, as previously described and maintained in same media as 157! dHepaRG [START_REF] Lecluyse | !Isolation!and!culture!of!primary!hepatocytes!from!resected!human! 507! liver!tissue.!Methods![END_REF]. Huh7 and HepG2 (ATCC) cells were also maintained in complete was produced in HepG2.2.15 cells and differentiated HepaRG infected as previously 163! described [START_REF] Hantz | Persistence! of! the! 509! hepatitis!B!virus!covalently!closed!circular!DNA!in!HepaRG!human!hepatocyteMlike!cells.!The!Journal! 510! of!general![END_REF].

164!

Nucleic acid extraction and RT-qPCR/qPCR

165! Total RNA was extracted from cells with the NucleoSpin RNA II kit according to 166! manufacturer's instructions (Macherey-Nagel). RNA reverse transcription was 167! performed using the Superscript III RT (Life Technologies). cDNA was then analyzed 168! by qPCR with the "Express SYBR GreenER™ qPCR SuperMix Universal" 169! (Invitrogen). RNA expression level was determined using the comparative cycle 170! threshold (Ct) method, where the amount of target DNA was normalized to 171! housekeeping genes rplp0 and β-actin cDNA (2 -ΔCt ). When cells were stimulated with 172! ligands the amount of target DNA was normalized to housekeeping genes and to the 173! unstimulated control cDNA (2 -ΔΔCt ). Primers used for the qPCR step are presented in was performed as indicated above using HBV specific primers.

177!

Toll-and RIG-like receptor agonists and stimulation

184!

Flow cytometry analysis

185!

To detect TLR expression, 10 6 HepaRG or PHH cells were fixed with 186! paraformaldehyde (2%; Sigma) and permeabilized with saponin (0.25%; Sigma).

187!

After saturation (saponin 0.25%, fraction-V albumin 3%; Sigma), cells were stained chemiluminescence (Pierce). Anti-actin (Sigma) was used as a loading control.

206!

Enzyme-linked immunoassay for IL-6, IP-10, and secreted HBV antigens

207!

Supernatant from stimulated culture was harvested and assayed for IL-6 and IP10

208!

protein by ELISA using the human IL-6 or human IP10 assay kit according to the 209! manufacturer's protocol (Invitrogen). Cut-offs for these ELISA were 2.5 pg/mL.

210!

HBeAg and HBsAg were detected in the supernatant of infected HepaRG by ELISA 211! using Autobio kits (Autobio, China). Cut-offs for these ELISA were respectively 1 212! NCU/mL (i.e. 1 NCU ≈ 13 ng) and 2.5 ng/mL.

213!

Statistical analysis

214!

Statistical analysis was performed using the nonparametric Mann-Whitney tests Huh7 [START_REF] Sainz | ! Production! of! infectious! hepatitis! C! virus! by! wellMdifferentiated,! 512! growthMarrested!human!hepatomaMderived!cells[END_REF] and HepG2 [START_REF] Jammart | ! VeryMlowMdensity! 514! lipoprotein! (VLDL)Mproducing! and! hepatitis! C! virusMreplicating! HepG2! cells! secrete! no! more! 515! lipoviroparticles!than!VLDLMdeficient!Huh7.5!cells[END_REF] cells were investigated for total RNA expression levels for expressed than other sensors in both dHepaRG and PHH (Fig. 1A and1B). The

Results

220!

Analysis of TLR and RLR RNA expression in dHepaRG cells and PHH at RNA

235!

Analysis of TLR and RLR protein production in HepaRG cells and PHH

236!

TLR protein expression in pHepaRG and dHepaRG was further analyzed (Fig.

283!

In PHH, low-level of gene activation was also observed with TLR-7/9-L (Fig. 4A),

284!

but as PHH cultures are not completely pure, we cannot exclude that residual 285! activation was due to the presence of other liver cells. but yet significant level of IL-6 production (i.e. between 300 and 400 pg/mL). Despite 296! a good RNA expression induction, TLR-8 ligands did not lead to a strong 297! accumulation of IL-6 in the supernatant (only around 30 pg/mL). This could be due to

286!

Secretion profile of IL-6, IP-10, and type-I IFN after stimulation with TLR/RLR

298! ! 12!
fast turnover of the secreted cytokine after a very early expression of IL-6 at RNA 299! level. In agreement with RNA expression data, TLR-7 and 9 ligands did not lead to 300! any detectable level of IL-6. The same ligands that were able to induce IL-6 301! secretion could also increase the IP-10 production . However, TLR-3 and RIG-I/MDA-302! 5 ligands led to the higher production, with a respective secretion of around 2800 and 303! 1400 pg/mL at the highest doses of ligands, as compared to the secretion of around were produced as compared to dHepaRG.

309!

In contrast, we could only detect biologically active type-I IFN, using a functional 5A/5B/5C), and iii) its specific agonisation by ligands leads to a strong secretion of 325! IL-10 (slightly stronger in PHH as compared to dHepaRG) (Fig. 5D). Thus this 326! pathway seems to be operational in hepatocytes.

315!

Exemplary comparison of expression and functionality of TLR-2 in dHepaRG

327!

Antiviral activity of some TLR/RLR ligands in HBV-infected dHepaRG

328!

To fully characterize the functionality of the TLR/RLR receptors that are expressed in when studying the interplay between pathogens and hepatocyte innate immunity.

349!

Indeed pathogen sensing, IFN response and NF-κB signaling are commonly altered 350! these cell types as compared to PHH [START_REF] Alexopoulos | ! Networks! 480! inferred! from! biochemical! data! reveal! profound! differences! in! tollMlike! receptor! and! inflammatory! 481! signaling!between!normal!and!transformed!hepatocytes[END_REF]. RNA levels for TLR receptors and a set of HepG2 cells while it suppressed viral replication in differentiated HepaRG cells [START_REF] Lucifora | Control!of!hepatitis!B!virus! 520! replication!by!innate!response!of!HepaRG!cells[END_REF].

355!

Furthermore it has been observed that the patterns of cytokine secretion were also 356! different even when a common transcription factor (NF-κB) was involved. The defects 357! in TLR signaling observed in hepatoma cells was consistent with the hypothesis that 358! innate immunity could play an important role in suppressing tumorigenesis [START_REF] Alexopoulos | ! Networks! 480! inferred! from! biochemical! data! reveal! profound! differences! in! tollMlike! receptor! and! inflammatory! 481! signaling!between!normal!and!transformed!hepatocytes[END_REF].

359!

These defects make them of limited interest to study innate immune response 360! induced by an hepatotropic pathogen [START_REF] Melen | .! InterferonMinduced! gene! expression! and! 476! signaling!in!human!hepatoma!cell!lines[END_REF][START_REF] Keskinen | .! Impaired! antiviral! 478! response!in!human!hepatoma!cells[END_REF].

361!

