Laure Mathews-Martin

Manon Namèche

Gwenaël Vourc'h

Sabrina Gasser

Isabelle Lebert

Valérie Poux

Séverine Barry

Séverine Bord

Jeremy Jachacz

Karine Chalvet-Monfray

Gilles Bourdoiseau

Sophie Pamies

Diana Sepúlveda

Sandrine Chambon-Rouvier

Magalie René-Martellet
email: magalie.renemartellet@vetagro-sup.fr

Questing tick abundance in urban and peri-urban parks in the French city of Lyon

Keywords: Ixodes, Dermacentor, Ticks, Park, Lyon, France

Background: In Europe, ticks are responsible for the transmission of several pathogens of medical importance, including bacteria of the Borrelia burgdorferi (s.l.) complex, the agents of Lyme borreliosis. In France, the Auvergne Rhône-Alpes region is considered a hot spot for human tick-borne pathogen infections, with an estimated annual rate of 156 cases of Lyme borreliosis per 100,000 inhabitants. Although several studies have assessed the abundance of ticks in rural areas, little consideration has been given thus far to urban green spaces in France.

Methods: This study aimed to estimate tick abundance in three parks, two urban (U1, U2) and one peri-urban (PU), in and around the city of Lyon (France). A forest in a rural area was used as a control (C). Tick sampling campaigns were performed in each site in April, May, June, July, and October 2019 using the dragging method. One hundred transects of 10 m 2 each were randomly chosen in each park in places frequented by humans. The sampling sessions were carried out under semi-controlled abiotic conditions. Ticks were stored in 70% ethanol and identified to species and developmental stage under a light microscope using morphological keys.

Results: A total of seven ticks (nymphs and adults) were collected in the two urban parks (six in U1 and one in U2), while 499 ticks were sampled in the peri-urban park. Of the 506 ticks collected, 504 were identified as Ixodes ricinus, one as Dermacentor marginatus, and one as Ixodes frontalis. In the peri-urban park, ticks were mainly collected under the forest cover and at forest edges. Tick density under forest cover was 7.1 times higher in the control site than in the peri-urban park throughout the survey period.

Conclusions: This study confirmed the presence of ticks in all of the parks surveyed, although their occurrence in the urban parks was very rare compared to the peri-urban park and the control site. These results should serve as a basis for the implementation of preventive measures.

Background

Ticks are the most important vectors of pathogens to humans and animals in Europe, especially in France. In particular, Ixodes ricinus, which is widespread in mainland France, is responsible for transmission of the agents of Lyme borreliosis, one of the most prevalent tick-borne zoonoses in Europe. A significant increase in human cases of Lyme borreliosis was noted in mainland France from 2015 to 2018, reaching 104 cases/100,000 inhabitants in 2018. The Auvergne-Rhône-Alpes region is one of the three most affected regions, with an estimated annual rate of 156 cases/100,000 inhabitants in 2016 [START_REF] Vaissière | Surveillance de la borréliose de Lyme[END_REF][START_REF] Fournier | Épidémiologie de la borréliose de Lyme en médecine générale, France métropolitaine[END_REF]. Although the risk of exposure to ticks, in particular to I. ricinus, has been previously assessed in several rural and peri-urban areas in France [START_REF] Paul | Environmental factors influencing tick densities over seven years in a French suburban forest[END_REF][START_REF] Marchant | Infection of Ixodes ricinus by Borrelia burgdorferi sensu lato in periurban forests of France[END_REF][START_REF] Papillon | Periurban forests shifting from recreation to wellness[END_REF][START_REF] Dernat | Tick bite risk as a socio-spatial representation -an exploratory study in Massif Central[END_REF], urban parks have only rarely been explored [START_REF] Dutraive | Etude de la corrélation entre la densité de la tique Ixodes ricinus et la densité des chevreuils en région toulousaine[END_REF][START_REF] Gilot | Bio-écologie des tiques induisant les pathologies les plus importantes en France[END_REF][START_REF] Pichot | Ixodes populations (xodes ricinus Linné, 1758; Ixodes hexagonus Leach, 1815) in the city of Lyon (France) and its outskirts[END_REF][START_REF] Rizzoli | Ixodes ricinus and its transmitted pathogens in urban and peri-urban areas in Europe: new hazards and relevance for public health[END_REF].

Mitigating the risk of human exposure to ticks and tickborne diseases has been a priority for the French public health authorities since the launch of the National Lyme Disease Plan in 2016 [START_REF]Plan national de lutte contre la maladie de Lyme et les maladies transmissibles par les tiques[END_REF]. At a local scale, the implementation of this plan relies on the involvement of territorial health authorities who are responsible for the management of urban areas. However, these efforts could potentially be complicated by 're-greening' initiatives in many cities. These actions aim to increase vegetated areas to improve the welfare of residents and enhance biodiversity levels. Such programmes could have the unintended side effect of expanding microhabitats suitable for tick development. Moreover, urban green spaces are highly frequented by humans. Thus, high tick densities in urban parks could lead to a high risk of human infection by tickborne pathogens.

The aim of this study was to provide a preliminary snapshot of questing tick abundance and diversity in urban and peri-urban parks in the city of Lyon (France) using the dragging method of sampling. Our results were compared to a control site situated in a natural environment in the same area. In each park, we paid special attention to environments with a higher risk of tick exposure in order to provide useful data for the implementation of preventive measures.

Methods

Study sites

The survey was performed in three parks (two urban and one peri-urban) in and around the city of Lyon. The location of the study sites is presented in Fig. 1 de Lacroix-Laval (PU), is situated in a north-western suburban area (11 km from the center of Lyon) at the interface of the city and the foothills of the Monts du Lyonnais. Information on flora and fauna composition was obtained from the management staff of each park.

U1 is composed of a recreational park of 105 ha (Parc de la Tête d'Or) and an area of 7 ha (Parc du Brétillod). The site contains a 16-ha lake. Vegetation cover is mainly lawn with small groups of trees and a wooded area of approximately 6.5 ha. More than 8800 trees have been inventoried, with 61% of those being deciduous and 36.5% coniferous. Small areas are left unmanaged to limit the impact of mowing and tree cutting on biodiversity. The Brétillod area is subjected to flooding along the River Rhône. The majority of the trees in this area are deciduous. It also includes a meadow cut once a year which was explored as part of the study. Only domestic animals (cats, dogs, and horses) and small wild animals (e.g. birds and squirrels) are generally reported in U1.

U2 is a recreational park of 188 ha, which is considered a 'green lung' in the highly urbanized environment of eastern Lyon. More than 75% of the park is covered by grass, which is frequently cut, with small groups of trees (mainly coniferous). Only 2% of the park is managed as a semi-natural environment, with deciduous trees and brambles on the ground. Two small ponds are present. The animal community is roughly the same as in U1.

PU is a partially enclosed recreational park of 115 ha. Approximately 70% of the park is covered by woods, composed mainly of deciduous trees (90%), of which 80% are oaks and hornbeams. The open areas are meadows that are either mowed on a regular basis or only once or twice a year to meet an Ecolabel standard. A "jardin à la française" (French formal garden) is also accessible. A stream runs through the forest and two small ponds are present. The park is highly frequented by domestic and wild animals, including wild ungulates such as roe deer.

The 4-ha control site (C) has been monitored for questing ticks on a monthly basis since 2016 as part of the CCEID-CLIMATICK project [START_REF] Chalvet-Monfray | Bilan de 5 ans de suivi mensuel d'un réseau d'observatoires d'Ixodes ricinus[END_REF]. The C site is characterized by wooded areas, forest edges, and roadside tracks. It is located 1 km away from the PU site, to which it is linked by an ecological corridor. Records collected in 2019 in the C site were used as the basis for comparison with the observations obtained for this study from the urban and peri-urban parks.

