Julia Hauck-Tiburski

Amauri Rosenthal
email: amauri.rosenthal@embrapa.br

Cyril Iaconnelli

Jean-Marie Perrier-Cornet

Patrick Gervais
email: gervais@u-bourgogne.fr

Cyril Iaconelli
email: cyril.iaconelli@yahoo.fr

Inactivation of dried spores of Bacillus subtilis 168 by a treatment combining high temperature and pressure

Keywords:

Specific treatments combining high temperatures of up to 150°C and moderate pressure of up to 0.6 MPa have been applied to Bacillus subtilis 168 spores conditioned at different a w levels (between 0.10 and 0.70) corresponding to different residual water contents within the spore core. The spores were treated as a dry powder in a pressurized nitrogen environment or in water/glycerol solutions.

These thermodynamic conditions were intended to prevent any water evaporation from the spore core during time/temperature treatments.

Our results clearly show that retaining liquid water in the core by applying pressure during the treatment resulted in greater spore destruction (between 2.4 and 4.9 log at 150°C, 120 s and a w 0.5 in powder) than the destruction observed after the treatment at atmospheric pressure (0.7 log), during which the water rapidly evaporated because its boiling point was reached. Moreover, we found that the water activity level of the spore had a significant impact on spore destruction: the higher the a w level, the greater the spore inactivation.

We obtained similar results from spores heat-treated in powder and in water/glycerol solution at the same a w , confirming the strong influence of this parameter. We hypothesized that the increased spore inactivation was related to the well-known thermal sensitivity of vital organic molecules such as proteins, enzymes, and ribosomes in the presence of water.

Introduction

Low water activity foods (a w <0.6), such as food powders, seeds, dried fruits, cakes, flakes, spices and aromatic herbs, represent a large amount of food products consumed directly or used as intermediate food products [START_REF] Cuq | Food powders engineering, between knowhow and science: Constraints, stakes and opportunities[END_REF]. These products contain vegetative and sporulated bacteria that cannot grow due to the low water activity level. Safety problems can arise after rehydration before or during ingestion. To prevent such incidents, these products must be decontaminated, but heat decontamination is more difficult for dry products than it is for liquid products. Indeed, dried microorganisms are more heat-resistant [START_REF] Grasso | Heat and steam treatments[END_REF][START_REF] Laroche | Unexpected thermal destruction of dried, glass bead-immobilized microorganisms as a function of water activity[END_REF] and heating without water could also rapidly cause burning damage to the product. Among dried microorganisms, sporulated bacteria are particularly resistant [START_REF] Tiburski | Water distribution in bacterial spores: A key factor in heat resistance[END_REF] and spores of Bacillus species can survive various drying and heat treatments used in the food industry [START_REF] Andersson | What problems does the food industry have with the spore-forming pathogens Bacillus cereus and Clostridium perfringens ?[END_REF]. These spores have also been seen to adhere to stainless steel and to resist cleaning in place (CIP) procedures in and around food factories [START_REF] Tauveron | Variability among Bacillus cereus strains in spore surface properties and influence on their ability to contaminate food surface equipment[END_REF]. These characteristics have obvious implications, from equipment bacteria could contaminate food and therefore cause high numbers of spores to be found in food products. Dried foods such as spices, milk powders and cereal products are often quite heavily contaminated with spores, and when water becomes available during food preparation these spores may germinate, leading to spoilage or food poisoning [START_REF] Logan | Bacillus and relatives in foodborne illness[END_REF]. However, destroying bacterial spores is rather difficult, and most of the techniques commonly used to treat dry foods result in very low spore inactivation [START_REF] Silva | Methods of destroying bacterial spores[END_REF].

The most frequently used decontamination techniques for dry products are steam thermal processing, irradiation and fumigation. However, steam adds undesirable moisture to dry foods and causes color changes. Fumigation has been banned in most countries due to its toxicity, and irradiation is strongly rejected by consumers. In addition, these decontamination techniques cause loss of quality and sometimes low inactivation rates [START_REF] Ghisleni | The microbial quality aspects and decontamination approaches for the herbal medicinal plants and products: an in-depth review[END_REF].

Therefore, there is a need to develop new technologies to produce high quality and safe dry food ingredients such as herbs and spices.

Though spices are non-perishable ingredients, once they are put in water-rich food products, their natural or contaminant flora can quickly develop and multiply, consequently putting the health of consumers at risk. This is an especially important issue for spices added to ready-toeat foods that are not subjected to further heat treatments [START_REF] Van Doren | Foodborne illness outbreaks from microbial contaminants in spices, 1973-2010[END_REF].

Moreover the existence of pathogenic spores like B. cereus strains, that may cause foodborne infections [START_REF] Glasset | Bacillus cereus-induced food-borne outbreaks in France, 2007 to 2014: epidemiology and genetic characterisation[END_REF] or like Clostridium botulinum strains that may sometimes cause infant infection through dairy products [START_REF] Doyle | Anaerobic sporeformers and their significance with respect to milk and dairy products[END_REF], is considered as a major risk for the food industry.

It is thus important to decontaminate food powders efficiently, especially if these powders are to be incorporated into a more complex preparation with a higher water content. The decontamination of dried powders is difficult, and the difficulty correlates with the increasing resistance of spores and vegetative forms to low water activity levels (Fine and Gervais, 2005b;[START_REF] Laroche | Water activity affects heat resistance of microorganisms in food powders[END_REF][START_REF] Laroche | Unexpected thermal destruction of dried, glass bead-immobilized microorganisms as a function of water activity[END_REF][START_REF] Tiburski | Water distribution in bacterial spores: A key factor in heat resistance[END_REF].

Many authors have already shown that the low water activity or low water content in spores could explain their high heat resistance and that water diffusion from or out of the spore core is slowed [START_REF] Knudsen | Water and small-molecule permeation of dormant Bacillus subtilis spores[END_REF][START_REF] Loison | Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: a molecular rotor/FLIM study[END_REF]Nguyen Thi Minh et al., 2010b). In a previous work, Bacillus subtilis spores have been reported to have a low water permeability and suggested that the coat structure may be necessary for maintaining this low a w [START_REF] Knudsen | Water and small-molecule permeation of dormant Bacillus subtilis spores[END_REF]. In another manuscript, using a molecular rotor, the high viscosity of the internal membrane of this spore was demonstrated and correlated this property with its barrier properties [START_REF] Loison | Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: a molecular rotor/FLIM study[END_REF].

In previous studies on spore thermal inactivation, Bacillus subtilis spores were shown to have D-values which fell in the same range of pathogen spores like B. cereus and B. anthracis [START_REF] Montville | Thermal resistance of spores from virulent strains of Bacillus anthracis and potential surrogates[END_REF]. Nevertheless, large interstrain variations in spore heat resistance has been regularly reported. For example, the presence of a specific operon (SPOVA2mob not present in strain 168 used in this work) leads to increase the D value more than 100 times [START_REF] Krawczyk | Spore heat-activation requirements and germination responses correlate with sequences of germinant receptors and with the presence of a specific spoVA 2mob operon in food-borne strains of Bacillus subtilis[END_REF].

B. subtilis is also used as a surrogate for some pathogenic spores [START_REF] Hu | Selection of surrogate bacteria for use in food safety challenge studies: a review[END_REF].

Moreover B. subtilis itself can induce food spoilage incidents particularly on bread [START_REF] Thompson | Spoilage of bread by Bacillus[END_REF].