In contrast, PHH and HepaRG cells, which are mainly non-cancerous, functional to hepatoma cells, and closer to that of liver biopsies [START_REF] Hart | !XMb.!A!Comparison!of!Whole!Genome! 522! Gene! Expression! Profiles! of! HepaRG! Cells! and! HepG2! Cells! to! Primary! Human! Hepatocytes! and! 523! Human!Liver!Tissues.!Drug[END_REF]. The relatively low 365! replication rate of hepatotropic viruses (low rate for HBV and virtually no permissivity 366! to HCV) in HepaRG cells might result, at least in part, from a cellular antiviral 367! response [START_REF] Marion | The!HepaRG!cell!line:!biological!properties!and!relevance!as! 525! a!tool!for!cell!biology,!drug!metabolism,!and!virology!studies.!Methods[END_REF]. Indeed, an experimentally strong HBV expression and replication after 368! baculoviral delivery of HBV was shown to induce a potent anti-HBV IFN response, 369! while in the same experimental conditions this IFN response did not show any 370! antiviral effect in transformed HepG2 cells [START_REF] Lucifora | Control!of!hepatitis!B!virus! 520! replication!by!innate!response!of!HepaRG!cells[END_REF]. Therefore HepaRG cells, because

371! ! 15!
physiologically related to PHH [START_REF] Guguenmguillouzo | .! General! review! on! in! vitro! hepatocyte! models! and! their! 527! applications.!Methods[END_REF], were expected to be a valuable tool to study 372! host/pathogen interactions. However, detailed features of this immune response

376!

In this respect, our study showed that RNA of all TLR as well as cytoplasmic

387!

These stimulations could also lead to the RNA induction of several cytokines such as 388! IL-6, with the exception of TLR-7 and 9. However, TLR-7 and 9 ligands could 389! somehow induce a weak IFN-α and β RNA expression implying that all these TLR 390! could be slightly functional, yet not leading to detectable secretion of IL-6 or IP-10. In 391! humans, the expression of TLR-7 and TLR9-is mainly confined to plasmacytoid 392! dendritic cells and B-lymphocytes. However, low levels of TLR7 and or TLR9 have

393!

been reported in other cell types, including hepatocytes [START_REF] Lee | !Activation!of!antiMhepatitis!C!virus! 529! responses!via!TollMlike!receptor!7.![END_REF] particularly in the setting 394! of chronic inflammation [START_REF] Takii | Enhanced! 531! expression! of! type! I! interferon! and! tollMlike! receptorM3! in! primary! biliary! cirrhosis[END_REF]. In addition, the functionality of TLR-3 and RIG-I/MDA-5 with prototypic ligands targeting PRR, using different doses of ligands, IL-6 could be 399! secreted and detected at high levels up to 1000 pg/ml with TLR-1/2, 4, and 6 400! stimulations, and between 100 and 500 pg/mL for TLR-3, 5, as well as RLR 401! stimulations. A weak IL-6 expression was obtained with TLR-8 stimulation, but no 402! secretion could be evidenced for TLR-9 and TLR-7 ligands. In contrast, dsRNA antiviral effect against HBV [START_REF] Hosel | ! Not! 534! interferon,!but!interleukinM6!controls!early!gene!expression!in!hepatitis!B!virus!infection[END_REF], many other cytokine may also have antiviral effect.

414!

Therefore, the engagement of PRR in HepaRG was expected to lead to conditioned 415! media having potent antiviral activity. This was strongly obtained with TLR-1/2 and 4 416! agonists, as well as with RIG-I/MDA-5 ligands, but surprisingly less with a TLR-3 417! ligand, despite its ability to better induce the production of IL-6 and IP-10 as by more advanced comparison done in both dHepaRG and PHH (Fig. 5). These appear to be lower in hepatoma cell lines than in primary hepatocytes [START_REF] Alexopoulos | ! Networks! 480! inferred! from! biochemical! data! reveal! profound! differences! in! tollMlike! receptor! and! inflammatory! 481! signaling!between!normal!and!transformed!hepatocytes[END_REF]. However, measured by FACS, and reported on graph as fold increase of MFI (using 571! cholangiocyte as reference, set at 1).

564!

Fig. 2. Protein expression of PRR in pHepaRG and dHepaRG cells. A and B) TLR

572!

Fig. 3. Induction of PRR, cytokine, and ISG gene expression in dHepaRG cells

573!

& secretion of IL-6 and IP-10 after stimulation with prototypic ligands. A) Gene

  medium and cultured in presence of DMSO for three days before RNA 159! extraction. The transfection of siRNA (SMARTpool TM from Darmacon/GE) into 160! dHeppaRg or PHH cells was performed using Darmafect-1 reagent, as 161! recommended by provider (Darmacon/GE). HBV recombinant virus (i.e. inoculum) 162!

  All agonists were purchased from InvivoGen and used according to provider's 179! recommendation. Pam3CSK4, LTA, Poly(I:C)-LMW, LPS, Flagellin (FLA-BS), FSL-1, 180! Imiquimod (R837) or CL264, R848, ssRNA40/lyoVec TM , CpG ODN-2395 (class-C + 181! CpG control), Poly(I:C)-LMW/LyoVec TM were respectively used to stimulate/engage 182! TLR-1/2, TLR-2, TLR-3, TLR-4, TLR-5, TLR-2/6, TLR-7, TLR7/8, TLR-8, TLR-9, and 183! RIG-I/MDA-5. The concentrations used are indicated in the figure legends.

  188!with purified anti-human TLR-2, 4, 5, 6 (sc-73181, sc-52062, sc-57461,! sc-30001; 189! Santa Cruz), anti-human TLR-7, 8, 9 (Alexis Biochemicals (ALX-210-874-C100, ALX-190! 804-376-C100) or Abcam (ab184943, ab85859, ab45371)), or anti-human TLR-3 191! (mAb hTLR-3; Invivogen), followed by incubation with the appropriate secondary 192! antibody conjugated to Alexa Fluor 488 (Invitrogen). For double staining, either anti-193! cytochrome P450-3A4 antibody (ab135813; Abcam) or anti-cytochrome P450-3A1 194! (ab22724; Abcam), which specifically stain hepatocytes and not cholangiocytes 195! (Sup.Fig. 1) were used in combination with anti-PRR antibodies from either mouse 196! or rabbit origin. Cells were analyzed using a FACScalibur flow cytometer and 197! analyzed using CellQuestPro software (BD Biosciences).

8 !

 8 Cells were harvested in lysis buffer (140 mM NaCl, 10 mM Tris pH 7.6, 1 mM 200! EDTA, 1% Triton X-100, 0.05% sodium dodecyl sulfate, 1x protease inhibitor cocktail 201! 5Roche). Clarified lysates were subjected to SDS-PAGE and Western blot transfer to 202! nitrocellulose membranes (Millipore). Membranes were probed with anti-RIG-I, anti-203! MDA5, or anti-TLR2 antibodies (Alexis Biochemicals and Santa Cruz) followed by 204! ! peroxidase-conjugated secondary antibodies (Life Technologies) and detection by 205!

