Jean-Marie Volland
email: volland_jeanmarie@hotmail.com

Paco Bustamante

Dalila Aldana Aranda

Olivier Gros
email: olivier.gros@univ-antilles.fr

The potential role of spherocrystals in the detoxification of essential trace metals following exposure to Cu and Zn in the fighting conch Strombus (Lobatus) pugilis

Keywords: exposure, Mollusca, phosphate granule, trace metal, ultrastructure

Crypt cells -one of the three cell types composing Strombidae digestive tubules -are characterized by the presence of numerous metal-containing phosphate granules termed spherocrystals. We explored the bioaccumulation and detoxification of metals in Strombidae by exposing wild fighting conch Strombus pugilis for nine days to waterborne CuSO4 and ZnSO4. The total amount of Cu and Zn was determined in the digestive gland and in the rest of the body by Inductively Coupled Plasma (ICP) analyses. The digestive gland spherocrystal metal content was investigated based on the semi-quantitative energy dispersive X-ray (EDX) elemental analysis. ICP analyses of unexposed individuals revealed that 87.0±5.9% of the Zn is contained in the digestive gland, where its concentration is 36 times higher than in the rest of the body. Regarding Cu, 25.8±16.4% of the metal was located in the digestive gland of the control individuals, increasing to 61.5±16.4% in exposed individuals. Both Cu and Zn concentrations in the digestive gland increased after exposures, pointing to a potential role of this organ in the detoxification of these metals. EDX analysis of spherocrystals revealed the presence of Ca, Cl, Fe, K, Mg, P, and Zn in unexposed individuals. No difference was found in the relative proportion of Zn in spherocrystals of exposed versus control individuals. Contrastingly, copper was never detected in the spherocrystals from controls and Zn-exposed individuals, but the relative proportion of Cu in spherocrystals of Cu-exposed individuals varied from 0.3% to 5.7%. Our results show the direct role of spherocrystals in Cu detoxification.

Introduction

Strombidae are benthic gastropods, some species of which represent a staple food and important trade commodity (Aldana [START_REF] Aranda | El Caracol Strombus gigas: Conocimiento Integral Para Su Manejo Sustentable En El Caraibe. Impressos profesionales Aranda[END_REF]. The fighting conch Strombus (Lobatus) pugilis (Linnaeus, 1758) is one of these species and is widely fished in the Caribbean region and especially consumed around the Yucatan coasts in Mexico. Strombidae recently received a special focus because their digestive gland hosts putative symbionts belonging to the Sporozoa group [START_REF] Cárdenas | Coccidian (apicomplexa) parasite infecting Strombus gigas Linné, 1758 digestive gland[END_REF][START_REF] Gros | Structural analysis of the digestive gland of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites[END_REF][START_REF] Volland | Occurrence of sporozoa-like microorganisms in the digestive gland of various species of Strombidae[END_REF].

Ultrastructural investigation of the digestive gland highlighted the presence of three cell types in the digestive tubules: digestive cells, vacuolated cells and crypt cells. It is now wellestablished that digestive cells are mostly involved in intracellular digestion (Volland and Gros 2012) whereas vacuolated cells store lipids and host the Sporozoan symbionts [START_REF] Gros | Structural analysis of the digestive gland of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites[END_REF]Volland and Gros 2012). In contrast, the function of the crypt cells remains unclear.