Study design

In the three parks, surveillance campaigns were performed monthly, a few days apart, during the main period of tick activity, which is well-known thanks to the active surveillance that has been carried out in the region since 2016 as part of the CCEID-CLIMATICK project.

In order to collect as many ticks as possible while maximizing the efficiency of collection, we selected days for sampling that had optimal meteorological conditions for the activity of ticks, I. ricinus in particular. Specifically, the days of sampling were chosen using the following criteria: rainless, windspeed less than 10 km/h, temperature under the forest cover between 12-25 °C, and humidity under the forest cover at or above 50%. For this, a weather station was placed 20 cm from the ground under tree cover in each park to monitor local temperature and hygrometry (HOBO ® , MX2300, MX2302, Onset Computer Corporation ® ). The time period during the day for the collection of questing ticks was determined using the temperature and hygrometry curves of the five previous days and systematically confirmed by temperature and hygrometry records at the beginning and the end of the collection.

Every month, 100 different transects of 10 m 2 were randomly selected in each park. Transects were not the same from month to month. The aim was to explore all environments that could be frequented by humans. In the control site, the same 10 transects of 10 m 2 were sampled every month.

Five types of vegetation were defined: forest, footpath/ track inside the forest, forest edge, footpath/track in an open area, and meadow. Forest and footpath/track inside the forest were described as closed environments (CE), forest edge as a transitional environment (TE), and footpath/track in an open area and meadow as open environments (OE). A GPS waypoint (Garmin, Dakota ® 10) and a picture were taken at each transect.

Tick collection, storage, and identification

Ticks were collected using a 1-m 2 white flannel cloth that was dragged on the ground [START_REF] Boyard | Local environmental factors characterizing Ixodes ricinus nymph abundance in grazed permanent pastures for cattle[END_REF]. Both sides of the cloth were closely examined for the presence of ticks. Only nymphs and adults were collected. All questing ticks were stored in Eppendorf tubes in 70% ethanol at ambient temperature until morphological identification was performed under a light microscope using identification keys [START_REF] Estrada-Peña | Ticks of Europe and North Africa: a guide to species identification[END_REF].

Data analysis

Positive transects (transects where at least one tick was collected) and tick densities (number of ticks per sampled area of 10 m 2 ) were mapped using QGIS software (Desktop 3.4.3 [START_REF]QGIS Geographic Information System[END_REF]). A Chi-square Test or Fisher's exact test was performed to compare the number of tick-positive transects between parks or between environments within a park. For all tests, the significance threshold was defined as P < 0.05. The statistical analyses were performed using R (4.0.0.) [START_REF] Development | R: a language and environment for statistical computing[END_REF].

Results

Tick collection in parks

In the three parks, surveillance campaigns were performed monthly, a few days apart, in April, May, June, July, and October 2019, when meteorological conditions fulfilled the criteria of the study design. No sampling was performed in August and September due to the particularly hot and dry weather conditions.

A total of 506 ticks were collected (499 in PU, 6 in U1, 1 in U2) from the 5000 m 2 area (100 × 10 m 2 × 5 visits) surveyed in each park during the study period. The majority of ticks were identified as I. ricinus (n = 504, of which 94% (n = 474) were nymphs and 6% (n = 30) were adults). We also identified one adult male of Dermacentor marginatus (in U1) and one nymph of Ixodes frontalis (in PU). The spatial distribution of tick-positive and -negative transects in the three parks surveyed, and their relative tick densities, are presented in Fig. 2. Tick densities observed in the peri-urban park (PU) were at least 100 times higher than those observed in the urban parks (U1, U2) (Table 1). Specifically, 29.2% (146/500 transects) of the transects yielded ticks in PU versus 1.2% in U1 (6/500 transects) and 0.2% in U2 (1/500 transects). The number of positive transects was significantly different among the parks (Chi-square test, χ 2 = 464.85, df = 3, P < 0.0001).

Tick densities in closed, transitional, and open environments in PU

Of the three parks surveyed, only the PU site contained all three distinct types of environments (CE, TE, and OE) and an adequate sample size of questing ticks from each type to permit an analysis of the habitat effect (Table 2). Tick density (corresponding to 498 Ixodes ricinus and 1 Ixodes frontalis in PU) was estimated to be 32 times higher in closed environments (CE) and 16 times higher in transitional environments (TE) than in open environments (OE). Similarly, a higher number of positive transects was found in CE (44.4%; 119/268 transects) than in TE (22.4%; 19/85 transects) or OE (5.4%; 8/147 transects). Throughout the study period, there was a significant difference among the environment types in PU concerning the number of positive transects (Chi-square test, χ 2 = 72.02, df = 2, P < 0.0001).

Comparison of PU with the control site (C)

A total of 493 ticks (all identified as I. ricinus) were collected in April, May, June, July, and October (10 transects per month) in the 500 m 2 area (10 × 10 m 2 × 5 visits) sampled at site C. Of the questing ticks collected, 98% (n = 483) were nymphs and 2% (n = 10) were adults. Only closed and transitional environments were present in this location. We thus compared tick densities and the number of positive transects between the PU and C locations, considering only ticks that were sampled in closed and transitional environments (Table 3). The highest tick density was observed in June in both sites (Fig. 3). Due to the low number of questing ticks collected in the urban parks (U1 and U2), these sites were excluded from comparisons to site C. In these parks, it was not possible to identify any seasonal pattern of tick activity. Tick density was higher in site C than in PU, regardless of the period assessed (all five months or only June) (Table 3). During the peak of activity in June, tick density was 5.5 times higher in site C (19.1 ticks/10 m 2 ) than in PU (3.5 ticks/10 m 2 ). Overall, tick density was 7.1 times higher in site C than in PU.

When examining the overall percentage of positive transects from closed and transitional environments, we detected a statistically significant difference between the two study sites. Specifically, the percentage of positive transects was two times higher in site C than in PU (Fisher's exact test, P < 0.0001). However, such a difference was not observed in June (Fisher's exact test, P = 0.11).

Discussion

The aim of the present study was to estimate tick abundances in urban and peri-urban parks in the city of Lyon (France). We confirmed the presence of ticks in all parks surveyed, mainly of I. ricinus, with significant differences in abundance among parks.