Recently [START_REF] Tros | Picosecond orientational dynamics of water in living cells[END_REF] have shown that a large fraction of "slow" intracellular water linked to proteins exists in the Bacillus subtilis spores and in a previous work [START_REF] Tiburski | Water distribution in bacterial spores: A key factor in heat resistance[END_REF], the role of water in the heat resistance of dried Bacillus subtilis spores was also investigated using Differential Scanning Calorimetry. The spore temperature was linearly increased in pans with different pressure resistances. The results demonstrated that even in dry external conditions, a water fraction stayed embedded in the spore core and that maintenance of this core water content during heating dramatically increased spore destruction. Infrared analysis of treated spores showed that the heat destruction was highly related to protein denaturation and dipicolinic acid (DPA) release, which were maximized in wet conditions. This work was intended to apply these results to optimizing dried spore decontamination based on preserving the initial water content of the spore core. The method used to prevent water evaporation was to pressurize a hermetic container and consequently change the water vapor equilibrium.

This study investigated the inactivation of dried Bacillus subtilis spores equilibrated at different water activities by a treatment using high temperature and absolute pressure levels from 0.1 to 0.6 MPa. Bacillus subtilis spores were already used as surrogates for pathogenic spores [START_REF] Hu | Selection of surrogate bacteria for use in food safety challenge studies: a review[END_REF]. The applied pressure prevented the water in the samples from boiling, thus allowing its initial water content to be maintained. The effect of time/temperature treatments under different pressure levels and for different initial water activities on final spore viability were compared.

Material and Methods

Bacterial strain and growth conditions

The reference strain B. subtilis 168 from the BGSC (Bacillus Genetic Stock Center, Department of Biochemistry, The Ohio State University, USA) was used in this study. B. subtilis sporulation was induced in a complex medium in a reactor at 37°C, pH 8.0, with an air flow of 4 L/min and with agitation (450 rpm) [START_REF] Nguyen Thi Minh | Effect of the osmotic conditions during sporulation on the subsequent resistance of bacterial spores[END_REF]. Sporulation was estimated by plating on Bromocresol Purple agar (Dextrose Tryptone agar, BIOKAR Diagnostics, Beauvais, France) before and after a heat treatment at 80°C for 10 min. When more than 95% of the population in the sample resisted this heat treatment, the spore suspension was harvested (3-5 days). The spore suspension was washed with sterile distilled water four times and was then spray-dried (Mini spray dryer B-290, Buchi, France) and stored in powder form in sterile receptacles at 4°C until use. The final microbial concentration of the powder was approximately 10 11 CFU/g.

Equilibration of spores at different water activities in gaseous and liquid media

Dried spores with water activity values of 0.10, 0.20, 0.30, 0.40, 0.50 and 0.70 were obtained by placing the spores (±500 mg) inside 1 L air-tight plastic boxes containing the water/glycerol solutions presented in Table 1 (the solution occupied 1/10 of the box volume) until equilibrium was reached (at least one week). Water activity determinations of the osmotic solutions and spore powders were performed in triplicate at 25°C using a Decagon-AQUALAB CX-2 water activity meter (Meter, WA, USA).

For liquid media, 10 mg of spray-dried spores previously equilibrated at different water activity levels were placed directly in water/glycerol solutions at concentrations predicted by the Norrish Equation to correspond to a w levels of 0.10 to 0.70 [START_REF] Norrish | An equation for the activity coefficients and equilibrium relative humidities of water in confectionery syrups[END_REF]. The experimental a w values were also checked with the osmometer previously described.

2.3 Treatment of dried spores in gaseous or liquid medium at high temperature and up to a pressure level of 0.6 MPa The reactor consisted of a small hermetic stainless-steel cylinder (1 cm 3 of volume) that could be pressurized with gas. Nitrogen was used to pressurize the reactor, after which the reactor's valve was closed to maintain the applied pressure.

Gaseous medium

In closed systems containing water, increasing pressure prevents water vaporization.

Therefore, if the pressure in the reactor is lower than the pressure of water vaporization (P vap ) at the considered temperature, water vaporization from the spores will occur until P vap is reached. On the other hand, as soon as the pressure exceeds P vap , no vaporization of the water present in the spores will occur. The final pressure inside the reactor is determined by the input controlled pressure of nitrogen and two modifying factors: i) the pressure increase resulting from increased temperature and ii) the pressure increase resulting from water evaporation from the sample. These factors have been considered to fit the evolution of the water vaporization in the reactor as a function of the total pressure evolution for different initial nitrogen pressures.

Figure 1 models the increasing pressure in the vessel for each initial nitrogen pressure level as a function of the increasing temperature and the water vaporization temperature evolution curve. As soon as the temperature/pressure curve crosses the temperature vaporization curve (which occurs for 0.1 MPa, 0.2 MPa and 0.3 MPa at 107, 130 and 145°C, respectively) any liquid water present in the vessel will evaporate from this intersection point until the end of the heat treatment. For the 3 higher initial pressure levels (i.e., 0.4, 0.5 and 0.6 MPa), the 3 curves did not cross the water saturation curve before a temperature of 150°C was reached in the experiment (see Figure 1), thus one can assume that there was no evaporation of any liquid water in the vessel from an initial pressure of 0.4 MPa.

For most experiments, 10 mg of dried spores were placed in aluminum foil that was folded and placed inside the reactor. The reactor was then pressurized between 0.2 and 0.6 MPa, closed and heated in an oil bath at 150°C. When atmospheric pressure was used, the valve was left open to allow water evaporation. After the treatment, the reactors were cooled in an ice bath, then the valve was opened to release the pressure.

Liquid medium

For one set of experiments, performed at 0.6 MPa initial pressure and 150°C, the reactor described previously was filled with water-glycerol suspensions at the same five a w values previously examined in gaseous medium, and 10 mg of spores were added immediately before heat treatment. Under these thermodynamic conditions, there was no vaporization of the different water/glycerol solutions or of pure water.

For liquid and gaseous treatment, it was verified that such a pressure level (0.6 MPa) does not alter spore viability. In fact pressure levels greater than 200 MPa are necessary to slightly inactivate spores with gas medium (Colas de la [START_REF] De La Noue | High gas pressure: an innovative method for the inactivation of dried bacterial spores[END_REF] and 100 MPa in liquid medium (Nguyen Thi Minh et al., 2010a).

Statistical analysis

On each viability point (at least 3 independent measurements) standard deviation was calculated and plotted as error bars on each figure. In order to confirm the effect of pressure level, an ANOVA and Tukey's honestly significant difference test (if p < 0.05) were performed to determinate whether significant differences existed among treatment at different pressure. Analyses were performed using the R software, version 3.1.2.

Results

Effect of temperature and pressure on the viability of dried spores in gaseous medium

Initial pressures between 0.2 and 0.6 MPa were used to study the effect of pressure combined with heat in Bacillus spore inactivation. Spores were equilibrated at a water activity of 0.50, which is a typical a w for dried foods such as flours, cereals and spices [START_REF] Lang | Modeling the heat inactivation of foodborne pathogens in milk powder: High relevance of the substrate water activity[END_REF]. The heat treatment (150°C, 120 s) was chosen after preliminary tests that showed these conditions to be discriminative on the ratio of spore viability to mortality.

In Figure 2, we show that heat treatment performed with no additional pressure, i.e., 150°C at atmospheric pressure, resulted in a slight destruction level of 0.3 log, which is not significantly different from the control sample maintained at 25°C and which certainly corresponds to the inactivation of pre-germinating or germinating spores.