Crypt cells, also known as basophilic cells, calcium cells or secretory cells, are described in the digestive gland of many mollusks [START_REF] Fretter | British prosobranch molluscs: Their functional anatomy and ecology[END_REF][START_REF] Merdsoy | Phasic activity in the digestive gland cells of the marine prosobranch gastropod, Littorina littorea (L.)[END_REF][START_REF] Voltzow | Gastropoda: Prosobranchia[END_REF]. They are characterized by their pyramidal shape and their location in the crypt of digestive tubules [START_REF] Taïeb | Histochemistry and ultrastructure of the crypt cells in the digestive gland of Aplysia punctata (Cuvier, 1803)[END_REF]. Other frequently reported characteristics of this cell type include the presence of a large nucleus with a prominent nucleolus and an abundant rough endoplasmic reticulum [START_REF] Fretter | British prosobranch molluscs: Their functional anatomy and ecology[END_REF][START_REF] Lutfy | The histology of the alimentary system of Marisa cornuarietis[END_REF][START_REF] Merdsoy | Phasic activity in the digestive gland cells of the marine prosobranch gastropod, Littorina littorea (L.)[END_REF][START_REF] Wigham | Feeding and digestion in the marine prosobranch Rissoa parva (da Costa)[END_REF][START_REF] Voltzow | Gastropoda: Prosobranchia[END_REF][START_REF] Lobo-Da-Cunha | Ultrastructural and cytochemical aspects of the basophilic cells in the hepatopancreas of Aplysia depilans (Mollusca, Opisthobranchia)[END_REF][START_REF] Taïeb | Histochemistry and ultrastructure of the crypt cells in the digestive gland of Aplysia punctata (Cuvier, 1803)[END_REF]Volland et al. 2012). Finally, one remarkable component of the crypt cells is spherical mineral inclusions termed spherocrystals. While not always present, they are often characteristic of mollusk crypt cells. Spherocrystals, also known as phosphate granules or metal-containing granules, are mineral inclusions amorphous to X-ray and electron diffraction [START_REF] Taylor | Metal environments in intracellular granules -iron[END_REF][START_REF] Taylor | Biotransformation of intracellular minerals by zinc ions in vivo and in vitro[END_REF][START_REF] Mitchell | Hydrated sites in biogenic amorphous calcium phosphates: An infrared, raman, and inelastic neutron scattering study[END_REF][START_REF] Masala | Analysis of metal-containing granules in the barnacle Tetraclita squamosa[END_REF]). They are present in various taxonomic groups and are particularly well described in arthropods (Ballan-Dufrançais 2002). Among mollusks, spherocrystals have been described from bivalves [START_REF] George | Isolation and elemental analysis of metal-rich granules from the kidney of the scallop, Pecten maximus (L.)[END_REF][START_REF] George | Characterization of metal-containing granules from the kidney of the common mussel, Mytilus edulis[END_REF][START_REF] George | Interspecies differences in heavy metal detoxication in oysters[END_REF], gastropods [START_REF] Howard | The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa)[END_REF][START_REF] Greaves | The local environment of metal sites in intracellular granules investigated by using X-ray-absorption spectroscopy[END_REF]Nott et al. 1993;[START_REF] Triebskorn | Localization of iron stores and iron binding sites in blood cells of five different priapulid species by energy-filtering electron microscopy (EFTEM)[END_REF][START_REF] Gibbs | The composition of phosphate granules in the digestive glands of marine prosobranch gastropods: Variation in relation to taxonomy[END_REF][START_REF] Taïeb | Histochemistry and ultrastructure of the crypt cells in the digestive gland of Aplysia punctata (Cuvier, 1803)[END_REF] and cephalopods [START_REF] Martoja | Localisation cytologique du cuivre et de quelques autres métaux dans la glande digestive de la seiche, Sepia officinalis L. (mollusque céphalopode)[END_REF][START_REF] Costa | Microstructural and histochemical advances on the digestive gland of the common cuttlefish, Sepia officinalis L[END_REF]. Spherocrystals have been observed in the crypt cells of various Strombidae species including S. pugilis, where they occupy a considerable part of the cytoplasm [START_REF] Gros | Structural analysis of the digestive gland of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites[END_REF][START_REF] Volland | Occurrence of sporozoa-like microorganisms in the digestive gland of various species of Strombidae[END_REF]Volland et al. 2012). These structures store minerals and trace metals and are thus thought to be involved in their detoxification [START_REF] Marigómez | Cellular and subcellular distribution of metals in molluscs[END_REF][START_REF] Delakorda | Application of elemental microanalysis to elucidate the role of spherites in the digestive gland of the helicid snail Chilostoma lefeburiana[END_REF]). Nevertheless, their function remains unclear.

A previous exposure experiment of S. pugilis to waterborne Cd and Pb suggested that spherocrystals are not involved in detoxifying these two nonessential trace metals in this species (Volland et al. 2012). We did, however, detect Al, Ca, Fe, Mg, Mn, P, and Zn in the spherocrystals of two Strombidae, S. gigas and S. pugilis, indicating that these structures play a role in regulating minerals and essential trace metals in these gastropods (Volland et al. 2012). Moreover, spherocrystals in barnacles accumulate considerable amounts of metals, whereby Cu and Zn concentrations can reach 3.7 mg g -1 and 27.1 mg g -1 , respectively [START_REF] Pullen | The composition of pyrophosphate heavy metal detoxification granules in barnacles[END_REF].