The earliest studies on the abundance of ticks in urban environments date back to the late 1980s in the USA [START_REF] Falco | Potential for exposure to tick bites in recreational parks in a Lyme disease endemic area[END_REF]. Lyme disease is endemic in the USA, and this type of survey has been used in many published studies [START_REF] Noden | Risk of encountering ticks and tick-borne pathogens in a rapidly growing metropolitan area in the US Great Plains[END_REF][START_REF] Mead | Risk factors for tick exposure in suburban settings in the northeastern United States[END_REF]. Likewise, the presence of ticks in urban parks has been investigated in several European countries [START_REF] Rizzoli | Ixodes ricinus and its transmitted pathogens in urban and peri-urban areas in Europe: new hazards and relevance for public health[END_REF][START_REF] Corrain | Study on ticks and tick-borne zoonoses in public parks in Italy. Zoonoses[END_REF][START_REF] Buczek | Threat of attacks of Ixodes ricinus ticks (Ixodida: Ixodidae) and Lyme borreliosis within urban heat islands in south-western Poland[END_REF][START_REF] Nelson | Tick abundances in south London parks and the potential risk for Lyme borreliosis to the general public[END_REF][START_REF] Oechslin | Prevalence of tick-borne pathogens in questing Ixodes ricinus ticks in urban and suburban areas of Switzerland[END_REF][START_REF] Heylen | Ticks and tick-borne diseases in the city : role of landscape connectivity and green space characteristics in a metropolitan area[END_REF]. In France, the first surveys of ticks in urban environments (specifically, in and around the city of Lyon) date back to the 1990s [START_REF] Pichot | Ixodes populations (xodes ricinus Linné, 1758; Ixodes hexagonus Leach, 1815) in the city of Lyon (France) and its outskirts[END_REF][START_REF] Quessada | Prevalence of Borrelia burgdorferi species and identification of Borrelia valaisiana in questing Ixodes ricinus in the Lyon region of France as determined by polymerase chain reaction-restriction fragment length polymorphism[END_REF], but have not been updated since. Recently, participatory tools have been developed to allow citizens to report tick bites. These declarations have indicated a high incidence of tick bites in urban and peri-urban areas. Specifically, 29% of tick bites reported via the "Signalement-Tique" application from July 2017 to December 2018 occurred, according to the declarants' assertions, in parks and gardens [START_REF] Durand | CiTIQUE, les sciences participatives au service de tous[END_REF]. Likewise, in Belgium, 38% of tick bites were recorded near the houses of the respondents, and 2.2% in a city park, respectively [START_REF] Tersago | Surveillance des morsures de tiques en Belgique[END_REF]. To further evaluate the risk of human-tick encounters, efforts have been made in Europe to develop active surveillance programmes of ticks in parks that are highly frequented by humans. Temperature and relative humidity are known to shape the host-seeking activity of ticks [START_REF] Ruiz-Fons | Factors driving the abundance of Ixodes ricinus ticks and the prevalence of zoonotic I. ricinus-borne pathogens in natural foci[END_REF][START_REF] Agoulon | Dynamique des populations de tiques et liaison avec les facteurs environnementaux[END_REF]. Thus, by using weather stations, we were able to choose days for collection that had appropriate abiotic conditions, in particular suitable temperature and humidity near the ground. In cities, abiotic factors are of primary importance for the survival of the off-host stages of ticks. The urban heat island effect has been linked to reduced tick abundance and activity, and therefore a lower prevalence of Lyme disease [START_REF] Buczek | Threat of attacks of Ixodes ricinus ticks (Ixodida: Ixodidae) and Lyme borreliosis within urban heat islands in south-western Poland[END_REF]. Previous tick abundance surveys performed in Lyon and its outskirts [START_REF] Pichot | Ixodes populations (xodes ricinus Linné, 1758; Ixodes hexagonus Leach, 1815) in the city of Lyon (France) and its outskirts[END_REF][START_REF] Quessada | Prevalence of Borrelia burgdorferi species and identification of Borrelia valaisiana in questing Ixodes ricinus in the Lyon region of France as determined by polymerase chain reaction-restriction fragment length polymorphism[END_REF] focused on a single sampling event (30 min) of 46 different sites in spring, in order to increase the coverage area. Here, instead, we chose to focus on only three parks, but with monthly surveillance and a higher number of samples in each park. The sampling effort targeted the areas that are most frequented by humans. Consequently, areas that were overgrown by vegetation or less accessible were not sampled.

Throughout the five months of the survey, we explored 5000 m 2 in each park (15,000 m 2 in total), which represents 0.27% of the area of U2, 0.43% of PU, and 0.52% of U1. As a comparison, a total of 12,420 m 2 were sampled in 15 urban and suburban plots in the outskirts of Antwerp, in Belgium, in 2014 and 2016. In that study, the sampling effort varied from 0.07% for the largest plot (132 ha) to 1.63% for a suburban plot of 10.3 ha [START_REF] Heylen | Ticks and tick-borne diseases in the city : role of landscape connectivity and green space characteristics in a metropolitan area[END_REF]. In terms of abundance, the values we obtained in PU were higher than those recorded in Italy in 2012, where the highest density was 0.63 ticks per 10 m 2 in a thickly forested natural reserve situated on the outskirts of Imola [START_REF] Corrain | Study on ticks and tick-borne zoonoses in public parks in Italy. Zoonoses[END_REF]. Tick density in PU was similar to that found in Belgium, where the abundance of I. ricinus in the largest suburban plot sampled (132 ha) was 1.5 ticks per 10 m 2 (nymphs and adults). It is, however, difficult to compare abundances among countries due partly to climatic differences, which have also been shown to influence tick abundance and activity [START_REF] Ruiz-Fons | Factors driving the abundance of Ixodes ricinus ticks and the prevalence of zoonotic I. ricinus-borne pathogens in natural foci[END_REF][START_REF] Agoulon | Dynamique des populations de tiques et liaison avec les facteurs environnementaux[END_REF].

The discrepancies regarding tick abundances between the three analyzed parks could be due to various factors. First, PU is regularly frequented by wild fauna, especially wild ungulates (confirmed by the observations of the park employees). Instead, these animals are extremely rarely observed in the intra-urban parks. Here, the low tick densities observed within the urban parks are likely the result of (i) altered ecological corridors that do not allow wild ungulates to easily enter these locations and (ii) the (probably) low contribution of other large animals (including domestic animals, which may be treated with acaricides on a regular basis) to the maintenance of tick life-cycles.

Another factor that likely contributed was the habitat composition of the sites. Of the three sites, PU has the most favorable habitat for Ixodes spp. ticks, with a forest cover that is mainly composed of hardwoods and brambles in the undergrowth [START_REF] Tack | The abundance of Ixodes ricinus ticks depends on tree species composition and shrub cover[END_REF]. The Parc du Brétillod (in U1) is a greenway that connects with the Parc de la Feyssine and Grand parc de Miribel-Jonage, which are located upstream on the right bank of the Rhône. This space is managed as naturally as possible. However, the soil is very sandy and does not retain much moisture, which probably limits tick establishment. The remainder of U1 (Parc de la Tête d'Or) and U2 have much sparser woodland cover and are regularly mowed, which is also unfavorable for tick presence.

As a control, we used a site on the north-western outskirt of the city of Lyon that has been surveyed for questing ticks since 2016; data from this site were used for comparison of both tick density and the seasonal peak of activity. During the surveillance period, similar patterns of Ixodes spp. activity were observed between the PU and C sites, but tick density was 7.1 times higher in C than in PU. These results suggest that tick abundance increases from an urban center to the outskirts, as previously reported in the same area [START_REF] Pichot | Ixodes populations (xodes ricinus Linné, 1758; Ixodes hexagonus Leach, 1815) in the city of Lyon (France) and its outskirts[END_REF][START_REF] Quessada | Prevalence of Borrelia burgdorferi species and identification of Borrelia valaisiana in questing Ixodes ricinus in the Lyon region of France as determined by polymerase chain reaction-restriction fragment length polymorphism[END_REF] and in recent studies performed in Europe [4, 20-22, 24, 31, 32]. In the 1990s, the north-eastern and western peri-urban areas of Lyon were highlighted as being more at risk with respect to tick abundance, given their proximity to the wooded mountainous region of Les Monts du Lyonnais [START_REF] Pichot | Ixodes populations (xodes ricinus Linné, 1758; Ixodes hexagonus Leach, 1815) in the city of Lyon (France) and its outskirts[END_REF].