  Prism software. For all tests, a p value ≤0.05 (*) was considered 216! as significant.

  Differentiated HepaRG cells, PHH, as well as DMSO-partially re-differentiated 223!

2

 2 to tlr-10, as well as rig-I and mda-5. All tlrs were similarly expressed in dHepaRG 225! and PHH, whereas levels of expression were significantly lower in the two 226! transformed hepatoma cell lines, except for tlr-8 expression, which was slightly 227! higher in the latters. Rig-I and mda-5 were well expressed in all cell types, but more 228! expressed in PHH as compared to other cells. Tlr-7, 8, 9, and tlr-10 were less 229!

  these PRR was further analyzed according to the HepaRG 231! differentiation status (Fig.1C). Differentiated HepaRG cells expressed slightly more 232! tlr-2, 3, 4 and tlr-6 as compared to proliferative HepaRG (pHepaRG) whereas the 233! expression level of the other sensors tested was not highly significantly changed in 234! dHepaRG versus pHepaRG.

  this led to a readjustment of the concentration in experiments 273! presented hereafter. Stimulation of TLR-3, 7, 9 and RIG-I/MDA-5 also slightly 274! induced IFN-α expression (Fig. 3A, panel IFN-α). Type-III IFN expression has been 275! shown to depend on the same stimulations, i.e. viral infection or ds-RNA ligands, and 276! signal transduction pathway, as those inducing type I IFN expression in epithelial 277! cells [30]. IL-28B (IFN-λ3) expression was only induced by TLR-3 or RIG-I/MDA-5 278! stimulations, although the kinetic of activation was different (i.e. earlier for TLR-3) 279! (Fig. 3A, panel IL-28B). The induction of IL-29 (IFN-λ1) was unexpectedly lower 280! than that of IL-28B (Fig. 3A, panel IL-29). The activation of all IFN transcripts was 281! associated to the activation of prototypic ISGs such as ISG56 induced after TLR-3 282! stimulation and RIG-I/MDA-5.

  The cell culture supernatants of HepaRG cells were first tested for the production 289! of IL-6 and IP-10 following a 24h TLR/RLR stimulation with increasing doses of 290! prototypic ligands (Fig.3B/C). Basically, all TLR/RLR ligands tested, but not TLR-291! 7/8/9 ligands, could induce a strong secretion of IL-6 in supernatants in a dose 292! dependent manner. The highest IL-6 secretions (between 500 and 1000 pg/mL) were 293! obtained with of TLR-1/2, 4, 6 ligands used at a high concentration, as expected.294! Double-stranded RNA ligands, engaging either TLR-3 or RIG-I/MDA-5, led to a lower, 295!

  mL with TLR-1/2 or 4, and respectively 200 and 500 pg/mL with TLR-5 and 6 305! ligands. Similarly to what was obtained with IL-6 secretion, TLR-7, 8 and 9 did not 306! lead to any detectable level of IP-10. Results obtained in PHH are shown in Figure 307! 4B. In PHH even more IP-10, but less IL-6 (with the exception of TLR-4 agonisation) 308!

  310! assay, in the supernatant of HepaRG cell stimulated only with very high (and rather 311! cytotoxic in the context of multiple dosing; data not shown) doses of RIG-I and 312! MDA/5 ligands (Sup.Fig. 3), thus suggesting that the interferon levels produced after 313! stimulation by other prototypic ligands might be under the detection threshold of the 314! assay.

  As a first attempt to compare the expression and functionality of all TLRs/RLRs in 318! dHepaRg and PHH, we studied TLR-2 expression and function in both cell type using 319! two additional batches of cells. Using Pam3CSK4 to stimulate and specific TLR-2 320! siRNA (siRNA against HCV was used as control) to control specificity, we have 321! shown that i) the basal level of expression (RNA and protein) of this sensor is rather 322! even in both cell types (Fig. 5A/5B; Sup.Fig. 2), ii) its expression can be strongly 323! induced by Pam3CSK4 agonisation and decreased by specific siRNA (Fig.

329 !

 329 HepaRG cells, we have treated cells with TLR-1/2, 3, 4, 7, 8, 9 or RIG-I/MDA-5 330! ligands with non-toxic doses (as determined by 3x exposure of HepaRG cells to 331! ligands; Fig. 6D) for 24 hour before infecting them with HBV. A 24 hours inoculation 332! with HBV was made in presence of cytokine produced during the 24h exposure to 333! ligands. Then, infected cells were washed and further retreated twice with the same 334! dose of ligands respectively at days 1 and 4 post-infection. The impact of TLR/RLR 335! ligands treatment on the establishment of HBV infection was analyzed at day 7 post-336! infection by measuring HBV viral protein secretion (HBsAg/HBeAg) and the 337! accumulation of intracellular HBV DNA by qPCR. A strong antiviral activity was 338!obtained with TLR-1/2 and TLR-4 ligands, as well as with a RIG-I/MDA-5 ligand (Fig.339!6A/B/C), whereas weaker or no antiviral activity was observed with a TLR-3, 7, and 8 340! ligands. Surprisingly, an antiviral activity was obtained with the TLR-9 ligand/agonist, 341! which did not correlate with any production of IL-6 or IP-10. However the same 342! antiviral activity was obtained with the control CpG ligand, thus suggesting that the 343! phenotype obtained was independent of innate functions.

  Hepatoma cell lines or transformed human hepatocytes are commonly used to 347! study hepatotropic pathogens, however the relevance of these models is limited 348!

  are lower in these cells compared to PHH, and not 352! modified in response to stimulation. Moreover, we previously showed that a type-I353!IFN response to HBV replication does not lead to an antiviral effect in transformed 354!

  362!for IFN signaling, and permissive to HBV replication, are more relevant models[20, 363! 21]. Their transcriptomes have been shown to be closer to each other, as compared 364!

  and the present study provides the first characterization and 374! functionality of PRRs gene expression in an HBV susceptible and non-cancerous cell 375! line, the HepaRG cell in comparison with PHH.

  I and MDA5 receptors, could be detected in either proliferative or 378! differentiated HepaRG cells, in similar amount than in PHH. While all RNA could be 379! detected, the level of expression of tlr-7, 8, 9 and tlr-10 proved to be very low in 380! hepatocytes. Protein expression analysis showed that all TLR were expressed in 381! both dHepaRG and PHH, with slightly higher levels of expression in HepaRG. The 382! detection of TLR-7, 8, and 9 at protein level contrasted with the low RNA expression, 383! and suggests that post-transcriptional mechanisms could be involved to allow 384! detectable protein expression. RIGI and MDA5 were also expressed at the protein 385! level and highly inducible after interferon stimulation. For most PRR, the stimulation 386! by their prototypic ligands could induce their own expression by positive feed-back.