Thus, this treatment did not inactivate any spores, which illustrates their significant thermoresistance. The results presented in Figure 2 also clearly show that when the applied pressure was increased, spore inactivation also increased. Below 0.3 MPa, the inactivation level was found to be less than 3 log, whereas above 0.3 MPa, the inactivation suddenly increased from 4 log CFU for 0.4 MPa to 5 log CFU for 0.6 MPa.

An ANOVA performed on these results confirmed that the inactivation levels corresponding to the two pressure level groups (0.2 and 0.3 MPa versus 0.4, 0.5 and 0.6 MPa) were significantly different (p < 0.05).

To further probe the previous results, the kinetics of spore inactivation was studied at a w 0.5 and 150°C. The pressure used was 0.6 MPa, which corresponded to higher spore destruction in Figure 2. The inactivation of spores with time is shown in Figure 3. Spores heated at atmospheric pressure exhibited only a slight inactivation of approximately 0.5 log after 240 s, whereas spores subjected to 0.6 MPa pressure exhibited an inactivation of 5 log between 60 s and 120 s of treatment.

Data from Figures 2 and3 show that pressure significantly affects the destruction of B. subtilis spores undergoing a 150°C heat treatment at an a w of 0.5. This pressure effect exists at all pressure levels but becomes more significant with a gap of 1 to 2 log destruction for initial pressure levels greater than 0.4 MPa.

3.2 Effect of water activity on the viability of dried spores at different times for a 150°C/0.6

MPa treatment

Dried food products and food powders normally have a water activity ranging from 0.20 to 0.50, and in exceptional cases, their a w may reach 0.70, thus it is important to investigate the effect of this variable on the proposed treatment.

In gaseous medium

The influence of water activity on spore inactivation is presented in Figure 4. These results clearly show that the water activity, and consequently the water content of the spores, plays a fundamental role in spore resistance. Spores equilibrated at an a w of 0.10 exhibited an inactivation of 5 log after 560 s, whereas spores at an a w of 0.20 exhibited an inactivation of 8 log after the same amount of time. When water activity increased, spore inactivation also increased, as shown by the decrease in logarithmic reduction time (D-values) presented in Table 2. Indeed, the D value is four times greater for an a w value of 0.10 than for an a w value of 0.7. Moreover, we found that the same increase in water activity level does not result in a proportional increase in spore inactivation. For example, there is only a small difference between spore inactivation at a w 0.30 and 0.50, but further increase to an a w of 0.70 resulted in complete inactivation after only 240 s.

It is important to emphasize that, with a pressure of 0.6 MPa, the treatment times are greatly reduced, and so, even for water activity levels as low as 0.10 and 0.20, it is possible to reduce the spore population by a factor of 10 to 5 in approximately 9 or 6 minutes, respectively.

In liquid medium

To build on the previous results, the protective effect of a w on spore thermal resistance was analyzed in a liquid aqueous medium in the same experimental chamber used for spore inactivation in a gaseous medium. Addition of glycerol in the treatment liquid medium was used to control the a w of the medium and therefore may have protected spores from heat.

However, osmolytes sometimes also have direct toxic effects on spores and interfere with the pure osmotic effect [START_REF] Mazas | Thermal inactivation of Bacillus cereus spores affected by the solutes used to control water activity of the heating medium[END_REF]. Nevertheless, spores are known for their low permeability to many toxic chemicals, in particular, those that can damage the spore DNA located in the central spore core [START_REF] Knudsen | Water and small-molecule permeation of dormant Bacillus subtilis spores[END_REF]. Moreover, glycerol, which was used in our experiment, is a compatible solute with very few interactions with other molecules, thus it is an almost ideal solute. Therefore, its action may be mainly related to the a w decrease.

The results shown in Figure 5 demonstrate that the water activity of the treatment medium had a major effect on spore heat inactivation. When heated in higher water activities, spores were more sensitive to heat. Spores treated at a w 0.10 or 0.20 presented a 2 log reduction after 240 s, whereas spores at a w 0.30, 0.40 and 0.50 presented a 6 log inactivation after only 180 s.

Spore inactivation began at different treatment times depending on the a w of the medium and later than we observed for spores treated in dried form (see Figure 3). This result can be explained by the heating kinetics inside the reactor. Indeed, previous experiments (data not shown) have allowed us to verify that the temperature inside the reactor reached 99% of the oil bath temperature in less than 10 s when air/nitrogen was the transmitting medium; however, more than 100 s were needed for pure glycerol and 60 s for pure water to reach the same 99% temperature response. Figure 5 shows that inactivation at an a w of 0.70 was noticeable between 60 s and 90 s, whereas for lower a w levels, corresponding to lower spore water content, inactivation started only after 120 s of treatment.

Comparing these results with those obtained from dried spores in gaseous conditions (see Figure 3), it can easily be noted that for the same water activity, the final spore inactivation was quite similar.

Discussion

Steam treatment is in extensive use today for microbial decontamination of dry foods, but its use presents problems since it results in water condensation on the product that must be removed to avoid subsequent mold growth and spoilage. On the other hand, thermal processes such as microwaves [START_REF] Kim | Destruction of Bacillus licheniformis spores by microwave irradiation[END_REF], infrared [START_REF] Eliasson | Infrared decontamination of oregano: effects on Bacillus cereus spores, water activity, color, and volatile compounds[END_REF] and High Temperature Short Time treatment (Fine and Gervais, 2005a) necessarily involve supplementary drying of the product. Moreover, alternative treatments available for use in aqueous media, such as high pressure and pulsed electric fields, are not efficient for the decontamination of dried foods [START_REF] Espinasse | High-Pressure inactivation of dried microorganisms[END_REF]. Therefore, new procedures for decontaminating dried foods that can destroy bacterial spores are needed.

This study investigates the inactivation of dried Bacillus subtilis spores equilibrated at different water activities by a treatment using high temperature and absolute pressure levels of nitrogen from 0.2 to 0.6 MPa. In this experiment, the applied pressure prevented water evaporation from the treated samples, allowing the initial water content of the spores, which is mainly situated in the spore core, to be maintained. and the initial pressure 0.4 MPa at 25°C (which generates 0.57 MPa at 150°C), there is a sudden increase in inactivation, in contrast to the slower increase observed at higher pressures. Based on these results, it can be concluded that when the pressure generated in the vessel exceeded the pressure corresponding to the water vaporization temperature of 150°C (0.47 MPa, see Figure 1) the spore inactivation significantly increased, as shown in Figure 2.

From this result, it can be proposed that i) maintaining water in the endospore favors bacteria inactivation and that ii) spore drying, which is initiated for pressure levels lower than 0.47 MPa, favors bacteria survival at 150°C. Moreover, the drying time and thus the final drying level decreased with increasing pressure until no further drying occurred, when the internal pressure reached the 0.47 MPa value, which corresponded to a nitrogen pressure between 0.3 and 0.4 MPa (see Figure 1).

One can propose a role of water in spore inactivation in agreement with numerous works [START_REF] Coleman | How moist heat kills spores of Bacillus subtilis[END_REF][START_REF] Sochava | Heat capacity and thermodynamic characteristics of denaturation and glass transition of hydrated and anhydrous proteins[END_REF][START_REF] Subramanian | Determination of spore inactivation during thermal and pressure-assisted thermal processing using FT-IR Spectroscopy[END_REF] in which the internal endospore macromolecules, mainly composed of DNA, DPA, and proteins (the last being the most delicate), are preferentially heat-damaged when mobile and unfolded in a water medium.

Moreover, a recent work [START_REF] Tros | Picosecond orientational dynamics of water in living cells[END_REF] has observed a large fraction of "normal" intracellular water in the spore core of B. subtilis.