In the Auvergne-Rhône-Alpes region, ticks from the genera Dermacentor and Ixodes are widespread; the former is responsible for transmission of the agents of canine and equine piroplasmosis, while the latter carries bacteria of the Borrelia burgdorferi (s.l.) complex, which cause Lyme disease [START_REF] Gilot | Bio-écologie des tiques induisant les pathologies les plus importantes en France[END_REF]. Previous studies in and around the city of Lyon reported only ticks of the genus Ixodes [START_REF] Gilot | Bio-écologie des tiques induisant les pathologies les plus importantes en France[END_REF][START_REF] Pichot | Ixodes populations (xodes ricinus Linné, 1758; Ixodes hexagonus Leach, 1815) in the city of Lyon (France) and its outskirts[END_REF]. This pattern of tick species diversity was also observed in the present study, where Ixodes spp. ticks represented the vast majority of collections. The only exception was a single Dermacentor marginatus tick collected in U1. Dermacentor ticks are generally found in rural areas, in open environments such as grazed meadows, roadsides, and forest edges [START_REF] Nosek | The ecology and public health importance of Dermacentor marginatus and D. reticulatus ticks in central Europe[END_REF], and, at the adult stage, often parasitize horses and dogs. Immature stages are endophilic (nidicolous) and can be found on rodents. The U1 park often hosts horses that are bred in meadows outside of the city and is also highly frequented by dogs. Ixodes frontalis, a bird-associated tick [START_REF] Pérez-Eid | Les tiques: identification, biologie, importance médicale et vétérinaire[END_REF], can also be occasionally found questing on vegetation [START_REF] Agoulon | Unravelling the phenology of Ixodes frontalis, a common but understudied tick species in Europe[END_REF]. However, there is to date no evidence of local (i.e. intra-urban) reproduction of either species.

Because this was a preliminary study, screening for potential tick-borne pathogens was not performed. Previous work has shown that the pathogen burden in ticks may be higher in developed urban areas compared to rural areas [START_REF] Buczek | Threat of attacks of Ixodes ricinus ticks (Ixodida: Ixodidae) and Lyme borreliosis within urban heat islands in south-western Poland[END_REF]. In a study conducted in this same region in the 1990s, 13.5% of the ticks analysed were infected by Borrelia burgdorferi (s.l.), reaching 14.5% in Les Monts du Lyonnais [START_REF] Quessada | Prevalence of Borrelia burgdorferi species and identification of Borrelia valaisiana in questing Ixodes ricinus in the Lyon region of France as determined by polymerase chain reaction-restriction fragment length polymorphism[END_REF]. In Switzerland, the infection rate of urban ticks was shown to be equivalent to that of rural areas, with a tendency toward co-infection (bacteria, viruses, parasites) [START_REF] Oechslin | Prevalence of tick-borne pathogens in questing Ixodes ricinus ticks in urban and suburban areas of Switzerland[END_REF]. Thus, tick-borne pathogen surveillance should be a priority in the future to evaluate the risk of human exposure to tick-borne pathogens. Although the overall human threat in urban and peri-urban parks appears to be low, the risk, which is the combination of the hazard and human exposure, could still be high due to increased visitation rates by humans, and must be taken into account in the design of preventive measures.

Conclusions

This study confirmed the presence of ticks, mainly of the species I. ricinus, in all three parks surveyed, although notable differences in tick density were observed. Such differences are probably linked with variations in landscape connectivity and the densities of wild fauna, especially ungulates. Notably, ticks were rarely found in intra-urban parks, particularly when compared to a periurban environment in the same period. Tick density was much higher in forests and forest edges than in lawns, where the number of ticks was low. A more-accurate description of green-space management, human activities, and a fauna inventory could help to improve our understanding of the factors that influence tick densities and tick bite risk within urban parks. Surveillance programs should be continued to assess the possible introduction of new tick species, particularly in the contexts of global warming and of urban policies of 're-greening' .

Fig. 1 .

 1 Fig. 1. Location of the parks (U1, U2, PU) and control site (C) in the region of Lyon. Map background BDOrtho ® 50 cm IGN. Abbreviations: U1, U2, urban parks; PU, peri-urban park; C, control site

Fig. 2 .

 2 Fig. 2. Distribution of ticks in PU, U1, and U2 sites. Key: red spot, transect positive for I. ricinus ticks; green spot, transect negative for I. ricinus ticks. Tick number is expressed per 10-m 2 transect. Map background BDOrtho ® 50 cm IGN. Abbreviations: U1, U2, urban parks; PU, peri-urban park

Fig. 3 .

 3 Fig. 3. Seasonality of Ixodes spp. activity in PU park and C site. 1000 m 2 in PU and 100 m 2 in C were sampled each month. Abbreviations: PU, peri-urban park; C, control site

Table 1 .

 1 Density of questing ticks and percentage of positive transects in the three parks throughout the study period

	Site	PU	U1	U2
	Tick number/area (m 2 )	499/5000	6/5000	1/5000
	Density/10 m 2	1.00	0.01	0.002
	Positive transects/total transects	146/500	6/500	1/500
	Percentage	29.2	1.2	0.2
	Notes: Tick number/area (m 2 ): total number of ticks/sampled area in m 2 ; positive
	transects/total transects: number of tick-positive transects/total number of
	transects

Table 2 .

 2 Density of questing ticks and percentage of positive transects in the three environments in the periurban (PU) location throughout the study period

	Environment type	CE	TE	OE
	Tick number/area (m 2 )	425/2680	65/850	8/1470
	Density/10 m 2	1.6	0.8	0.05
	Positive transects/total transects	119/268	19/85	8/147
	Percentage	44.4%	22.4%	5.4%

Notes: Tick number/area (m 2 ): total number of ticks/sampled area in m 2 ; positive transects/total transects: number of tick-positive transects/total number of transects Abbreviations: CE, closed environment, within forests and along footpaths or tracks in forests; TE, transitional environment, forest edge; OE, open environment, meadow or along footpaths or tracks in open areas

Table 3 .

 3 Density of questing ticks and percentage of positive transects in closed and transitional environments in periurban (PU) and control (C) sites

	Time frame	Entire period	June
	Site	PU	C	PU	C
	Tick number/area (m 2 )	490/3530 493/500 223/640 191/100
	Density/10 m 2	1.4	9.9	3.5	19.1
	Positive transects/total	138/353 40/50	48/64	10/10
	transects
	Percentage	39	80	75	100

Notes: Entire period: cumulative data for all five months; Tick number/area (m 2 ): total number of ticks/sampled area in m 2 in closed and transitional environments; positive transects/total transects: number of tick-positive transects/total number of transects in closed and transitional environments

Acknowledgements

We thank everyone who participated in the collection of ticks from the field: Claudine Gadoulet (weather data collection), Didier Augros, Paul Nougaret, Morgane Boespflug, Ombeline Triau, Héloïse Dinette, Elisa Guerineau, and Margot Le Tacon. We also thank Lindsay Higgins for her English language revisions.

Funding

The study was funded in equal measure by the INRAE, VetAgro Sup, the Ville de Lyon, and the Metropole de Lyon.

Availability of data and materials

The datasets used and/or analysed in the present study are available from the corresponding author upon reasonable request.

Abbreviations

U1: Urban park 1 (Tête-d'Or and Brétillod, Lyon, France); U2: Urban park 2 (Parilly, Bron, France); PU: Peri-urban park (Lacroix-Laval, Charbonnières-les-Bains, France); C: Control site (La Tour de Salvagny, France); GIS: Geographical information system.

Authors' contributions

MRM: conceptualization, study design and management, field campaigns, writing original draft. LM: field campaigns, writing original draft, data analysis. MN: field campaigns, data analysis. IL: field campaigns, data analysis (in particular GIS analyses), review of the original draft. JJ: statistics, writing original draft. KCM: methodology and access to the results from the control site, thanks to the CCEID-CLIMATICK project supported by the Adaptation of Agriculture and Forests to Climate Change (AAFCC) metaprogram of INRAE. VP: field campaigns and tick identification. GV, SG, GB, SP, DS, SCR, SBa and SBo-conceptualization and/or field campaigns. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Ph.D Francesca Branzoli
email: francesca.branzoli@icm-institute.org

PhD Małgorzata Marjańska

Magnetic Resonance Spectroscopy of IDH-mutated gliomas: current knowledge on the neurochemical profile

Keywords: IDH mutation, glioma, brain metabolites, treatment effect, progression

Purpose of review: Magnetic resonance spectroscopy (MRS) may play a key role for the management of patients with glioma. We highlighted the utility of MRS in the noninvasive diagnosis of gliomas with mutations in isocitrate dehydrogenase (IDH) genes, by providing an overview of the neurochemical alterations observed in different glioma subtypes, as well as during treatment and progression, both in vivo and ex vivo.