  395! pathway was demonstrated after cell stimulation with extracellular dsRNA (poly(I:C)) 396! and cytoplasmic dsRNA (poly(I:C)-lyovec) respectively showing an induction of 397!IFNα, β and λ as well as ISGs (such as ISG56). More importantly, after stimulation

  TLR-3 or RIG-I/MDA-5 were more prone to induce secretion 404! of IP-10 (i.e. with production of 2800 and 1400 pg/mL respectively), an IFN-related 405! chemokine. Other functional PRR, based on their ability to induce IL-6 secretion, 406! were also capable to induce IP-10. Interestingly, type-I interferon could only be 407! detected in cell supernatants after stimulation with high, yet rather toxic doses of 408! RIG-I and MDA-5 ligands, thus suggesting that it is difficult to measure the production 409! of type-I IFN by hepatocytes. 410! An important part of the demonstration of PRR functionality in HepaRG relies on 411! the antiviral effect observed with some ligands regarding the establishment of HBV 412! infection in these HepaRG cells. Indeed, while it was shown that IL-6 could have an 413!

  RIG-I/MDA5 agonist. This result could only be explained by the ability 419! of RIG-I/MDA-5 agonists to induce measurable amount of type-I IFNs compared to 420! TLR-3 agonists. The strongest antiviral activity was obtained by Pam3CSK4 421! agonisation of TLR-1/2, a pathway which is well functional in hepatocytes, as shown 422!

  423!interesting results warrant further investigation on this pathway and its antiviral Infections by hepatotropic pathogens can be cleared once innate immune 426! activation and pathogen-specific T cell responses predominate over pathogen 427! immune escape mechanisms, as it occurs in HAV infections, and in cases of 428! resolution of acute HBV or HCV infections. Viral persistence in hepatocytes can 429! occur by escaping both innate and adaptive immune responses, as observed in 430! chronic HBV or HCV infections. HepaRG exhibits functional TLR and dsRNA-431! activated signaling pathways in contrast to cultured hepatoma cells [38]. It appears 432! unclear why hepatoma cell lines are unresponsive to a wide range of TLR agonists, 433! but RNA levels for TLR receptor and a set of downstream signaling molecules 434!

  435!low responsiveness to ligands was observed even when the cognate TLR was 436! expressed at apparently normal levels, suggesting that other downstream factors 437! must be involved. Our data show that HepaRG cells express functional PRRs. The 438! activation of these PRRs leads to the production of cytokines/chemokines, including 439! cytokines bearing antiviral activity. This study opens doors to relevant and detailed 440! analyses of hepatocyte-like cell response to hepatotropic viruses or other 441! hepatotropic pathogens in the absence of non-parenchymal or liver resident immune 442! cells. Therefore, the HepaRG cell line that maintains the functional properties of 443! primary human hepatocytes provides a unique tool for the understanding of the 444! interplay between the innate immune response and hepatotropic pathogens. This 445! work may have potential applications to study the activation of hepatocyte innate 446!immunity by these pathogens, the evasion to innate immunity by these pathogens,

  which innate responses could be restored.

Fig. 1 .

 1 Figure legends

  FACS. Black histograms represent staining with the 566! indicated TLR antibody, shaded gray histograms represent the isotype control, in 567! pHepaRG, dHepaRG and PHH. C) Western-Blot analysis of RIGI and MDA5 in 568! dHepaRG (and PHH), with or without IFNα (1000 UI/mL) induction. D) Expression of 569! some TLRs in hepatocyte-like cells as compared to cholangiocyte-like cells 570!

Fig. 4 .

 4 Fig. 4. Induction of IL-6 and IFNs gene expression in PHH cells & secretion of

Fig. 5 .

 5 Fig. 5. Comparison of the functionality of TLR-2 in dHepaRG and PHH cells. A)

Fig. 6 .

 6 Fig. 6. Effect of stimulation of some PRR receptors by ligands on HBV infection

Table 1 .

 1 Total DNA was extracted from HepaRG using the MasterPure™ Complete

	174!
	175!
	176!	DNA/RNA Purification Kit (Epicentre). Quantitative PCR for intracellular HBV DNA

DD and FZ have received research grants from Hoffmann-La-54! Roche; SF and HJ was/is employees at Hoffmann-La-Roche; SL and MAG were post-55! doctoral fellows at INSERM at the time the research was performed, but are now employees 56! at Hoffmann-La-Roche. staff from Pr Michel Rivoire's surgery room for providing us with liver resection. This work 66! was supported by grants from ANRS (French national agency for research on AIDS and viral 67! hepatitis; several grants from CSS4), FINOVI (Foundation for innovation in infectiology; 68! project call n°#4), FRM (Foundation for medical research; DEQ20110421327), Hoffmann-La-69! Roche Ltd (Switzerland division) and by INSERM core grants. This work was also supported 70! by the DEVweCAN LABEX (ANR-10-LABX-0061) of the "Université de Lyon", within the 71! program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National 72! Research Agency (ANR). 73! 74! !

Supplementary figures and figure legends

Supplementary

Twenty four hours after stimulation, supernatant were tested along with non stimulated cells (NS) for type I interferon activity following procedure described in supplementary materials and methods.

Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3


Maria Cristina Vitelli Queiroz

Martial Douin

Mario Eidi Sato

Marie-Stéphane Tixier
email: marie-stephane.tixier@supagro.fr

Maria Cristina Vitelli Quei

Molecular variation of the cytochrome b DNA and protein sequences in Phytoseiulus macropilis and P. persimilis (Acari: Phytoseiidae) reflect population differentiation

Keywords: Resistance, Cytochrome B mtDNA, Protein structure, Phytoseiidae, Taxonomy

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Many Phytoseiidae mites are efficient predators of pest mites and little insects [START_REF] Gerson | The genome of Tetranychus urticae reveals herbivorous pest adaptations[END_REF]. Some species are commercialized all over the world for biological control issues. Among these species, two belong to the genus Phytoseiulus: Phytoseiulus persimilis Athias-Henriot and Phytoseiulus macropilis Banks. Both species are specialist predators [START_REF] Mcmurtry | Revision of the life styles of phytoseiid mites (Acari: Phytoseiidae) and implications for biological control strategies[END_REF]. They preferentially prey on Tetranychus urticae Koch (Acari: Tetranychidae), a polyphagous and cosmopolitan species, feeding on-and damaging-more than 1100 plant species (140 families) [START_REF] Associates | The effect of the rate of successful dispersal of a phytoseiid mite, Phytoseiulus persimilis Athias-Henriot (Acarina: Phytoseiidae) on the persistence in the interactive system between the predator and its prey[END_REF][START_REF] Zhang | Mites in greenhouse: identification, biology and control[END_REF][START_REF] Escudero | Life-history of predatory mites Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae) on four spider mite species as prey, with special reference to Tetranychus evansi (Acari: Tetranychidae)[END_REF][START_REF] Gerson | The genome of Tetranychus urticae reveals herbivorous pest adaptations[END_REF]. Phytoseiulus persimilis is the oldest commercialized predatory mite and is more widely spread than P. macropilis [START_REF] Demite | Phytoseiidae Database. Available from www. lea. esalq. usp. br/ phyto seiid ae[END_REF]. This latter species, also commercialized, is reported in various continents, but is most frequently encountered in South America, the supposed origin centre of the genus Phytoseiulus [START_REF] Kanouh | Phylogenetic and biogeographic analysis of the genus Phytoseiulus (Acari: Phytoseiidae)[END_REF][START_REF] Demite | Phytoseiidae Database. Available from www. lea. esalq. usp. br/ phyto seiid ae[END_REF]. Phytoseiulus macropolis and P. persimilis are morphologically very close, only differing by the number of setae on the ventrianal shield [START_REF] Mcmurtry | Phytoseiid mites from Guatemala, with descriptions of two news species and redefinitions of the genera Euseius, Typhloseiopsis, and the Typhlodromus occidentalis species-group (Acari: Mesostigmata)[END_REF]Takahashi and Chant 1993a;[START_REF] Kanouh | Phylogenetic and biogeographic analysis of the genus Phytoseiulus (Acari: Phytoseiidae)[END_REF]. Several studies-including morphological and molecular approaches, and crosses-seem to show that they are distinct species (Takahashi and Chant 1993b;[START_REF] Okassa | Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae)[END_REF]). However, one question remains about the identity of these two species in Brazil.