Even for very low water activity levels, noticeable spore inactivation was obtained in liquid as well as in gaseous medium. These results agree with previous work [START_REF] Tiburski | Water distribution in bacterial spores: A key factor in heat resistance[END_REF], which has shown a residual high water content in the core even for low a w -equilibrated spores. This phenomenon was attributed to specific high barrier properties of the internal spore membrane slowing down the thermodynamic balance. Thus, it could be assumed that some water remaining in the core caused macromolecule denaturation.

Inactivating one or more key spore proteins is believed to be important in spore heat killing [START_REF] Coleman | Mechanism of killing of spores of Bacillus cereus and Bacillus megaterium by wet heat[END_REF]. When heat surpasses a threshold level, especially in aqueous media, changes in the structure of vital growth proteins become irreversible, causing spore inactivation [START_REF] Grinshpun | Thermal inactivation of airborne viable Bacillus subtilis spores by short-term exposure in axially heated air flow[END_REF]. It is also known that in low moisture states the temperature of protein denaturation increases [START_REF] Sochava | Heat capacity and thermodynamic characteristics of denaturation and glass transition of hydrated and anhydrous proteins[END_REF], making inactivation of these key proteins more difficult. In this context, the high spore heat resistance at low water content can be explained by the low mobility and changes in the conformations of proteins and enzymes. Indeed, most spore proteins are rotationally immobilized, which may contribute to heat resistance by preventing heat-denatured proteins from aggregating irreversibly [START_REF] Sunde | The physical state of water in bacterial spores[END_REF]. Consequently, when the original water content of the spore is maintained, protein damage is increased during heat treatment, and such damage is certainly relevant to the differences in spore inactivation found in our experiments. This hypothesis has been directly verified through FTIR measurement in a previous work [START_REF] Tiburski | Water distribution in bacterial spores: A key factor in heat resistance[END_REF]. Nevertheless, the present results were obtained on B. subtilis strain 168 and it is possible that at the pressure and temperature levels used the spore inactivation mechanisms could vary for other bacterial spores.

Conclusion

The model system developed in this work can be extended to treating low water content foods. This system is based on the application of temperature under pressure, which prevents any water evaporation from bacterial spores embedded in the food product. This model has the advantage of not adding moisture to the product, and it results in high spore destruction rates (approximately 5 log). The efficiency of this process is related to the high water permeability barrier of the internal membrane of the spore, which has been shown to allow maintenance of a core water content greater than the outer membrane long after drying. Thus, this new process is particularly efficient for dried bacteria spore destruction because of their high residual core water content. Further studies must first verify this effect on other sporulated strains and especially pathogens. In a second phase, we must integrate the impact of such pressure/temperature/time treatments on the organoleptic and nutritional properties of different dried food products. Indeed, optimal treatments should conserve the functional properties of food in addition to assuring maximal bacteria spore inactivation. Following this work, this process could then be tested to treat dried food products such as spices. The normal a w of dried food ranges between 0.30 and 0.50, and in this work, we have shown that spore inactivation higher than 5 log can be expected from a 3 minute treatment at these water activities. Therefore, if the products already possess a similar a w , no previous equilibration will be needed; however, if the product to be processed has a very low a w , an equilibration step would be necessary. In this case, the dried food could be stored in an atmosphere with a relative humidity of 50% for a few weeks prior to treatment. Certainly, different conditions of time, temperature and pressure adapted to each product will need to be tested to optimize this new decontamination procedure.

The next step would be to develop a scaled-up version of the system to perform tests in real food products to evaluate both microbiological decontamination and changes in quality parameters.

Figure 1: Calculated variations of internal pressure during isochoric heating of pressurization devices (internal volume of 1.7 ml) loaded with a 10 mg spores sample initially equilibrated at a w 0.5 and at initial pressure ranging from 0.1 MPa to 0.6 MPa, in comparison with water boiling temperature of a model saline solution initially at a w 0.5. (NIST Chemistry Webbook has been used for water data calculation).

  dried spores and spores in liquid medium were serially diluted with physiological saline solution (0.9% NaCl w/v), and 100 µl of each dilution was seeded in triplicate onto BCP agar (Dextrose Tryptone Agar, Biokar Diagnostics, Beauvais, France) plates. Colonies were counted after 24 hours of incubation at 37°C. Control samples contained untreated spores. All experiments were performed at least in triplicate. Spore inactivation was expressed using a logarithmic reduction factor log (N 0 -N)/N 0 , with N 0 representing the number of developing spores before treatment and N representing the number of developing spores after treatment.

Figure 2 :

 2 Figure 2: Thermal inactivation of B. subtilis 168 dried spores initially equilibrated at a water activity of 0.50 treated at 150°C / 120s in a gaseous medium at different initial pressure levels. (Error bars are SD, Mean comparison using Tukey's test: same letters indicate no significant difference between treatments P>0.95)

Figure 4 :

 4 Figure 4: Inactivation of B. subtilis 168 spores equilibrated at different water activities (0.10, 0.20, 0.30, 0.50, and 0.70) after a treatment at 150°C and 0.6 MPa. Error bars are SD of at least 3 independent measures

Table 1 :

 1 Glycerol concentration (from Norrish equation) used to equilibrate the water activity in water/glycerol binary solutions.Table 2: Decimal reduction times (in minutes) for B. subtilis 168 dried spores according to the initial water activity of gaseous environment for a treatment at 150°C and a pressure of 0

Acknowledgments

This work is supported by the Conseil Régional de Bourgogne through the plan d'actions régional pour l'innovation (PARI) and the European Union through the PO FEDER-FSE


Nadia Amanzougaghene

Florence Fenollar

Bernard Davoust

Félix Djossou

Muhammad Ashfaq

Idir Bitam

Didier Raoult
email: didier.raoult@gmail.com

Dr Oleg Mediannikov

Prof Raoult Didier

Mephi

Mitochondrial diversity and phylogeographic analysis of Pediculus humanus reveals a new Amazonian clade "F"

Keywords: Pediculus humanus, phylogeography, genetic diversity, Clade F, Amazonia

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Introduction

Sucking lice (Phthiraptera: Anoplura) are obligate blood-feeding ectoparasites of placental mammals that have coevolved with their hosts for more than 65 million years [START_REF] Durden | The sucking lice (Insecta, Anoplura) of the world : a taxonomic checklist with records of mammalian hosts and geographical distributions[END_REF][START_REF] Light | Evolutionary history of mammalian sucking lice (Phthiraptera: Anoplura)[END_REF]. Humans are parasitized by two species of sucking lice, the pubic louse (Pthirus pubis) and head/body lice (Pediculus humanus) [START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. The association between the P. humanus and its human host can be traced back to at least 6 million years ago (MYa) to a common ancestor of humans and chimpanzees [START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. P. humanus includes two ecotypes, head lice (P. h. capitis) and body lice (P. h. humanus), that are morphologically and biologically almost similar but ecologically distinct [START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF][START_REF] Veracx | Biology and genetics of human head and body lice[END_REF]. Head lice are confined to the scalp and feed on human blood every 4-6 hours [START_REF] Veracx | Biology and genetics of human head and body lice[END_REF]. Body lice live in clothing and feed less frequently but take larger blood meals than head lice [START_REF] Veracx | Biology and genetics of human head and body lice[END_REF]. Aside from their role as pests [START_REF] Chosidow | Scabies and pediculosis[END_REF], body lice are the main vectors of serious human pathogens; Rickettsia prowazekii (the causative agent of epidemic typhus), Bartonella quintana (trench fever), Borrelia recurrentis (relapsing fever) and probably Yersinia pestis (plague) [START_REF] Raoult | A Personal View of How Paleomicrobiology Aids Our Understanding of the Role of Lice in Plague Pandemics[END_REF][START_REF] Raoult | The body louse as a vector of reemerging human diseases[END_REF]. It was once believed that only body lice could transmit disease. However, recently several combined epidemiological and laboratory studies have strongly implicated head lice as a vector of infectious agents, although its vectorial capacity is lower as compared to body lice [START_REF] Amanzougaghene | Detection of bacterial pathogens including potential new species in human head lice from Mali[END_REF](Amanzougaghene et al., , 2016a;;[START_REF] Angelakis | Altitude-dependent Bartonella quintana genotype C in head lice, Ethiopia[END_REF][START_REF] Diatta | Prevalence of Bartonella quintana in patients with fever and head lice from rural areas of Sine-Saloum, Senegal[END_REF][START_REF] Goldberger | The Transmission of Typhus Fever, with Especial Reference to Transmission by the Head Louse (Pediculus capitis)[END_REF][START_REF] Kim | Comparison of the proliferation and excretion of Bartonella quintana between body and head lice following oral challenge[END_REF][START_REF] Murray | Virulence of Rickettsia prowazeki for head lice[END_REF][START_REF] Sangaré | Detection of Bartonella quintana in African body and head lice[END_REF].