Recent findings: 2-hydroxyglutarate (2HG) decrease during anti-cancer treatments was recently shown to be associated with altered levels of other metabolites, including lactate, glutamate and glutathione, suggesting that tumor treatment leads to a metabolic reprogramming beyond 2HG depletion. In combination with 2HG quantification, cystathionine and glycine seem to be the most promising candidates for higher specific identification of glioma sub-types and follow-up of disease progression and response to treatment.

Summary:

The implementation of advanced MRS methods in the routine clinical practice will allow the quantification of metabolites that are not detectable with conventional methods and may enable immediate, accurate diagnosis of gliomas, which is crucial for planning optimal therapeutic strategies and follow-up examinations. The role of different metabolites as predictors of patient outcome still needs to be elucidated.

Introduction

In 2009, mutations of the genes encoding the enzymes isocitrate dehydrogenase 1 and 2 (IDH1/2) were discovered in gliomas [START_REF] Yan | IDH1 and IDH2 mutations in gliomas[END_REF]. IDH mutations are found in ~90% of diffuse grade II and 60% of diffuse grade III gliomas, 5% of primary glioblastomas (GBM), and are absent in other brain tumors [START_REF] Picca | Current therapeutic approaches to diffuse grade II and III gliomas[END_REF]. IDH-mutated gliomas form a biologically independent subset of tumors associated with a 2-to 4-fold longer median survival compared to wild-type (WT) tumors [START_REF] Houillier | IDH1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas[END_REF]. Own to its high diagnostic and prognostic value, IDH mutational status was integrated into the 2016 World Health Organization classification of gliomas [START_REF] Louis | The 2016 World Health Organization classification of tumors of the central nervous system: a summary[END_REF]. Diffuse glioma classification requires histologic evaluation and assessment of the IDH mutational status and codeletion status of chromosomal arms 1p and 19q (1p/19q codeletion) [START_REF] Reifenberger | Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and 1p[END_REF]. 1p/19q codeletion is found only in IDH-mutated gliomas, is linked with the oligodendroglial histologic subtype, and is associated with the best prognosis. IDH-mutated 1p/19q-noncodeleted gliomas are classified as diffuse astrocytomas and are associated with intermediate outcome, while IDH-WT have the poorest prognosis [START_REF] Picca | Current therapeutic approaches to diffuse grade II and III gliomas[END_REF].

Magnetic resonance imaging (MRI) plays a vital role in the diagnosis and management of patients with brain tumors [6**]. Among different MRI modalities, in vivo 1 H MR spectroscopy (MRS) provides unique metabolic information through the measurement of the neurochemical concentrations in tissue.

The aim of this review is to provide an overview of the metabolic alterations observed in different glioma subtypes both in vivo and ex vivo, and to highlight the possible role of 1 H MRS in the noninvasive diagnosis of IDH-mutated gliomas, as well as in monitoring the effect of tumor progression and anti-cancer treatments on the neurochemical profile.

D-2-hydroxyglutarate

IDH mutations result in the overproduction of D-2-hydroxyglutarate (2HG) (Figure 1) and have been suggested to cause changes in global cellular metabolism, either in consequence of increased 2HG or through inhibition of isocitrate metabolism [START_REF] Reitman | Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome[END_REF]. In vivo MRS enables the noninvasive detection of 2HG, potentially providing a great impact on patient management.

Clinical relevance

Noninvasive 2HG detection by MRS is a powerful tool for immediate, pre-surgical diagnosis of IDH-mutated gliomas. Indeed, 2HG MRS allows in first place to differentiate noninvasively gliomas from metastases, primary brain lymphomas or other non-neoplastic lesions. In vivo assessment of IDH status is highly beneficial for neurosurgical planning as maximal surgical resection has been shown to confer a better prognosis in IDH-mutated gliomas compared to WT [START_REF] Beiko | IDH1 mutant malignant astrocytomas are more amenable to surgical resection and have a survival benefit associated with maximal surgical resection[END_REF]. Thus, although in vivo MRS is not meant to replace surgery or biopsy, which are needed for histological confirmation of the IDH status and assessment of the tumor genetic profile, pre-operative MRS would be extremely useful as part of the clinical routine practice as noninvasive identification of IDH mutation may influence surgical decision-making regarding the extent of the resection. Moreover, 2HG quantification may be useful for the identification of minor IDH mutations undetectable by immunohistochemistry, given that DNA sequencing is not available in all clinical centers. 2HG may also serve as a biomarker of IDH mutation if surgery is not feasible or associated with high risk, and as a marker of treatment efficacy, treatment response, and tumor progression, especially when evaluation based on other imaging methods is inconclusive, assisting clinicians in the choice of the best therapeutic strategy.

In vivo MRS methods

Since 2012, several in vivo 1 H MRS methods have been proposed for 2HG detection [START_REF] Andronesi | Detection of oncogenic IDH1 mutations using magnetic resonance spectroscopy of 2-hydroxyglutarate[END_REF][START_REF] Kim | In-Vivo proton magnetic resonance spectroscopy of 2hydroxyglutarate in isocitrate dehydrogenase-mutated gliomas: a technical review for neuroradiologists[END_REF][START_REF] Suh | 2-Hydroxyglutarate MR spectroscopy for prediction of isocitrate dehydrogenase mutant glioma: a systemic review and metaanalysis using individual patient data[END_REF]**]. These can be grouped into three types: short echo-time (TE) MRS [START_REF] Pope | Non-invasive detection of 2hydroxyglutarate and other metabolites in IDH1 mutant glioma patients using magnetic resonance spectroscopy[END_REF]; long-TE MRS optimized for 2HG detection [START_REF] Choi | 2-hydroxyglutarate detection by magnetic resonance spectroscopy in IDH-mutated patients with gliomas[END_REF][START_REF] Emir | Noninvasive quantification of 2hydroxyglutarate in human gliomas with IDH1 and IDH2 mutations[END_REF]; edited MRS [15,16*]. Examples of spectra acquired with these three methods have been shown previously [START_REF] Andronesi | Detection of oncogenic IDH1 mutations using magnetic resonance spectroscopy of 2-hydroxyglutarate[END_REF]. Short-TE (TE = 30 ms) MRS is typically performed using the point resolved spectroscopy (PRESS) sequence, available on clinical scanners. This method has been shown to produce the highest rate of false-positives, which is the worst outcome for clinical evaluation [11**]. This is because the quantification of the 2HG signal at 2. Optimized PRESS uses T E = 97 ms and has been shown to better quantify 2HG and reduce false-positive results compared to short-TE MRS [START_REF] Choi | A comparative study of short-and long-TE 1H MRS at 3 T for in vivo detection of 2-hydroxyglutarate in brain tumors: MRS detectability of 2hydroxyglutarate[END_REF]. Nevertheless, a recent study suggested that necrosis and treatment status were associated with false-positive 2HG measurements using optimized PRESS in GBM [START_REF] Suh | False-positive measurement at 2-hydroxyglutarate MR spectroscopy in isocitrate dehydrogenase wild-type glioblastoma: a multifactorial analysis[END_REF]. A semi-localization by adiabatic selective refocusing pulse sequence with T E = 110 ms was also proposed as alternative approach for 2HG detection at 7 T [START_REF] Emir | Noninvasive quantification of 2hydroxyglutarate in human gliomas with IDH1 and IDH2 mutations[END_REF].