Phytoseiulus persimilis is reported all over the world, including South America, but not in Brazil, whereas P. macropilis is present in Brazil. The absence of P. persimilis in Brazil thus seems strange and we can wonder whether some of the specimens identified as P. macropilis in Brazil actually belong to P. persimilis. The present study aims to provide additional elements to the study of [START_REF] Okassa | Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae)[END_REF], including many more specimens of both species. The second objective of this study is to assess genetic variation at the intraspecific level. Genetic variation within a species can be affected by several factors, such as host plants (as shown for Phytoseius finitimus Ribaga; [START_REF] Tixier | Great molecular variation questions the status of the species Phytoseius finitimus (Acari: Phytoseiidae) and the barcoding decision diagnosis[END_REF], prey (as shown for Phytoseiulus longipes Evans; Tixier et al. 2010a), and geographical distribution (as shown for Amblyseius largoensis (Muma); [START_REF] Bowman | Molecular discrimination of phytoseiids associated with the red palm mite Raoiella indica (Acari: Tenuipalpidae) from Mauritius and South Florida[END_REF][START_REF] Navia | Reproductive compatibility and genetic and morphometric variability among populations of the predatory mite, Amblyseius largoensis (Acari: Phytoseiidae), from Indian Ocean islands and the Americas[END_REF]Barbosa Lima et al. 2018). Here, we investigate how molecular genotype reflects the differentiation of the populations according to geographical distances and resistance to pesticides, as one of the P. macropilis populations was resistant to pyrethroids.

There is a great challenge in early characterization of resistance in field populations for determining resistance persistence and dissemination. For this, we choose to use a neutral marker, the cytochrome b mtDNA (Cytb) fragment. Mutation in this DNA fragment has been associated with resistance to bifenazate, acequinocyl and other mitochondrial Qo inhibitors, in Tetranychidae mites [START_REF] Van Leeuwen | Mitochondrial heteroplasmy and the evolution of insecticide resistance: non-mendelian inheritance in action[END_REF], 2011;[START_REF] Van Nieuwenhuyse | Mutations in the mitochondrial cytochrome b of Tetranychus urticae Koch (Acari: Tetranychidae) confer cross-resistance between bifenazate and acequinocyl[END_REF][START_REF] Fotoukkiaii | Identification and characterization of new mutations in mitochondrial cytochrome b that confer resistance to bifenazate and acequinocyl in the spider mite Tetranychus urticae[END_REF]). In the present case, a population is resistant to pyrethroids and the resistance mechanisms are obviously different to Qo inhibitor acaricide resistance. The Cytb marker was considered as neutral for this strait, and was chosen because of its high evolution rate and great intraspecific variation within Phytoseiidae species (Dos [START_REF] Santos | Molecular markers for analysing phylogenetic relationships within the mite family Phytoseiidae (Acari: Mesostigmata)[END_REF][START_REF] Tixier | Great molecular variation questions the status of the species Phytoseius finitimus (Acari: Phytoseiidae) and the barcoding decision diagnosis[END_REF][START_REF] Tixier | Phytoseiid mites of the genus Typhlodromus (Acari: Phytoseiidae) from Southern France: combined morphological and molecular approaches for species identification[END_REF]. We thus assumed that this marker would be appropriate to capture intraspecific variation.

3

Material and methods

Species and populations studied

The characteristics of the species and populations considered are described in Table 1. Eight populations of P. macropilis collected in two states of Brazil (Sao Paulo and Rio Grande do Sul) and in Argentina were considered. Some populations were collected in open-field crops and greenhouses (population A was collected on gerbera, I and L on strawberry, and V on Sechium elude). One population is a commercialized population (P, obtained from Promip, Limeira, SP, Brazil). Population R, initially collected in 2010 from a strawberry field in State of São Paulo, is maintained in the Acarology Laboratory of the Instituto Biológico (Campinas, SP). This population is known for its resistance to fenpropathrin [START_REF] Queiroz | Pyrethroid resistance in Phytoseiulus macropilis (Acari: Phytoseiidae): cross-resistance, stability and effect tof synergists[END_REF] and has been kept in the laboratory under selection pressure. It is now approximately 5300 × , 738 × and 735 × more resistant to fenpropathrin, bifenthrin and deltamethrin than a susceptible population, respectively [START_REF] Queiroz | Pyrethroid resistance in Phytoseiulus macropilis (Acari: Phytoseiidae): cross-resistance, stability and effect tof synergists[END_REF]. Population C has colonized the rearing units of T. urticae in the Acarology Laboratory of Instituto Biológico and its origin is unknown. Four DNA sequences (deposited in GenBank) of a population of P. macropilis collected in Argentina were also considered [START_REF] Okassa | Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae)[END_REF].

The 26 specimens of P. persimilis considered were collected in the South of Europe and Northern Africa (France, Spain, Tunisia and Italy) on crops and weeds. One population is a commercial one. Twenty-two sequences were those used in [START_REF] Okassa | Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae)[END_REF] and four were newly obtained.

Molecular analysis

DNA extraction

DNA extraction was carried out on a single female as described by [START_REF] Kanouh | Phylogenetic and biogeographic analysis of the genus Phytoseiulus (Acari: Phytoseiidae)[END_REF] using a DNeasy tissue kit (Qiagen, Hilden, Germany) adapted for total DNA extracting of mites. Mites were retrieved after DNA extraction and mounted on slides as described by Tixier et al. (2010b).

DNA amplification and sequencing

Cytochrome b mtDNA was chosen as this DNA fragment allows assessing recent evolutionary history and is sensitive to species and population differentiation (i.e., Dos Santos and Tixier 2017).