In 2010, reproductive failure in a Strombus gigas population from Florida Keys nearshore waters was attributed to heavy metal exposure; the offshore population not exposed to the pollution reproduced normally [START_REF] Spade | Queen conch (Strombus gigas) testis regresses during the reproductive season at nearshore sites in the Florida keys[END_REF]. Those authors found an inverse correlation between Cu and Zn concentrations in the digestive gland and the spermatogenic index (which reflects the fecundity level). The nearshore population showed a repression in the expression of many genes related to spermatogenesis and mitochondrial functions. The transcription of small GTPase-related signaling genes was affected and reflected in the lack of testis development they observed histologically. The authors hypothesized that heavy metals contribute to the reproductive failure of the conchs. They proposed that the elevated concentrations of Zn, and possibly Cu, in the digestive gland affect the development of the conch testis.

In this context, the present study investigates the potential role of the digestive gland in storing and detoxifying the two essential metals Cu and Zn. We analyzed unexposed and waterborne-exposed individuals of the fighting conch Strombus pugilis maintained in controlled conditions in the laboratory. The total amount of these metals was determined in the digestive gland and in the rest of the body by ICP analyses. This was accompanied by ultrastructural observations of the digestive gland coupled with the EDX-ray elemental analysis of spherocrystals.

Materials and methods

Sample collection and exposure

Specimens of Strombus pugilis were collected by hand on sandy areas in Guadeloupe, French West Indies (F.W.I), and in Campeche bank, Mexico. Ultrastructural observations were performed on the two populations, whereas the exposure experiment with trace metals was performed on the Mexican population only. Once collected, organisms were rapidly brought to the laboratory. Many specimens (n=740) were processed for histological examination and 30 individuals were starved for 24 h and placed in five 40 L aquaria filled with 5µm filtered seawater in order to depurate their gut. Two aquaria each were used for the exposure to Cu and Zn. A stock solution of CuSO4.5H2O at 3.20 mmol L -1 was used to obtain final concentrations of 0.40 µmol L -1 and 0.80 µmol L -1 . A stock solution of ZnSO4.7H2O at 2.78 mmol L -1 was used to obtain final concentrations of 0.35 µmol L -1 and 0.70 µmol L -1 . A fifth aquarium was used as negative control without addition of Cu and Zn. The water was oxygenated and renewed every three days. Individuals were fed daily with artificial food developed and provided by the CINVESTAV-IPN of Mérida (Nutrition and Aquaculture of Mollusks Laboratories). After nine days of waterborne exposure (and to a lesser degree dietary-borne because food was in contact with the contaminant in the water), the individuals were killed.

From each of the five aquaria, the six individuals were killed: three were processed for the ICP quantitative analyses of trace metals and the other three for the EDX-ray analysis of spherocrystals from the digestive gland.

Histology and transmission electron microscopy

The digestive glands were dissected and fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer (0.1 M; 1100 mOsm; pH: 7.2) for 24 h at 4°C and rinsed with an iso-osmotic buffer. For histology, tissue samples were dehydrated in an ascending ethanol series and infiltrated with Paraplast wax before embedding. Wax sections, 7 µm-thick, were obtained from each sample and stained with a modified Goldner trichrome method that included Alcian blue at pH 2.5 in order to stain proteoglycans [START_REF] Gabe | Techniques histologiques[END_REF]). For TEM, samples were post-fixed for 1 h in 1% osmium (OsO4) in the same buffer. The samples were then dehydrated through an ascending ethanol series and embedded in epoxy resin according to [START_REF] Volland | Occurrence of sporozoa-like microorganisms in the digestive gland of various species of Strombidae[END_REF]. Semi-thin sections were stained with 0.5% toluidine blue in 1% borax for photonic microscopy observations. Ultra-thin sections (60 nm) were contrasted with uranyl acetate and lead citrate before observation in a Leo 912 transmission electronic microscope.