Finally, edited MRS measures the 2HG signal at 4.02 ppm by completely removing the signals from other metabolites (Figure 2) and provided no false positives in a recent prospective 3 T study [16*]. These results point towards the need of implementation of advanced MRS methods for clinical examinations.

Both single-voxel (SV) MRS and MRS imaging (MRSI) have been employed for the detection of 2HG, yet the majority of the studies employed SV MRS, own to the shorter acquisition times and the higher reproducibility of the results with respect to MRSI.

Regardless the method employed, a few studies have reported lower sensitivity and specificity in post-operative compared to pre-operative cohorts [START_REF] De La Fuente | Integration of 2-hydroxyglutarate-proton magnetic resonance spectroscopy into clinical practice for disease monitoring in isocitrate dehydrogenase-mutant glioma[END_REF][START_REF] Zhou | Diagnostic accuracy of 2-hydroxyglutarate magnetic resonance spectroscopy in newly diagnosed brain mass and suspected recurrent gliomas[END_REF]. This could be due to technical challenges related to the presence of post-surgical cavities, size of the residual tumor or effect of previous treatments. The evaluation of the full metabolic profile may thus become crucial for follow-up examinations and evaluation of tumor progression.

Correlation with cellularity and grade

Previous reports showed that 2HG concentrations detected by MRS were positively correlated with tumor cellularity [START_REF] De La Fuente | Integration of 2-hydroxyglutarate-proton magnetic resonance spectroscopy into clinical practice for disease monitoring in isocitrate dehydrogenase-mutant glioma[END_REF][START_REF] Choi | Prospective longitudinal analysis of 2hydroxyglutarate magnetic resonance spectroscopy identifies broad clinical utility for the management of patients with IDH -mutant glioma[END_REF]. 2HG concentrations were also found to be significantly different between low-and high-grade, consistent with differences in tumor cellularity in different grades [START_REF] Choi | Prospective longitudinal analysis of 2hydroxyglutarate magnetic resonance spectroscopy identifies broad clinical utility for the management of patients with IDH -mutant glioma[END_REF] and confirming ex vivo findings obtained using 1 H high-resolution magic angle spinning (HR-MAS) spectroscopy [START_REF] Elkhaled | Magnetic resonance of 2-hydroxyglutarate in IDH1-mutated low-grade gliomas[END_REF]: mean ex vivo 2HG levels were shown to be significantly higher in grade IV compared to grade II gliomas. When normalized by average cellularity, 2HG levels did not differ significantly between grades II and IV, suggesting that 2HG differences between glioma grades reflect differences in cellularity [START_REF] Elkhaled | Magnetic resonance of 2-hydroxyglutarate in IDH1-mutated low-grade gliomas[END_REF].

Conversely, another short-TE MRS 3 T study suggested that tumor grade had a significant influence on glutamate (Glu), but not on 2HG concentration [START_REF] Nagashima | Diagnostic value of glutamate with 2hydroxyglutarate in magnetic resonance spectroscopy for IDH1 mutant glioma[END_REF]. This negative result could be due to the technical challenges associated with 2HG quantification using short-TE MRS, as discussed above.

In line with these findings, a recent study reported increased ex vivo 2HG levels in gliomas undergoing malignant progression from grade III to grade IV [START_REF] Jalbert | Metabolic profiling of IDH mutation and malignant progression in infiltrating glioma[END_REF]. Additionally, 2HG levels were correlated with increased mitotic activity, axonal disruption, vascular neoplasia, and with several metabolites (see section "Correlation with other metabolites") [START_REF] Jalbert | Metabolic profiling of IDH mutation and malignant progression in infiltrating glioma[END_REF]. While in vivo studies did not report significant differences in 2HG levels between different histological subtypes [START_REF] Choi | Prospective longitudinal analysis of 2hydroxyglutarate magnetic resonance spectroscopy identifies broad clinical utility for the management of patients with IDH -mutant glioma[END_REF][START_REF] Fellah | Multimodal MR imaging (diffusion, perfusion, and spectroscopy): is it possible to distinguish oligodendroglial tumor grade and 1p/19q codeletion in the pretherapeutic diagnosis?[END_REF], elevated ex vivo 2HG levels were reported in oligodendrogliomas undergoing progression to grade III and IV compared to astrocytomas [START_REF] Jalbert | Metabolic profiling of IDH mutation and malignant progression in infiltrating glioma[END_REF].

Longitudinal changes

A 2HG concentration decrease during treatment has been shown previously [21,26,27**]. A prospective longitudinal study employing SV optimized PRESS at 3 T reported stable absolute 2HG levels in untreated patients with indolent disease, while 2HG showed a marked increase at progression. In contrast, 2HG levels decreased during radio-and chemo-therapies in most patients under investigation, and the decrease during treatment was accompanied by a measurable decrease in tumor volume in almost all gliomas. 2HG concentration decreased more rapidly in oligodendrogliomas compared to astrocytomas or mixed gliomas, consistent with a better response to treatment of the first sub-type. In patients included up to 24 months after treatment, 2HG levels were very close to the detectability threshold set to 1 mM [START_REF] Choi | Prospective longitudinal analysis of 2hydroxyglutarate magnetic resonance spectroscopy identifies broad clinical utility for the management of patients with IDH -mutant glioma[END_REF].

An edited 3D MRSI sequence was employed at 3 T to evaluate the effect of both standard

Correlation with other metabolites

The median value of 2HG/tCr was found positively correlated with that of lactate(Lac)/tCr in pre-treatment scans, possibly resulting from the Warburg effect due to silencing of lactate dehydrogenase A (LDHA) in IDH-mutated gliomas, while it was negatively correlated with that of Glu + glutamine (Glx)/tCr in post-treatment scans, as a possible direct consequence of 2HG depletion during treatment [START_REF] Andronesi | Treatment response assessment in IDHmutant glioma patients by noninvasive 3D functional spectroscopic mapping of 2hydroxyglutarate[END_REF]. These findings require further corroboration from larger patient cohorts. Similarly, the inhibition of IDH1 activity through a novel anti-IDH1 treatment was suggested to induce a reprogramming of tumor metabolism, showing decreased 2HG levels, reported relative to tCr quantified in healthy tissue (tCr healthy ), associated with increased Glx/tCr healthy , glutathione (GSH)/tCr healthy and Lac/tCr healthy levels. The trend to an inverse correlation between 2HG/tCr healthy and Glx/tCr healthy in IDH-mutated gliomas after treatment suggested the 2HG/Glx ratio as a more sensitive biomarker of IDH mutation, given that Glu levels were found to be lower in IDH1-mutated compared to WT gliomas, as measured by the mass spectrometry in tumor biopsies [START_REF] Nagashima | Diagnostic value of glutamate with 2hydroxyglutarate in magnetic resonance spectroscopy for IDH1 mutant glioma[END_REF]. However, the inverse correlation between 2HG and Glx in mutated gliomas has not yet been shown in vivo at baseline.

Increased GSH levels after anti-IDH1 treatment may lead to a reduced sensitivity of IDHmutated gliomas to radiotherapy, providing evidence against a benefit of simultaneous administration of IDH1 inhibitors and radiotherapy [27**]. The increase in Lac levels posttreatment were suggested to be linked with reversible downregulation of LDHA activity associated with inhibition of mutant IDH1.