Primers and thermal cycling were those described in [START_REF] Tixier | An integrative morphological and molecular diagnostics for Typhlodromus pyri (Acari: Phytoseiidae)[END_REF]. The PCR reactions were performed in a volume of 25 μL, containing 4 μL of mite DNA, 2.5 μL (1 mM) of 10 × buffer, 0.5 μL (25 mM) of MgCl 2 , 0.5 μL (2.5 mM) DNTPs, 0.175 μL (10 μmM) of each primer, 0.5 (10 mg/mL) BSA, 0.125 μL (5 U) of Taq Qiagen and 13.375 μL of water. Electrophoresis was performed on 1.5% agarose gel in 0.5 × TBE buffer for 30 min at 100 V. PCR products were sequenced using the dynamic ET terminator cycle sequencing kit. DNA Purification was carried out with Exosap-IT (Amersham). The sequencer used was the Megabase 1000 apparatus. All DNA fragments were sequenced along both strands. A preliminary analysis was conducted to check for the absence of stop codons. The sequences obtained were compared to those included in the GenBank database to identify possible contaminations. The sequences were aligned and analysed within MEGA v.6.0.6 [START_REF] Tamura | MEGA6: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Genetic distances (using the Kimura 2 parameter) were calculated to compare DNA sequences. This genetic distance is the most used for molecular species identification and its use will permit to compare the present results with those of previous studies. A maximum likelihood tree was constructed; the best-fit-substitution model (TrN + G) was determined by Modeltest v.3.07 [START_REF] Posada | Modeltest: testing the model of DNA substitution[END_REF] Amino acid sequences of the Cytb protein were also studied. The amino acid sequences of the partial Cytb fragment herein considered were obtained using http:// insil ico. ehu. es/ trans late/. The total amino acid sequences of two T. urticae specimens retrieved from Gen-Bank (YP_001795379, ACI30848) were aligned with those of one specimen of P. persimilis and all the specimens of P. macropilis (using MEGA). Then mutations in amino acid associated to pesticide resistance in T. urticae were searched. Because we found mutations in amino acid sequences between two clades of P. macropilis, we wanted to determine the impact of those mutations on the protein structure and function. For this, we used Swissmodel (https:// swiss model. expasy. org/) and DynaMut (http:// biosig. unime lb. edu. au/ dynam ut/) online tools [START_REF] Rodrigues | DynaMut: predicting the impact of mutations on protein conformation, flexibility and stability[END_REF]. The structure of the protein was obtained as well as the location of the mutations. The ΔΔG index (Gibbs free energy, i.e., the difference in folding free energy between wild type and mutant) was used to assess the impact of mutations on protein stability and dynamics.

Results and discussion

Molecular results are based on the alignment of 443 bp for the Cytb fragment considered.

Identity of the specimens of Phytoseiulus macropilis from Brazil

The specimens of P. macropilis and P. persimilis are included in two different well-sustained clades (Fig. 1). As clades could correspond to populations or species (both monophyletic groups) [START_REF] Moritz | DNA barcoding: promise and pitfalls[END_REF][START_REF] Collins | The seven deadly sins of DNA barcoding[END_REF], we also analysed the genetic distances within and between these phylogenetic groups. The genetic distances between the 64 DNA sequences of P. macropilis specimens ranged from 0 to 9.5% (mean ± SE = 3.3 ± 0.6%). The genetic distances between the 26 DNA sequences of the P. persimilis specimens ranged from 0 to 1.1% (0.1 ± 0.07%). These values clearly correspond to the intraspecific variation range already observed for this DNA fragment within the family Phytoseiidae (Dos Santos & Tixier 2017;[START_REF] Tixier | Great molecular variation questions the status of the species Phytoseius finitimus (Acari: Phytoseiidae) and the barcoding decision diagnosis[END_REF]. The specimens of P. macropilis collected in Brazil thus belong to the same species as the ones collected in Argentina.

The genetic distances between the DNA sequences of P. persimilis and P. macropilis range from 16.5 to 19.9% (mean ± SE = 17.7 ± 3.06%) and no overlap between genetic distances of P. macropilis and P. persimilis specimens is observed. According to the 'barcoding gap' hypothesis [START_REF] Hebert | Biological identifications through DNA barcodes[END_REF], it seems thus that all the P. macropilis specimens herein considered do not belong to P. persimilis. An intraspecific genetic distance higher than 19% has been reported in P. finitimus (23%) and Typhlodromus (Anthoseius) 1 3

Fig. 1 Neighbour joining trees based on K2P genetic distances between the specimens of Phytoseiulus persimilis and P. macropilis with the Cytb mtDNA. The numbers at nodes correspond to bootstrap values 1 3 rhenanoides (Athias-Henriot) (21.7%) [START_REF] Tixier | Great molecular variation questions the status of the species Phytoseius finitimus (Acari: Phytoseiidae) and the barcoding decision diagnosis[END_REF][START_REF] Tixier | Phytoseiid mites of the genus Typhlodromus (Acari: Phytoseiidae) from Southern France: combined morphological and molecular approaches for species identification[END_REF]. In both cases, these high intraspecific distances only concerned some specimens, whereas in the present case the high genetic distances between P. macropilis and P. persimilis concern all the specimens considered. Furthermore, the comparison with the interspecific distances obtained until now is quite difficult, as these distances are usually higher than 22%, concern morphologically different species, and many species of another Phytoseiidae subfamily (Typhlodrominae)-P. macropilis and P. persimilis belong to the subfamily Amblyseiinae [START_REF] Tixier | Phytoseiid mites of the genus Typhlodromus (Acari: Phytoseiidae) from Southern France: combined morphological and molecular approaches for species identification[END_REF]. Within the Amblyseinae, a genetic distance of 20% has been reported between Neoseiulus idaeus and N. californicus [START_REF] Tixier | Is Neoseiulus wearnei the Neoseiulus californicus of Australia?[END_REF]. [START_REF] Okassa | Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae)[END_REF] found the distance between P. persimilis and P. macropilis to be about 14%, lower than the distance obtained in the current study-this is likely due to the much higher number of specimens presently considered.

As already stated for other arthropod groups, it seems difficult to apply a 'same decision threshold' based on intra-and interspecific overlap for all species of a single family because of very different biological features, evolution rates and speciation events (e.g., [START_REF] Hajibabaei | DNA barcodes distinguish species of tropical Lepidoptera[END_REF][START_REF] Van Velzen | DNA barcoding of recently diverged species: relative performance of matching methods[END_REF][START_REF] Chapple | A retrospective approach to testing the DNA barcoding method[END_REF]. The genus Phytoseiulus constitutes a quite particular Phytoseiidae group because of prey specificity, high reproductive parameters (rapid development cycle, high fecundity) and high dispersal ability among prey patches [START_REF] Mcmurtry | Life-styles of phytoseiid mites and their roles in biological control[END_REF]. These biological traits could be associated to a higher gene flow than in other Phytoseiidae groups, a more recent speciation and, thus, to lower interspecific distances between two sister species [START_REF] Avise | Principles of genealogical concordance in species concepts and biological taxonomy[END_REF][START_REF] Papadopoulou | Speciation and DNA barcodes: testing the effects of dispersal on the formation of discrete sequence clusters[END_REF].