The genetic diversity of human lice has been extensively studied using mitochondrial genes (mainly cytochrome b [cytb] and cytochrome oxidase subunit 1 [cox1] genes) revealing the presence of five highly divergent mitochondrial clades (A, D, B, C and E) (Amanzougaghene et al., 2016b;[START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF][START_REF] Drali | A New Clade of African Body and Head Lice Infected by Bartonella quintana and Yersinia pestis-Democratic Republic of the Congo[END_REF][START_REF] Kittler | Molecular evolution of Pediculus humanus and the origin of clothing[END_REF][START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. In addition to this inter-clade diversity, human lice also present intra-clade diversity, illustrated by several distinct haplotypes for each clade (Amanzougaghene et al., 2016a;[START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF]. Body lice belong to clades A and D, while head lice encompass the full genetic diversity of clades (Amanzougaghene et al., 2016a;[START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF][START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF]. Clade A is the most common and widely distributed across all continents, whereas the other clades are geographically restricted (Amanzougaghene et al., 2016b;[START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF][START_REF] Raoult | Molecular identification of lice from pre-Columbian mummies[END_REF]. Clade D is restricted to Africa and is found in the Democratic Republic of Congo (DR Congo), the Republic of Congo (Congo-Brazzaville), Ethiopia and Zimbabwe (Amanzougaghene et al., 2016a;[START_REF] Drali | A New Clade of African Body and Head Lice Infected by Bartonella quintana and Yersinia pestis-Democratic Republic of the Congo[END_REF]. Clade B is found in America, Europe, Australia, Algeria, South Africa, Saudi Arabia and has recently been found among the remains of Israeli head lice, which date back about 2,000 years [START_REF] Al-Shahrani | Genetic diversity of the human head lice, Pediculus humanus capitis, among primary school girls in Saudi Arabia, with reference to their prevalence[END_REF]Amanzougaghene et al., 2016b;[START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF][START_REF] Boutellis | Evidence that clade A and clade B head lice live in sympatry and recombine in Algeria[END_REF][START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF][START_REF] Raoult | Molecular identification of lice from pre-Columbian mummies[END_REF]. Clade C has been found in some African and Asian countries including Ethiopia, Congo-Brazzaville, Nepal, Pakistan and Thailand (Amanzougaghene et al., 2016a;[START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF][START_REF] Kittler | Molecular evolution of Pediculus humanus and the origin of clothing[END_REF][START_REF] Raoult | Molecular identification of lice from pre-Columbian mummies[END_REF][START_REF] Sunantaraporn | Molecular survey of the head louse Pediculus humanus capitis in Thailand and its potential role for transmitting Acinetobacter spp[END_REF]. Finally, clade E appears to be specific to West African lice, including Senegal and Mali, where it is highly prevalent, and has recently been identified in the head lice from Nigerian refugees arriving in Algeria and from migrant communities living in Bobigny, France [START_REF] Amanzougaghene | Detection of bacterial pathogens including potential new species in human head lice from Mali[END_REF][START_REF] Candy | Molecular Survey of Head and Body Lice, Pediculus humanus, in France[END_REF][START_REF] Louni | Detection of bacterial pathogens in clade E head lice collected from Niger's refugees in Algeria[END_REF].

Lice are highly host specific and fast-evolving parasites that have evolved in tandem with their primate hosts for thousands of years [START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF][START_REF] Reed | Pair of lice lost or parasites regained: the evolutionary history of anthropoid primate lice[END_REF].

Previous studies have shown that the time of divergence among lice clades (around 2 MYa) was far anterior to the time of modern human divergence, about 200,000 years ago (Light et al., 2008;[START_REF] Tishkoff | The Genetic Structure and History of Africans and African Americans[END_REF]. Previous reports have also suggested that different lice clades have evolved on other archaic hominids, likely those known from 2.3 to 0.03 MYa (such as Homo erectus, Neandertal and Denisovan), and have only switched to modern humans during the recent period of overlaps [START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF][START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. Moreover, the presence of highly divergent clades and their geographical isolation can yield important information regarding the evolutionary history of the lice as well as their human hosts [START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF]. Therefore, a more detailed analysis of genetic diversity in P.

humanus and current distributions of its major clades will provide a more detailed picture of evolutionary pattern of this parasite and will clarify additional events in human evolution.

In the present study, we obtained and analyzed head lice collected from an isolated Native American population from the Wayampi community in Trois-Sauts, French Guiana.

These results are integrated with all prior mitochondrial data from over the world to expand perspectives on the number, distributions and diversification rates of clades of Pediculus lice.

Materials and methods

1 Ethical approval

The Amazonian head lice were collected from infested Amerindians after obtaining their verbal consent or that of their legal representatives in the case of children, because most subjects were illiterate. Local authorities approved and were present at the lice collection. The study was validated by the ethics committee of the Institut Fédératif de Recherche 48, Faculty of Medicine, Marseille, France, under agreement12-017.

2 Louse samples

The Amazonian head louse specimens were recovered in 2013 from Amerindians of the Wayampi community living in Trois-Sauts (2°15'0" N and 52°52'60" W, 122 m), a remote and isolated village in French Guyana. A total of 98 head lice were recovered from 22 individuals. No body lice were found during the examination. Collected lice were then preserved in 70% ethanol before being sent to our laboratory in Marseille (France). All the samples were photographed with a camera (Olympus DP71, Rungis, France) prior molecular analysis. In addition, a total of 327 louse specimens were also included in this study, corresponding to body and head lice collected from several countries. These samples were obtained from the private frozen collection of world lice belonging to our laboratory.

Additional supporting information including, collection locations and numbers of lice tested are described in Table S1.

Moreover, because there were no available 12S sequences of New World monkey louse Pediculus mjobergi in the GenBank database, we have also amplified the 12S gene for this louse. In total three P. mjobergi lice were amplified in this study targeting 12S gene. These samples were obtained from the same P. mjobergi collection, from our laboratory, that was previously used by [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF]. More specifically, the louse specimens were collected from a wild howler monkey, Alouatta caraya, (#B2188) from the Iguazú National Park, Misiones Province [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF].