A previous 1 H HR-MAS experiments also reported a positive correlation between 2HG and Lac levels in IDH-mutated recurrent glioma biopsies, as well as correlations of 2HG with several other metabolites commonly associated with tumor, such as free choline (Cho), phosphocholine (PCho), glycerophosphocholine (GPC), tCho, aspartate (Asp), gammaaminobutyric acid (GABA), threonine (Thr), hypotaurine (hTau), creatine and phosphocreatine (Cr, PCr), betaine (Bet), glycine (Gly), GSH, phosphoethanolamine (PE), Glu, and Gln. Conversely, 2HG was negatively correlated with the myo-inositol (mIns)/tCho ratio, an index associated with the relative contribution of tumor cells and treatment-induced gliosis [START_REF] Elkhaled | Magnetic resonance of 2-hydroxyglutarate in IDH1-mutated low-grade gliomas[END_REF]. However, these correlations have not been yet corroborated by other ex vivo or in vivo studies. Correlations between 2HG and other metabolites observed both in vivo and ex vivo are summarized in Table 1.

Other metabolites

Differences in several other metabolites have been reported in IDH-mutated vs. WT gliomas, as well as across different glioma genetic sub-types and tumor grades, using in vivo and ex vivo MRS. Main findings are summarized in Table 2.

IDH vs. WT

IDH-mutated tumors exhibited a significantly lower Glu/tCr compared to WT gliomas from ultra-high-field short-TE MRS experiments [START_REF] Bisdas | MR spectroscopy for in vivo assessment of the oncometabolite 2-hydroxyglutarate and its effects on cellular metabolism in human brain gliomas at 9.4T: MRS of IDH Mutant Gliomas at 9.4T[END_REF]. The same study also reported a significantly lower GSH/tCr and significantly elevated tCho/tCr in mutated gliomas with respect to WT. However, a small cohort of patients was investigated and the basis set for metabolic quantification did not include the experimentally measured macromolecular spectrum, which may bias the results. Decreased Glu, Gln and GSH levels in IDH-mutated vs. WT gliomas were also reported from short-TE 3-T experiments [START_REF] Nagashima | Diagnostic value of glutamate with 2hydroxyglutarate in magnetic resonance spectroscopy for IDH1 mutant glioma[END_REF]. However, the experimentally measured macromolecular spectrum was not used for the quantification, and Glu, Gln and GSH levels associated with a CRLB > 30% were excluded, possibly introducing a bias [START_REF] Kreis | The trouble with quality filtering based on relative Cramér-Rao lower bounds: the trouble with quality filtering based on relative CRLB[END_REF].

Significantly lower Glx levels and a trend to lower GSH levels were observed in a small cohort of IDH-mutated vs. WT GBM at 3 T [START_REF] Natsumeda | Reliable diagnosis of IDH-mutant glioblastoma by 2-hydroxyglutarate detection: a study by 3-T magnetic resonance spectroscopy[END_REF]. Despite technical limitations, these results are in line with the reported lower Glu, Gln and GSH levels measured in mutant vs. WT IDH cell cultures [START_REF] Reitman | Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome[END_REF]. Lower GSH levels in mutant IDH vs. WT may be explained by the selective depletion in these gliomas of nicotinamide adenine dinucleotide phosphate (NADPH), which is used by IDH for 2HG production and is thus less available for GSH recycling. However, lower total GSH levels in mutated gliomas were not observed by liquid chromatography-mass spectrometry (LC-MS) in a recent study [31**].

Other more recent studies reported decreased Glu, but not Gln, levels in IDH-mutated glioma cells compared to WT, as well as in IDH-mutated vs. WT glioma tissue samples using gaschromatography-mass spectrometry [START_REF] Nagashima | Diagnostic value of glutamate with 2hydroxyglutarate in magnetic resonance spectroscopy for IDH1 mutant glioma[END_REF] and 1 H HR-MAS [START_REF] Jalbert | Metabolic profiling of IDH mutation and malignant progression in infiltrating glioma[END_REF]. Lower GABA concentration was also observed in the IDH-mutated group in one of these studies [START_REF] Jalbert | Metabolic profiling of IDH mutation and malignant progression in infiltrating glioma[END_REF]. Depletion of Glu levels were suggested to compensate for the altered flux of alphaketoglutarate to 2HG in IDH-mutated gliomas [START_REF] Nagashima | Diagnostic value of glutamate with 2hydroxyglutarate in magnetic resonance spectroscopy for IDH1 mutant glioma[END_REF]. Significant differences in both Glu and Gln levels between IDH-mutated and WT glioma biopsies were observed in another study and were both linked to replenishment of alpha-ketoglutarate by glutaminolysis [START_REF] Ohka | Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation[END_REF].

NAA and N-acetyl-aspartyl-glutamate (NAAG) levels were also found to be significantly lower in IDH-mutated vs. WT human glioma tissues, yet, the biological mechanism for NAA and NAAG depletion and whether it contributes to tumor pathogenesis remains unclear [START_REF] Reitman | Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome[END_REF].

Very recently, maximum tCho/N-acetylaspartate (NAA) and tCho/tCr values measured using short-TE MRSI in lesions were shown to be significantly higher in IDH-mutated tumors compared to WT, whereas mean tCho/NAA and tCho/tCr were not significantly different between two groups. In addition, maximum tCho/NAA was significantly higher in grade IV gliomas compared to lower grade gliomas. In contrast, in the peritumoral area, the mean tCho/tCr was found to be significantly elevated in IDH-mutated vs. WT gliomas [START_REF] Goryawala | The association between whole-brain MR spectroscopy and IDH mutation status in gliomas[END_REF]. These results are in line with the elevated tCho/tCr measured in vivo in human mutated gliomas at 9.4 T [START_REF] Bisdas | MR spectroscopy for in vivo assessment of the oncometabolite 2-hydroxyglutarate and its effects on cellular metabolism in human brain gliomas at 9.4T: MRS of IDH Mutant Gliomas at 9.4T[END_REF], and with the observed higher levels of free Cho, GPC, and PCho in IDH-mutated vs. WT gliomas measured ex vivo using 1 H HR-MAS [START_REF] Jalbert | Metabolic profiling of IDH mutation and malignant progression in infiltrating glioma[END_REF]. Given the very low concentration of free Cho in the brain, elevated tCho levels are very likely dominated by increased GPC and PCho. Lower PE and higher GPC levels in IDH-mutated vs. WT gliomas were reported in a study combining in vivo 31 P MRSI in mouse xenograft models, ex vivo 31 P HR-MAS measurements in human biopsies of IDH-mutated tumors, and cell line 31 P MRS experiments, suggesting altered phospholipid metabolism caused by IDH mutation [START_REF] Esmaeili | IDH1 R132H mutation generates a distinct phospholipid metabolite profile in glioma[END_REF].

IDH1 vs. IDH2

Higher 2HG levels in gliomas harbouring mitochondrial IDH2 R172K mutations with respect to cytosolic IDH1 R132H mutations were measured at 7 T [START_REF] Emir | Noninvasive quantification of 2hydroxyglutarate in human gliomas with IDH1 and IDH2 mutations[END_REF], confirming previous cell culture findings [START_REF] Ward | The potential for isocitrate dehydrogenase mutations to produce 2-hydroxyglutarate depends on allele specificity and subcellular compartmentalization[END_REF]. The same group also reported higher 2HG/tCho and mIns/tCho, and a trend to increase in citrate/tCho and tCr/tCho, in IDH2 vs. IDH1-mutated gliomas [36*].

However, given the variable tCho levels associated with IDH mutation, normalization of metabolic concentrations over tCho may represent a possible source of bias.