Finally, observations of the morphology of the specimens used for the molecular analysis showed that all the specimens of P. macropilis had the setae JV2 on the ventrianal shield whereas the P. persimilis specimens had not, also suggesting that they are different species. The conclusion is the same as in [START_REF] Okassa | Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae)[END_REF] and Takahashi and Chant (1993b), who showed complete reproductive incompatibility between P. persimilis and P. macropilis. We thus conclude that P. macropilis and P. persimilis are two different species and the presently analysed P. macropilis specimens from Brazil do not belong to the species P. persimilis.

Genetic variation between the populations of Phytoseiulus persimilis and P. macropilis

The genetic variation between the DNA sequences of P. persimilis is very low. All specimens considered are similar to each other, including the commercialised ones. Only one specimen (from Tunisia) is a bit different (distance of 1.1%). It can thus be hypothesized (i) that the specimens collected might be issued from commercial releases, that spread in the environment (maybe associated to a sampling bias in places where this species has been frequently released), or (ii) that very little variation exists within the species P. persimilis in the considered area. However, because of our previous statement hypothesizing higher evolution rate in species of the genus Phytoseiulus (because of particular biological features), a high genetic diversity between populations was expected. For this reason, our first hypothesis seems the most probable, emphasizing the difficulty to find 'natural' specimens for species massively released for commercial issues. Finally, the low genetic distance might also show that the West Palearctic area-despite being the area where P. persimilis was first described by Athias-Henriot in 1957 (in Algeria)-is not the centre of origin of 1 3 this species. To test this hypothesis, it would be interesting to consider DNA sequences of P. persimilis from South America.

The genetic variation between the DNA sequences of P. macropilis is much higher than that observed for P. persimilis, even if all populations were collected in Brazil and sometimes in the same state (in very close areas). Globally, the intra-population distances are very low with a mean ranging from 0 to 0.42%, whereas the inter-population distances are much higher (see below). The phylogenetic tree shows two distinct clades (Fig. 1). Clade 1 contains specimens collected in Argentina on Solanaceae and specimens of the population 'L' collected in Rio Grande do Sul on strawberry (Rosaceae). Clade 2 contains the remaining populations all collected in the Sao Paulo state on different plants. The mean genetic distance between these two clades is 8.4% (range: 7.7-9.5%), the within-clade genetic distance mean being 0.5% (0-1.1%) for clade 1 and 0.63% (0-2.1%) for clade 2. No overlap between intra and inter-clade distances is observed, but the inter-clade distances clearly correspond to intraspecific variation (compared to previous references for Phytoseiidae mites), leading to the conclusion that these two clades belong to the same species even if no overlap in genetic distance is observed between intra and inter-population distances [START_REF] Tixier | Great molecular variation questions the status of the species Phytoseius finitimus (Acari: Phytoseiidae) and the barcoding decision diagnosis[END_REF][START_REF] Tixier | Phytoseiid mites of the genus Typhlodromus (Acari: Phytoseiidae) from Southern France: combined morphological and molecular approaches for species identification[END_REF].

This genetic differentiation could be associated to geographic location as populations of clade 1-although separated by 570 km-are geographically less distant to each other than to the populations of clade 2-all collected in Sao Paulo state (1300 km remote from specimens of clade 1). A differentiation within these two clades is also observed. In clade 1, specimens of the two populations ('L' and Argentina) are separated in two groups (by a weak mean genetic distance of 1.04%); this could again be explained by a geographical effect and/or by rearing pressure effects. Phytoseiulus macropilis, like all the species of the genus Phytoseiulus, has a high fecundity and a relatively high dispersion capacity especially to colonise new prey patches [START_REF] Rosenheim | Herbivore population suppression by an intermediate predator, Phytoseiulus macropilis, is insensitive to the presence of an intraguild predator: an advantage of small body size?[END_REF]). These biological features could explain founder effects and differentiation of populations in remote areas.

In clade 2, populations collected in the field and assumed to be susceptible to pesticides are included in a separate clade to the specimens resistant to pyrethroids ('R') and to the specimens of population 'C' whose origin was unknown. Because of the similarity between specimens 'R' and 'C', it seems that population 'C' is issued from some specimens of population 'R' that contaminated the T. urticae rearings. Two specimens among the nine of population 'V' are included in the 'resistant' clade even if a bit distant from the other specimens of this group. Population 'V' was collected in an area close to the initial collection locality of population 'R'. These two specimens could belong to the same population 'R' collected 10 years ago.

The bootstrap supporting this clade differentiation is not high; the mean genetic distance between resistant and susceptible groups is 1.12% and the within-population mean distances are 0% for the field populations and 0.06% for the resistant populations. This weak structuring reflects resistant vs. susceptible phenotypes, but with no functional association. Mutations have been reported on the Cytb amino acid sequence to explain resistance to bifenazate, acequinocyl and Qo inhibitors for T. urticae and Panonychus ulmi Koch (G132A, G126S, A133T, P262T, I136T, S141T, I260V, N326S; [START_REF] Van Leeuwen | Mitochondrial heteroplasmy and the evolution of insecticide resistance: non-mendelian inheritance in action[END_REF], 2011;[START_REF] Van Nieuwenhuyse | Mutations in the mitochondrial cytochrome b of Tetranychus urticae Koch (Acari: Tetranychidae) confer cross-resistance between bifenazate and acequinocyl[END_REF][START_REF] Fotoukkiaii | Identification and characterization of new mutations in mitochondrial cytochrome b that confer resistance to bifenazate and acequinocyl in the spider mite Tetranychus urticae[END_REF]. All specimens of P. macropilis have a wild-type amino acid sequence (a 'G' in position 132, an 'A' in position 133, a 'P' in position 262, an 'I' in position 136, an 'S' in position 141 and an 'I' in position 260). As expected, this shows that the specimens of P. macropilis are not resistant to bifenazate, acequinocyl and other Qo inhibitors which is in line with laboratory trials only showing resistance of the population 'R' to pyrethroids [START_REF] Queiroz | Pyrethroid resistance in Phytoseiulus macropilis (Acari: Phytoseiidae): cross-resistance, stability and effect tof synergists[END_REF]. No diagnostic mutation was observed between 'susceptible' and 'resistant' populations.