3 DNA extraction, PCR amplification and sequencing

Genomic DNA was isolated from louse specimens using the DNeasy tissue kit (Qiagen, Courtaboeuf, France) as described previously [START_REF] Amanzougaghene | Detection of bacterial pathogens including potential new species in human head lice from Mali[END_REF]. Three mitochondrial genes cytb, cox1 and 12S ribosomal RNA were targeted for sequencing. PCR amplifications were conducted in a Peltier PTC-200 thermal cycler (MJ Research Inc., Watertown, MA, USA) with the Hotstart Taq-polymerase (Qiagen) in accordance with the manufacturer's instructions. The success of PCR amplification was then verified by electrophoresis of the PCR product on a 1.5% agarose gels. All primers used for these experiments and PCR conditions are described in Table 1. All PCR products were purified using the PCR filter plate Millipore NucleoFast 96 PCR kit (Macherey-Nagel EURL, Hoerdt, France) following the manufacturer's recommendations. The sequence reaction was carried out using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA) as per the manufacturer's instructions. Sequencing was performed with an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Biosystems). Finally, all the generated sequences were assembled and corrected using ChromasPro 1.7 software (Technelysium Pty Ltd., Tewantin, Australia).

4 Sequence analysis.

All the 98 Amazonian head louse sequences for the three mitochondrial genes (cytb, cox1 and 12S) obtained in this study were combined with all the available mitochondrial sequence data of P. humanus (1402 cytb, 743 cox1 and 344 12S) from other parts of the world to generate a global dataset to examine clade diversity in P. humanus. In addition, the newly amplified 12S sequence of P. mjobergi in this study, as well as, seven P. mjobergi sequences (six cytb and one cox1 sequences) reported by [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF], were also included in the analysis (Table S2).

The DNA sequences obtained from the literature varied in length, so sequences were trimmed to produce a dataset that maximized the number of sequences incorporated. The sequences between nucleotide positions 433-705 of cytb (272-bps, according to Genbank Accession KC685778), 748-1031 of cox1 (283-bps, according to Genbank Accession KC685838) and 109-666 of 12S (557-bps, according to Genbank Accession KC685877) were used for analysis. ClustalW alignments were performed in MEGA6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF].

Haplotypes were identified using DnaSP v5.10 software [START_REF] Librado | DnaSP v5: a software for comprehensive analysis of DNA polymorphism data[END_REF]. Finally, we created three datasets for which a total of 1500, 841 and 442 sequences were included, respectively, for cytb, cox1 and 12S (Tables S3-S5).

5 Phylogenetic analysis

Neighbor-joining (NJ) analysis was performed in MEGA6 using the K2P model with pairwise-deletion and 500 bootstrap replicates. The Maximum-likelihood (ML) analysis was also performed in MEGA6 using the Kimura 2-parameter model for nucleotide sequences under 500 bootstrap replicates. The subtree for each clade of lice was collapsed with the "compress/expand subtree" function. Cytb, cox1 and 12S sequences from P. schaeffi (AY695999, KC241887, AY696067, KC241883 and KR706169) were employed as outgroups.

6 Genetic diversity and haplotype analysis

For each dataset, population genetic indices including number of sequences (n), number of polymorphic sites (S), average number of pairwise nucleotide differences (k), nucleotide diversity (π), number of haplotypes (H) and haplotype diversity (Hd) were calculated using DNASP v5.10 software [START_REF] Librado | DnaSP v5: a software for comprehensive analysis of DNA polymorphism data[END_REF]. Kimura-2-parameter (K2P) pairwise distances among the cytb, Cox1 and 12S haplotypes were calculated using MEGA6 with pairwise deletion of gaps and missing data [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. Neutrality tests (Fu & Li's D and Tajima's D) were calculated with DNASP v5.10 ( [START_REF] Librado | DnaSP v5: a software for comprehensive analysis of DNA polymorphism data[END_REF]. In order to investigate the possible relationships between the haplotypes, networks haplotypes for each of the three genes were constructed with the median joining method of Bandelt available in NETWORK5.0 (www.fluxus-engineering.com/sharenet.htm) using equal weights for all mutations [START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF].

Results

A total of 98 head lice collected from 22 Amazonian individuals were analyzed, targeting three mitochondrial genes (cytb, cox1 and 12S). We obtained 11 haplotypes of cytb, 8 haplotypes of cox1 and 13 of 12S that were defined by 31, 25 and 41 polymorphic sites, respectively (Fig. S1-S4). The distribution of the head lice haplotypes identified in this study, according to mitochondrial genes, among the 22 infested Amazonian individuals are presented in Table S6. The generated Amazonian sequences (98 sequences for each gene) were then combined with all available sequences for cytb, cox1 and 12S. The number of haplotypes in each dataset within each clade and their distributions were determined for each gene. The details of the identified haplotypes, their GenBank accession numbers and geographic locations are described in Tables S3-S5.

In addition, six cytb and one cox1 P. mjobergi sequences reported by Drali et al. [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF], as well as, the three P. mjobergi 12S sequences amplified in this study were also included in the analysis (Table S2).

For the cytb dataset, 1506 sequences were included (1500 sequences of P. humanus and 6 sequences of P. mjobergi) from which 105 haplotypes, including two haplotypes from P. mjobergi, were identified from 45 countries on five continents. For the cox1 dataset, 842 sequences were included (841 sequences of P. humanus and one sequence of P. mjobergi) from which 57 haplotypes, including one haplotype from P. mjobergi, were identified from 27 countries on five continents. For the 12S dataset, 445 sequences were included (442 sequences of P. humanus and 3 sequences of P. mjobergi) from which 49 haplotypes, including one haplotype from P. mjobergi, were identified from 18 countries on five continents.

Neighbor-joining (NJ) and Maximum-likelihood (ML) analyses, including all haplotypes, were performed for each of the three mtDNA genes, consistently recovered six highly divergent and well-supported monophyletic clades (Fig. 2 and Fig. S5). Five clades corresponded to the mitochondrial clades previously recognized A, D, B, C and E, while the sixth was novel, here named "clade F". This novel clade consists mainly of Amazonian head lice (a total of 84 out of 98 [85.7%] Amazonian lice sequences belonged to this clade) as well as one haplotype from the New World monkey louse P. mjobergi, while the remaining 14 of the 98 (14.3%) Amazonian lice sequences belonged to clade A.

More precisely, for the 12S gene, the clade F consisted of nine haplotypes; eight haplotypes were for Amazonian lice (referred to here as F19-F26) whose F9 haplotype was the most common (83.3% of 84 sequences), while the ninth haplotype was from P. mjobergi.

For the cox1 gene, 8 haplotypes were identified, six haplotypes were from Amazonian lice (referred to here as F29-F34), one haplotype named F18 consisted of sequences reported by Ascunce et al. [START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF] from Argentina (10 cox1 sequences) and Mexico (2 cox1 sequences), the eighth haplotype was P. mjobergi. Lastly, for the cytb gene, the clade F also included nine haplotypes; eight haplotypes were from Amazonian lice (named here F54 and F1-F7) of which haplotype F54 was the most widespread (84.3% of 89 sequences were from Amazonian head lice sequenced in this study, while 15.7% of 89 sequences also from Amazonian lice and were recovered from Genbank database, the remaining haplotype was from P. mjobergi. It is important to note that the second cytb haplotype of P. mjobergi has the P. humanus' haplotype A5 within clade A (see [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF].