1p/19q codeletion

Higher cystathionine levels in brain tumors compared to normal tissue were previously reported from ex vivo tissue analysis [START_REF] Lefauconnier | Free amino acids and related substances in human glial tumours and in fetal brain: comparison with normal adult brain[END_REF][START_REF] Wróbel | Is development of high-grade gliomas sulfur-dependent?[END_REF]. A recent in vivo study reported significantly elevated cystathionine in patients with IDH-mutated 1p/19q codeleted gliomas compared with their non-codeleted counterparts [31**]. In vivo results obtained using SV edited MRS (Figure 2) were corroborated by ex vivo LC-MS analysis in tumor samples. Ex vivo experiments showed significantly decreased levels of serine (Ser), Gly, Thr, and ornithine (Orn), and a trend for lower GSH in 1p/19q codeleted vs. non-codeleted tumors. Interestingly, cystathionine, Gly and serine are involved in the glutathione biosynthesis pathways and their altered levels were specifically attributed to the deletion of two serine-and cystathioninepathway genes located on chromosome 1p -phosphoglycerate dehydrogenase (PHGDH) and cystathionine gamma-lyase (CTH) -possibly leading to a compensatory anti-oxidant mechanism in gliomas with 1p/19q codeletion (Figure 3

Grade and progression

Elevated in vivo tCho concentration was reported in high-grade compared to lower-grade gliomas [START_REF] Wenger | In vivo metabolic profiles as determined by 31P and short TE 1H MR-Spectroscopy: no difference between patients with IDH wildtype and IDH mutant gliomas[END_REF][START_REF] Grech-Sollars | Imaging and tissue biomarkers of choline[END_REF], suggesting that tCho could be used as a marker of cellularity. However, since tCho is not a specific tumor cell marker, and its levels may change due to other pathological processes such as inflammation or gliosis, these results need to be taken with caution.

Choline containing compounds as measured using 1 H HR-MAS were found to be significantly higher in tumors that had undergone malignant progression [START_REF] Jalbert | Metabolic profiling of IDH mutation and malignant progression in infiltrating glioma[END_REF]. Interestingly, oligodendrogliomas showed higher levels of GPC and tCho, while astrocytomas had higher levels of PCho and Cho. The associations of oligodendrogliomas with elevated GPC and astrocytomas with elevated PCho and Cho were suggested to reflect differences in the phospholipid metabolism found in the choline kinase pathway between these tumor subtypes.

In the same study, positive correlations of 2HG levels with all choline-containing compounds were reported, corroborating previous findings suggesting that both 2HG and choline compounds levels are linked to cellularity. Given the reported differences of choline compounds between different tumor grades and histological subtypes (see below), care must be taken when reporting metabolic levels as ratios over tCho.

Several other metabolites measured using 1 H HR-MAS were found to be significantly elevated in gliomas undergoing malignant progression [START_REF] Jalbert | Metabolic profiling of IDH mutation and malignant progression in infiltrating glioma[END_REF]. These included taurine (Tau), hTau, Gly, Glu, GSH, alanine (Ala), Asp, and Bet, Gln and glucose. 2HG was positively correlated with PE, GSH, Tau, Glu, Gln, Asp, myo-I, SI, GABA, PCr/Cr, Gly, Bet, and Thr. Among these metabolites, increases in PE, Glu and Gln were specifically associated with anaplastic astrocytomas, while Tau, Gly, Gln, and Bet were increased in anaplastic oligodendrogliomas.

Gln and Gly, which can be quantified using in vivo MRS, may be useful markers of tumor malignant progression. A very recent study reported a strong association between Gly levels measured by 3 T MRS and the presence of gadolinium enhancement. In addition, Gly levels, but not 2HG levels, were positively correlated with tumor cell proliferation as quantified by MIB-1 labelling index and high Gly/2HG was strongly associated with shorter survival [42**]. The correlation between Gly/2HG and patient outcome observed in this study may reflect the better outcome of IDH vs. WT gliomas (higher 2HG) combined with the better outcome of 1p/19q codeleted vs. non-codeleted gliomas (lower Gly [31**]). However, no information on the codeletion status was reported.

Conclusions

Noninvasive 2HG detection has a strong clinical utility due to its high diagnostic and prognostic power. While conventional in vivo MRS methods available in clinical settings have shown low specificity to IDH mutation, the implementation of advanced in vivo MRS methods enabling reliable 2HG quantification is highly desirable for routine clinical examinations and follow-up of treatment response. Lower Glu, Gln and GSH levels seem to be associated with IDH mutation and abnormal production of 2HG, while elevated tCho suggests altered phospholipid metabolism, which may depend on tumor sub-type, grade and cellularity. Anti-cancer treatments have been suggested to affect tumor metabolism beyond 2HG production, yet the role of different metabolites in predicting patient outcome requires further elucidation. The recent discovery of cystathionine detectability in glioma using in vivo MRS opens up the possibility to exploit a potential novel biomarker of tumor response to treatment and progression. Although at the moment the quantification of the full metabolic profile does not have a direct implication for clinical evaluation, we believe that it provides a better understanding of the tumor pathophysiology and, in the future, it may be crucial for achieving higher specificity in glioma sub-type identification and for evaluation of tumor response to treatment and patient outcome. Elevated glycine was strongly associated with presence of gadolinium enhancement, indicating more rapidly proliferative disease. Glycine concentration was positively correlated with MIB-1, and levels higher than 2.5 mM showed significant association with shorter patient survival, irrespective of IDH status. Concentration of 2HG did not correlate with MIB-1 index. A high glycine/2HG concentration ratio, >2.5, was strongly associated with shorter survival.

  25 ppm is complicated by the presence of other signals, including metabolites, macromolecules and baseline. Long-TE MRS optimized for 2HG detection partially removes the overlap of 2HG at 2.25 ppm with other resonances, by employing the TE that provides minimal contribution of other signals. These methods require modified sequences and are rarely available in the clinic.

  treatments and a novel IDH1 inhibitor on 2HG levels [26,27**]. 2HG levels measured relative to total creatine (creatine + phosphocreatine, tCr) were found to decrease significantly in patients receiving adjuvant radiation and chemotherapy and correlated with clinical status. Results from a phase 1 clinical trial indicated a rapid 2HG/tCr decrease by 70% after only 1 week of treatment with a novel IDH1 inhibitor [27**].

  ) [31**]. Although cystathionine can be reliably detected using the same edited MRS protocol used for 2HG, possible further acquisition improvements have been reported [39*].



  Reliable 2HG quantification for clinical examinations requires implementation of advanced MRS methods.  IDH mutation leads to tumor metabolic reprogramming beyond 2HG overproduction.  Cystathionine and glycine may play a crucial role for glioma sub-type identification and follow-up of glioma progression.  The evaluation of the full neurochemical profile may help achieving higher specificity in glioma sub-type identification and prediction of patient outcome. metabolism in diffuse adult glioma: 18F-fluoromethylcholine PET/CT, Magnetic Resonance Spectroscopy, and choline kinase α. Cancers 2019, 11:1969. 42.**Tiwari V, Daoud EV, Hatanpaa KJ, et al. Glycine by MR spectroscopy is an imaging biomarker of glioma aggressiveness. Neuro Oncol 2020, Epub ahead of print.

Figure 1 :

 1 Figure 1: Schematic diagram of the 2HG metabolic pathway. Illustration of IDH function

Figure 2 :

 2 Figure 2: In vivo detection of 2HG and cystathionine. An in vivo spectrum acquired in an

Figure 3 :SHMT1/ 2 :

 32 Figure 3: Schematic diagram of the cystathionine metabolic pathway. Solid arrows =

Acknowledgements

Acknowledgements: none.

Francesca Branzoli acknowledges the support from the programs 'Institut des neurosciences translationnelles' ANR-10-IAIHU-06 and 'Infrastructure d'avenir en Biologie Santé' ANR-11-INBS-0006. Małgorzata Marjańska acknowledges the support of the following NIH grants: BTRC P41 EB027061, and P30 NS076408.