Scanning electron microscopy and EDX-ray analysis

Three individuals from each condition (control, exposure to Cu at 0.8 µmol L -1 , exposure to Zn at 0.7 mmol L -1 ) were used for the EDX-ray analysis of spherocrystals. The digestive glands were dissected and fixed as described for TEM. They were then dehydrated through an ascending acetone series, critical point dried, and manually fractured before observation with a Quanta 250 scanning electron microscope (FEI®) at an acceleration voltage of 20 kV under low vacuum (650 Pa). Energy-dispersive X-ray analysis (EDX) of individual spherocrystals was carried out using an M-max 50mm 2 Oxford detector, monitored by an INCA system, in the point mode.

Trace metal analysis

The digestive gland and remaining parts of the body of S. pugilis exposed to Cu (0.40 µmol L - 1 , n=3 and 0.80 µmol L -1 , n=3) and Zn (0.35 µmol L -1 , n=3 and 0.70 µmol L -1 , n=3) and from the control (n=3) were prepared for Cu and Zn analysis using a Varian Vista-Pro ICP-OES and a Thermo Fisher Scientific X Series 2 ICP-MS (following [START_REF] Kojadinovic | Multi-elemental concentrations in the tissues of the oceanic squid Todarodes filippovae from Tasmania and the Southern Indian Ocean[END_REF]. Briefly, the tissues were freeze-dried for several days then ground with a Fritsch® Planetary ball mill.

Aliquots of the biological samples (100-300 mg) were digested with 6 ml 67-70% HNO3 and 2 ml 34-37% HCl (Fisher Scientific, trace element grade quality). Acidic digestion was carried out overnight at room temperature and then in a Milestone microwave (30 min with constantly increasing temperature up to 120°C, and finally 15 min at this maximal temperature). Each sample was completed to 50 ml with milli-Q water. Three control samples (two Certified Reference Materials (CRM), and one blank) treated and analysed in the same way as the samples were included in each analytical batch. CRMs were Dolt-4 dogfish liver (NRC, Canada) and TORT-2 Lobster Hepatopancreas (NRC, Canada). The results were in good agreement with the certified values, with a mean recovery rate of 90-104% for DOLT-4 and 92-102% for TORT-2. Trace element concentrations are expressed in μg g -1 dry weight (dw).

Results

Digestive gland structure

The digestive gland of individuals from Guadeloupe (F.W.I.) was observed under the light microscope (semi-thin sections) and under the transmission electron microscope. The digestive gland of controls from Mexico was observed under the light microscope after histological staining and under the scanning electron microscope. Based on many observations in recent years (F.W.I: n=40 and Mexico: n=700), digestive gland structure of individuals from the two locations did not differ. The gland of the individuals investigated here represented on average 14.4±3.2% of the whole body mass (n=15). It is composed of an assemblage of digestive tubules and ducts. The digestive tubule epithelium composes three cell types: digestive cells, vacuolated cells and crypt cells (Fig. 1). The vacuolated cells together with the crypt cells form a pseudo-stratified epithelium. The vacuolated cells are on average 30 µm long and 10 µm wide. They are characterized by the presence of an intracellular sporozoan symbiont and their cytoplasm is full of lipid droplets which appear metachromatic (green) on osmificated samples (Figs. 1A,B). The digestive cells are long columnar cells averaging 80 µm in length and 7 µm in width. They are the most abundant cells in the digestive tubules. They are packed together in well-aligned groups of cells which form a uni-stratified epithelium. A basal nucleus is surrounded by lipid droplets. The median third of their cytoplasm usually contains one large granule (~ 7 µm in diameter) with a dense heterogeneous content stained by Alcian blue. The apical third of digestive cells contains vesicles and granules of various diameters from 0.5 µm near the apical pole to 5 µm near the median part of the cells (Figs. 1A-C). The third cell type of the digestive tubules is the crypt cell. These cells are characterized by a pyramidal shape and a cytoplasm full of spherical inclusions identified as spherocrystals (Figs. 1B,C). Crypt cell size varies from 10 to 50 µm long and from 10 to 50 µm wide. The nucleus is large (Fig. 1B) and the rough endoplasmic reticulum abundant (Fig. 1D).

The spherocrystals are spherical inclusions about 1 µm in diameter. They are composed by alternating electron-dense and electron-lucent concentric layers organized around a matrix core (Fig. 1D). EDX analysis of spherocrystals revealed the presence of Ca, Cl, Fe, K, Mg, P, and Zn, whereby P, Mg, Zn and Ca were most abundant (Fig. 2). Between-cell variability of Fe, K and Zn was high, within-cell variability low (Fig. 3).