Finally, one should be careful in associating the clades observed in resistant phenotypes, as the Cytb is a neutral marker for the pyrethroid resistance trait (no functional relationship between pyrethroid resistance and Cytb mutations); other selection pressures could explain the population differentiation within clade 2 (rearing pressure, biological features). The 'R' population has been reared-and insecticide resistance was selected for-for 10 years in the laboratory, so founder effects, genetic drift events and/or inbreeding could have lead to this particular differentiation from these other populations of P. macropilis from Sao Paulo state (e.g., [START_REF] Harshman | Laboratory selection experiments using Drosophila: what do they really tell us?[END_REF][START_REF] Roderick | Genes in new environments: genetics and evolution in biological control[END_REF]. DNA variation does not lead to amino acid change, showing rather synonymous mutations, not affecting the protein structure and function. Furthermore, no differentiation of field populations from the commercial population was observed, questioning the origin of these field populations (natural or released), with a low diversity potentially due to founder effects during the mass rearing process [START_REF] Roderick | Genes in new environments: genetics and evolution in biological control[END_REF].

Four diagnostic mutations were observed between the South Brazil population ('L' and Argentina) and the other populations located in the state of Sao Paulo (N180Y, Y204S, N216Y, V224M). At position 180, the south populations have a leucine (L) (as for T. urticae and a T for P. persimilis) whereas the remaining specimens have a methionine (M). At position 204, the south populations have a serine (S) (as in T. urticae and F in P. persimilis) whereas all the remaining specimens have a tyrosine (Y). At position 216, the south populations have a tyrosine (Y) whereas all the remaining specimens have an asparagine (N) (F in T. urticae and S in P. persimilis). At position 224, the south populations have a methionine (M) whereas all the remaining specimens have a valine (V) (as in P. persimilis and L in T. urticae). We can speculate about the meaning of four mutations and their impact on protein function. We have not found examples in the literature of such mutations in mites (nor in arthropods in general). It seems that in insects founder effects and specific geographic locations may lead to accelerated divergence rates in amino acids, or that parasitic life-styles cause an increase in mutation rates (Page et al. 1998). Accelerated rate of Cytb nucleotide and amino acid evolution in bees may be correlated with increased metabolic rates associated with facultative endothermy (Simmon and Weller 2001). Because of the geographic location of P. macropilis populations of the two clades (south and São Paulo populations) in two different climates, we can hypothesize that these mutations may be involved in energy used for thermoregulation. Figures 2 and3 show the structural modification of the Cytb protein between the wild type (São Paulo populations) and mutant types (south populations). Table 2 reports the ΔΔG index (Gibbs free energy). The four mutations have different impacts on the protein stability. It seems, according to the Dyna-Mut model, that mutations N180Y N216Y and V224M have a stabilising effect, whereas mutation Y204S has a destabilizing effect. Changes in binding affinity caused by mutations may affect a molecule's functional activity [START_REF] Seddigh | Functional, structural, and phylogenetic analysis of mitochondrial cytochrome b (cytb) in insects[END_REF][START_REF] Geng | Finding the ΔΔG spot: are predictors of binding affinity changes upon mutations in protein-protein interactions ready for it?[END_REF]. There is no literature on Cytb modification stability in arthropods. Several mutations in the human Cytb have been related to diseases. [START_REF] Aledo | Thermodynamic stability explains the differential evolutionary dynamics of cytochrome b and COX I in Mammals[END_REF] compares Cytb stability in mammals. These authors concluded that from the thermodynamic point of view, cytochrome b is much more robust to mutations than COX 1 and stated that more stable proteins can tolerate better a decrease in stability, which in turn allows them to evolve faster. The present study supports these findings, as more stabilizing than destabilizing mutations were observed.

Conclusion

The present paper shows the correct identity of P. persimilis and P. macropilis, even if lower genetic distances than for other Phytoseiidae species were observed, certainly due to the singular biological features of species of the genus Phytoseiulus (inducing rapid evolution rates) and the difficulty to dress a general threshold rule for species differentiation for the whole of the Phytoseiidae family. As stated for insects, it appears that molecular identification should use specific thresholds depending on families, sub-families and even genera concerned. A very low intraspecific variation was observed among the specimens of P. persimilis, most likely because of the 'invasion' of the mass-released commercial strains and/or a sampling bias in areas where this species was frequently released. Further comparisons of DNA sequences of P. persimilis from South America would permit to test this hypothesis. The intraspecific variation within P. macropilis shows population separation mainly according to geographical factors and in a lesser extent according to pesticide resistance. However, additional resistant populations should be tested to determine whether such a separation is not due to other factors, especially founder and drift effects [START_REF] Roderick | Genes in new environments: genetics and evolution in biological control[END_REF], as the resistant population is laboratory-reared for 10 years. Furthermore, no diagnostic mutation in amino acid sequence was observed. On the opposite, four mutations were identified between geographically distant P. macropilis populations. This finding clearly opens new research lines on the effect of mutations on protein functioning in mites. It is the first time that such mutations are reported and that stability of proteins after mutation is investigated. However, much more work is required, especially for better characterizing the biological features of the populations (for instance, in relation to temperature requirements) and to better assess the functional positive or negative impact of the mutations in relation to thermodynamic characteristics of the protein.

Fig. 2

 2 Fig. 2 Visual representation of vibrational entropy energy due to mutations N180Y, Y204S, N216Y and V224M in the Cytb protein of Phytoseiulus macropilis. Amino acids are coloured according to the vibrational entropy change upon mutation-blue represents a rigidification of the structure, red a gain in flexibility. (Color figure online)

Fig. 3

 3 Fig. 3 Prediction of atomic interactions due to mutations N180Y, Y204S, N216Y and V224M in the Cytb protein of Phytoseiulus macropilis between populations collected in 'south' (Argentina and Rio Grande do Sul, Brazil) and in Sao Paulo state, Brazil. South and Sao Paulo residues are coloured in light-green and are also represented as sticks alongside with the surrounding residues which are involved in any type of interactions. (Color figure online)

Table 1

 1 Characteristics of the various populations of Phytoseiulus macropilis

	considered, with plant support species and accession numbers in the GenBank
	and P. persimilis

Table 2

 2 ΔΔG index (Gibbs free energy) provided by DynaMut a concerning the four diagnostic mutations between the populations of Phytoseiulus macropilis (from Sao Paulo state and the south of Brazil and Argentina) a http:// biosig. unime lb. edu. au/ dynam ut/

	Mutations
	N180Y	Y204S	N216Y	V224M
	ΔΔG (kcal/mol) 0.181 (stabilizing) 80.689 (destabilizing) 0.112 (stabilizing) 0.562 (stabilizing)

Acknowledgements

We thank Pr Liana Johann (Laboratório de Acarologia, Tecnovates, Universidade do Vale do Taquari -Univates) for providing the L population of P. macropilis. Our thanks also go to FAPESP

1 3 (São Paulo Research Foundation) for the Research Internship Scholarship Abroad (BEPE) to the first author (Process # 2019/04387-3) and the Master Grant (Processo #2019/07238-9) for the third author. Finally, we are very grateful to the two anonymous reviewers for their valuable comments.

Funding This study was supported by Mrs Maria Cristina Queiroz Vitelli, Fapesp.

Declarations

Conflict of interest There is no conflict of interest concerning the results provided in this manuscript. The authors declare no conflicts of interest.

Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.