The median-joining networks for all cytb, cox1 and 12S haplotypes corroborated the neighbor joining and Maximum-likelihood phylogenetic reconstructions, with all the recovered haplogroups forming separate clusters represented by six connected subnetworks corresponding to clades A, D, B, C, E and "F" (Fig. 345).

The maximum intra-clades distances at cytb were 1.2%, 1.9%, 1.4%, 1.4%, 1.5% and 1.1% for clades A (n= 34 haplotypes), D (n= 17), B (n= 9), C (n= 13), E (n= 23) and "F" (n= 8), respectively. The maximum intra-clades distances at cox1 were 1.2%, 1.0%, 1.3%, 1.3%, 1.3% and 1.1% for clades A (n= 17), D (n= 7), B (n= 12), C (n= 6), E (n= 7) and "F" (n= 7), respectively. The maximum intra-clades distances at 12S were 0.9%, 1.6%, 0.4%, 1.4%, 0.3% and 0.5% for clades A (n= 15), D (n= 8), B (n= 3), C (n= 10), E (n= 4) and "F" (n= 8), respectively. The nearest neighbors (NN) distances between clades and the nodal supports are presented in Fig. 2A (cytb), Fig. 2B (cox1) and Fig. 2C (12S). The new clade "F" shows divergence from its NN clade B of 5.4%, 3.7% and 3.6%, respectively, at cytb, cox1 and 12S.

Estimates of genetic diversity indices and the results of neutrality tests for cytb, cox1

and 12S are presented in Table 2. The average number of nucleotide diversity (π), pairwise nucleotide differences (k) and haplotype diversity (Hd) varied among the clades of three genes. The highest haplotype diversity was found within clade D in both cytb and 12S genes (Hd= 0.831 and 0.899, respectively, in cytb and 12S), while in cox1 gene, the highest haplotype diversity was found within clade B (Hd = 0.899). Overall, both (k) and (π) were similar in cytb, cox1 and 12S.

Discussion

In this study, we analyzed the genetic diversity of head lice collected from Amazonian individuals of the Wayampi community living in Trois-Sauts, a remote and isolated village.

In total, three mitochondrial genes were targeted (cytb, cox1 and 12S). By coupling these results with all available mitochondrial data of P. humanus from 45 countries, the present study has expanded understanding of its levels, sequence divergence pattern and revealed higher levels of mtDNA diversity in P. humanus corroborating results reported by others [START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF]. To the best of our knowledge, this is the most geographically widespread study to evaluate the mitochondrial genetic diversity in human lice based on three mtDNA genes. Previous studies on P. humanus showed that the maximum distances within clades were 1.4% at cytb and 1.9% at cox1, while the NN distances at these genes were 4.6% and 2.3%, respectively [START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF]. In the present study, the maximum distances within clades were almost similar (cytb 1.9%, cox1 1.3% and 1.6% 12S), while NN distances were higher (cytb 5.6% and cox1 6.5%). These results reflect the extended geographic coverage and the large sample sizes, which allowed the recovery of additional clades and haplotypes within each clade.

Three phylogenetic methods (NN, ML and MJ) at three mtDNA genes, have consistently recovered six highly divergent and well-supported monophyletic clades. Five clades corresponded to the previously recognized mitochondrial clades A, D, B, C and E (Amanzougaghene et al., 2016b;[START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF], while the sixth (clade F) was novel, consisting mainly of head lice from Amazonian individuals analyzed in this study and 12 sequences of head lice from Argentina and Mexico reported by Ascunce et al. [START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF]. In that study, the authors found these sequences to be highly derived B-haplotypes that are separated from the main group by more than seven mutational steps [START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF].

Interestingly, the P. mjobergi 12S and cox1 haplotypes, as well as, one of the two P. mjobergi cytb haplotypes previously classified within clade B by [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF] belong to this new clade F. This result suggests that P. mjobergi may not be a distinct species of P. humanus, but probably an evolutionary lineage of P. humanus species within clades A and F. A similar suggestion was offered by Drali et al. [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF]. Indeed, these authors argued that P. mjobergi was originally a human louse and was switched to new world monkeys from the first humans who reached the American continent thousands of years ago [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF]. Further studies are needed to confirm this hypothesis.

Clade "F" shows a divergence from its NN clade B of 5.4%, 3.7% and 3.6%, respectively, at cytb, cox1 and 12S. Clade B was first described in contemporary lice in the American continent, where it was highly prevalent and diversified [START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF][START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. This discovery and its identification in the lice of pre-Columbian mummies led researchers to deduce initially an American origin for this clade [START_REF] Boutellis | Evidence of sympatry of clade a and clade B head lice in a pre-Columbian Chilean mummy from Camarones[END_REF]. However, its recent discovery among head lice remains from Israel, dating back about 2,000 years, has challenged this hypothesis (Amanzougaghene et al., 2016b). In that study, the authors strongly argued in favor of an Asian origin of clade B, that resulted probably from a recent host switch from Neanderthals or Denisovans to modern humans which was followed by its introduction into the New World with the migration of early peoples (Amanzougaghene et al., 2016b;[START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF][START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. Because clade F was found only in head lice from Mexico and Argentina and it was the dominant lineage found in the Amazonian lice, knowing that Amazonia is one of the few places in the world that has not been strongly affected by globalization. This clade F may be the descendants of a pre-Columbian population and was derived from clade B brought by the first humans who had reached the American continent via the Bering straits thousands of years ago, thus representing Native America louse mitochondrial diversity. Interestingly, Ascunce et al. [START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF] have also proposed a similar suggestion for their sequences from Mexico and Argentina that we have reclassified here as members of clade F.

Previous studies reported that clade A is the most common and has a global distribution [START_REF] Amanzougaghene | Detection of bacterial pathogens including potential new species in human head lice from Mali[END_REF][START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF], results supported by its detection in approximately 46% of the analyzed lice from 49 countries on the five continents. Furthermore, the clade A subnetworks at the three analyzed mitochondrial genes (Fig. 345) were star-like in structure, combined with its significant negative Tajima's D value (-2.322, -1.866 and -2.055, respectively at cytb, cox1 and 12S; P<0.05), indicating the signature of population expansion for this clade [START_REF] Aris-Brosou | The impact of population expansion and mutation rate heterogeneity on DNA sequence polymorphism[END_REF], and corroborating the results reported by others [START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. In addition, [START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF] estimated that the demographic expansion of this clade occurred about 100,000 years ago, coinciding with the out-of-Africa expansion of Homo sapiens, thus reflecting a codemographic pattern between lice and humans [START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. Clade D (referred as clade E in [START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF] diverged from clade A between 0.37 and 0.54 MYa [START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF] and is restricted to Central Africa including DR Congo and Congo-Brazzaville, where it was detected mainly among indigenous Pygmy populations (Amanzougaghene et al., 2016a;[START_REF] Drali | A New Clade of African Body and Head Lice Infected by Bartonella quintana and Yersinia pestis-Democratic Republic of the Congo[END_REF]. This clade has also been reported in lice in Ethiopia (Amanzougaghene et al., 2016a) and we identified in this study its occurrence, for the first time, in body lice from Zimbabwe.