Zinc

The analysis of Zn in unexposed Strombus pugilis (controls) revealed that 87.0±5.9% of the total metal body burden was located in the digestive gland (Fig. 4A). The concentration in the digestive gland (1796±174 µg g -1 dw) was 36 times higher than in the rest of the body (50 ±10 µg g -1 dw) (Fig. 4B). After nine days of exposure to waterborne Zn, the proportions of the metal in the digestive gland and the rest of the body did not change significantly (Fig. 4A) but the concentration in the digestive gland was higher after the exposure (Fig. 4B). The Zn concentration in the rest of the body remained in the same range at the end of the exposure period for both concentrations and in controls (Fig. 4B). In parallel, the semi-quantitative elemental analysis of spherocrystals in the crypt cells of the digestive gland showed no differences in the relative proportion of Zn in exposed versus control individuals.

Copper

The analysis of Cu in S. pugilis tissues revealed that 25.8±16.4% of the total metal body burden was located in the digestive gland of the control individuals (Fig. 4C), with slightly higher concentrations compared to the rest of the body (32±11 µg g -1 dw vs 20±4 µg g -1 dw, respectively; Fig. 4D). After nine days of exposure to waterborne Cu, the distribution of the metal within the organism changed significantly. The percentage of the total Cu located in the digestive gland was higher after the exposure (Fig. 4C). The digestive gland of individuals exposed to 0.40 µmol L -1 of Cu contained 46.8±17.5% of the whole body burden whereas those exposed to 0.80 µmol L -1 contained 61.5±16.4% (Fig. 5). At the same time, the digestive gland Cu concentration increased almost nine fold to 316±153 µg g -1 dw in exposed individuals (Fig. 4D). Cu concentrations in the rest of the body were not significantly different from the control after the 0.40 µmol l -1 exposure to waterborne Cu (16±6 µg g -1 dw) but increased to 33±6 µg g -1 dw after the 0.80 µmol l -1 exposure. In the controls, no Cu was ever detected in any of the spherocrystals (n=42) analyzed by EDX. Likewise, no Cu was detected in any of the spherocrystals (n=29) from the three individuals contaminated with Zn. However, after nine days of exposure to Cu 0.80 µmol L -1 , the metal was detectable in spherocrystals (n=22) of all three individuals. The relative proportion of Cu in spherocrystals of exposed individuals varied from 0.3% to 5.7% (average 1.5±1.5%).

Discussion

Digestive gland structure and ultrastructure

The structure and ultrastructure of the digestive gland of mollusks have been extensively studied, especially in bivalves, pulmonate gastropods and cephalopods. While the overall organization (primary and secondary ducts connected to digestive tubules) is the same in all groups, the digestive tubule epithelium composition is group specific and sometime varies within the groups. Bivalves usually exhibit only two cell types: digestive (also termed acidophilic) and crypt cells (also termed basophilic, pyramidal or secretory cells) [START_REF] Pal | The fine structure of the digestive tubules of Mya arenaria L. basiphil cell[END_REF][START_REF] Pal | The fine structure of the digestive tubules of Mya arenaria L. II digestive cell[END_REF]Henry 1984a;1984b;[START_REF] Morse | Bivalvia[END_REF]. The digestive tubules of cephalopods consist of a single digestive cell type [START_REF] Purchon | The biology of the Mollusca[END_REF][START_REF] Semmens | Changes in the digestive gland of the loliginid squid Sepioteuthis lessoniana (lesson 1830) associated with feeding[END_REF][START_REF] Swift | The digestive gland of the southern dumpling squid (Euprymna tasmanica): Structure and function[END_REF]. Few studies have examined polyplacophoran digestive tubules, but at least two cell types have been described, digestive and basophilic cells (Lobo-da-Cunha 1997). Among gastropods, the epithelium seems to be more complex, involving three to five cell types in pulmonates [START_REF] Walker | The cytology, histochemistry, and ultrastructure of the cell types found in the digestive gland of the slug, Agriolimax reticulatus (Müller)[END_REF][START_REF] Luchtel | Gastropoda: Pulmonata[END_REF]. Three to four cell types are described in opistobranchs (digestive, calcium, excretory, and thin cells) [START_REF] Coelho | Processing of defensive pigment in Aplysia californica: Acquisition, modification and mobilization of the red algal pigment, r-phycoerythrin by the digestive gland[END_REF][START_REF] Taïeb | Histochemistry and ultrastructure of the crypt cells in the digestive gland of Aplysia punctata (Cuvier, 1803)[END_REF][START_REF] Lobo-Da-Cunha | The digestive cells of the hepatopancreas in Aplysia depilans (Mollusca, Opisthobranchia): Ultrastructural and cytochemical study[END_REF][START_REF] Taïeb | Distribution of digestive tubules and fine structure of digestive cells of Aplysia punctata (Cuvier, 1803)[END_REF]). Finally, the caenogastropods, to which the Strombidae, have at least two cell types, the digestive and the pyramidal crypt cells. The latter contains various cell types described and differently named as secretory, excretory, basophilic, and calcium cells [START_REF] Fretter | British prosobranch molluscs: Their functional anatomy and ecology[END_REF][START_REF] Lutfy | The histology of the alimentary system of Marisa cornuarietis[END_REF][START_REF] Purchon | The biology of the Mollusca[END_REF][START_REF] Merdsoy | Phasic activity in the digestive gland cells of the marine prosobranch gastropod, Littorina littorea (L.)[END_REF][START_REF] Boghen | Phasic activity in the digestive gland cells of the intertidal prosobranch, Littorina saxatilis (olivi) and its relations to the tidal cycle[END_REF][START_REF] Wigham | Feeding and digestion in the marine prosobranch Rissoa parva (da Costa)[END_REF][START_REF] Devi | Observations on the histology and cytochemistry of the digestive gland in Pila virens (Lamarck)(Mollusca: Gastropoda)[END_REF][START_REF] Voltzow | Gastropoda: Prosobranchia[END_REF]).