Our sampling did not encounter any new specimens of clade C, so it remains restricted to Africa and Asia (Amanzougaghene et al., 2016a;[START_REF] Ascunce | Mitochondrial diversity in human head louse populations across the Americas[END_REF][START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF][START_REF] Sunantaraporn | Molecular survey of the head louse Pediculus humanus capitis in Thailand and its potential role for transmitting Acinetobacter spp[END_REF]. Given its early divergence in the Pediculus tree around 2 MYa, this clade may have evolved on archaic hominids in Asia or Africa such as H. erectus [START_REF] Light | Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data[END_REF][START_REF] Reed | Genetic Analysis of Lice Supports Direct Contact between Modern and Archaic Humans[END_REF]. Lastly, clade E (referred as clade D in [START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF] diverged from the MRCA of clade C between 0.28 and 0.42 MYa [START_REF] Ashfaq | High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera)[END_REF]. This clade consists of head lice from West Africa including Senegal and Mali where it was highly prevalent [START_REF] Amanzougaghene | Detection of bacterial pathogens including potential new species in human head lice from Mali[END_REF]. Its recent detection in the head lice of Nigerian refugees arriving in Algeria and migrant communities living in Bobigny (France) is probably the result of a recent migratory flow from West African countries [START_REF] Candy | Molecular Survey of Head and Body Lice, Pediculus humanus, in France[END_REF][START_REF] Louni | Detection of bacterial pathogens in clade E head lice collected from Niger's refugees in Algeria[END_REF].

Conclusion

Our study underlines the importance of the use of mitochondrial genes in the analysis of phylogeographic patterns and genetic diversity of P. humanus. Six highly divergent and well-supported monophyletic clades were identified. Five clades corresponded to the previously recognized mitochondrial clades A, D, B, C and E, while the sixth "clade F'' was novel. The new clade F was found mainly in Amazonia, where it is also shared with the monkey louse P. mjobergi and could therefore represent Native American louse mitochondrial diversity.

The recovery of additional haplotypes within each of the five clades (A, D, C, E and B) along with the discovery of new clade F, demonstrate that levels of genetic diversity in P.

Figure captions

Figure 1 .

 1 Figure 1. Map showing the head lice collection site from the Amerindians of the

Figure 2 .Figure 3 .

 23 Figure 2. Neighbor-joining cluster analysis of Cytb (A), Cox1 (B) and 12S (C)

Figure 4 .Figure 5 .

 45 Figure 4. Cox1 haplotype networks of human body and head lice. Each circle indicates a

Table 1 . Primer sequences used in this study

 1

	Target gene	Primer	Primer sequences (5′-3′)	Product	Tm	source
		name		size (bp)
	Cytochrome b	cytbF	GAGCGACTGTAATTACTAATC 348	56°C (Li et al.,
		cytbR	CAACAAAATTATCCGGGTCC			2010)
	Cytochrome	cox1F	GGAGTGAGTTCGATTTTAG	828	55°C This study
	oxidase subunit 1	cox1R GTGCTGAGGAAAGAAAGTC
	12S ribosomal	12SF	CAGCACTAGCGGTCATACAT 596	56°C This study
	RNA	12SR	AATGACGGGCGATATGTAC

Table 2 . Analysis of genetic diversity indices and neutrality tests (Fu & Li's D and 423 Tajima's D) on mitochondrial cytb, cox1 and 12S sequences

 2 : number of sequences; S: number of polymorphic sites; k: average number of pairwise nucleotide differences; π: nucleotide diversity; h: number of haplotypes; Hd: haplotype diversity. Tajima's D: A negative Tajima's D signifies an excess of low frequency polymorphisms relative to expectation. A positive Tajima's D signifies low levels of both low and high frequency polymorphisms. Statistical significance: Not significant, P > 0.10.

	424
		n	S	K	π	h	Hd	Fu & Li's D	Tajima's D
	Cytb all	1500	96	20.341	0.075	105	0.999	-2,878 (P <0.05) S*	-0.172 (P> 0.1) NS
	Clade A	769	33	3.098	0.011	34	0.750	-3.751 (P <0.02) S**	-2.322 (P < 0.01) S**
	Clade D	69	20	5.044	0.018	17	0.831	-1.425 (P> 0.1) NS	-0.583 (P> 0.1) NS
	Clade B	200	13	3.694	0.013	9	0.789	-1.633 (P> 0.1) NS	-1.357 (P> 0.1) NS
	Clade F	104	11	2.750	0.010	9	0.700	-1.924 (P < 0.05) S*	-1.757 (P < 0.05) S*
	Clade C	205	16	3.744	0.014	13	0.792	-1.471 (P> 0.1) NS	-1.151 (P> 0.1) NS
	Clade E	153	26	4.075	0.015	23	0.803	-2.747 (P < 0.05) S*	-1.752 (P > 0.05) NS
	COI all	842	67	17.255	0.061	57	0.946	-0.065 (P > 0.1) NS	0.457 (P> 0.1) NS
	Clade A	443	21	3.294	0.012	17	0.769	-2.244 (P > 0.05) NS	-1.866 (P < 0.05) S*
	Clade D	48	8	2.762	0.010	7	0.739	-0.971 (P > 0.1) NS	-1.318 (P> 0.1) NS
	Clade B	157	12	3.697	0.013	12	0.786	-0.564 (P > 0.1) NS	-0.292 (P > 0.1) NS
	Clade F	139	8	2.952	0.010	8	0.738	-0.473 (P > 0.1) NS	-0.503 (P > 0.1) NS
	Clade C	25	8	3.667	0.012	6	0.782	0.457 (P > 0.1) NS	0.274 (P > 0.1) NS
	Clade E	30	8	3.524	0.012	7	0.781	0.321 (P > 0.1) NS	0.415 (P > 0.1) NS
	12S all	442	110	26.928	0.048	49	0.964	-0.332 (P > 0.1) NS	-0.130 (P > 0.1) NS
	Clade A	145	30	4,886	0.009	15	0.830	-2.502 (P < 0.05) S*	-2.055 (P < 0.05) S*
	Clade D	62	23	8.821	0.016	8	0.899	0.337 (P > 0.1) NS	-0.029 (P > 0.1) NS
	Clade B	36	3	0.271	0.001	3	0.679	-0.319 (P > 0.1) NS	-1.399 (P > 0.1) NS
	Clade F	87	10	2.893	0.005	9	0.746	-1.612 (P > 0.1) NS	-1.589 (P > 0.05) NS
	Clade C	69	24	7.622	0.014	10	0.885	-0.096 (P > 0.1) NS	-0.483 (P > 0.1) NS
	Clade E	43	3	1.500	0.003	4	0.606	-0.754 (P > 0.1) NS	-0.754 (P > 0.1) NS

n

Acknowledgments

The authors thank the Amerindian populations of the Wayampi community living in Trois-Sauts for allowing them to collect the louse specimens.

Funding: This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the program « Investissements d'avenir », reference ANR-10-IAHU-03, the Région Provence-Alpes-Côte d'Azur and the European funding FEDER PRIMI.

humanus is higher than previously thought, reinforcing the importance of continuing to survey and phylogeographically characterize human lice. Table S1. Additional louse specimens included in this study, obtained from the private frozen collection of world lice belonging to our laboratory.

Supporting Information

Table S2. Pediculus mjobergi sequences from new world monkey (Alouatta caraya)

included in this study. The cox1 and cytb P. mjobergi sequences analyzed in this study were those reported by [START_REF] Drali | Host switching of human lice to new world monkeys in South America[END_REF] collected from two monkey individuals B2188 and B1395. The 12S sequences were amplified in this study from three P. mjobergi specimens from monkey individual B2188. Table S3. Geographic occurrences and frequencies of cytb haplotypes of human head and body lice. Table S4. Geographic occurrences and frequencies of cox1 haplotypes of human head and body lice. Table S5. Geographic occurrences and frequencies of 12S haplotypes of human head and body lice. Table S6. Distribution of the head lice haplotypes identified in this study, according to mitochondrial genes, among the 22 infested Amazonian individuals.