In the digestive gland tubules of S. pugilis, three cell types are identified: the digestive, vacuolated, and crypt cells -consistent with those described in other Strombus species [START_REF] Gros | Structural analysis of the digestive gland of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites[END_REF][START_REF] Volland | Occurrence of sporozoa-like microorganisms in the digestive gland of various species of Strombidae[END_REF]Volland and Gros 2012). While the digestive and crypt cells present the classic features of the taxonomic group, the vacuolated cells seem to be more specific to this family. To our knowledge only one study has reported similar cells in the digestive tubules of the caenogastropod Maoricrypta monoxyla [START_REF] Nelson | Cyclic activity and epithelial renewal in the digestive gland tubules of the marine prosobranch Maoricrypta monoxyla (lesson)[END_REF].

Three types of granules are present in the digestive gland of S. pugilis. The biggest ones are the brownish spherules previously identified as sporozoan symbionts in the vacuolated cells [START_REF] Gros | Structural analysis of the digestive gland of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites[END_REF][START_REF] Volland | Occurrence of sporozoa-like microorganisms in the digestive gland of various species of Strombidae[END_REF]. The digestive cells present proteoglycan-rich granules of ~ 7 µm diameter as shown by Alcian blue specific staining. Those first two types of intracellular granules are also observed free in the lumen of tubules, in the digestive gland ducts and in the feces of S. gigas [START_REF] Gros | Structural analysis of the digestive gland of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites[END_REF]. We made similar observations in S. pugilis (data not shown). The third type of granules in the digestive gland is the spherocrystals in the crypt cells. These granules are too small to be observed in the feces using the light microscope, but SEM used in other mollusks showed they are expelled with the feces [START_REF] Nott | Transfer of metal detoxification along marine food chains[END_REF]1996). Spherocrystals have already been reported in the tissues of mammals [START_REF] Gallien | Cadmium-containing granules in kidney tissue of the atlantic white-sided dolphin (Lagenorhyncus acutus) off the Faroe islands[END_REF]), arthropods (Ballan-Dufrançais 2002), annelids [START_REF] Jenkins | The digestive system of the hydrothermal vent polychaete Galapagomystides aristata (Phyllodocidae): Evidence for hematophagy?[END_REF][START_REF] Mouneyrac | Trace-metal detoxification and tolerance of the estuarine worm Hediste diversicolor chronically exposed in their environment[END_REF]) and many mollusks. A study focusing on prosobranchs reported spherocrystals in 40 species belonging to 20 families [START_REF] Gibbs | The composition of phosphate granules in the digestive glands of marine prosobranch gastropods: Variation in relation to taxonomy[END_REF]. Taylor et al. (1988) proposed that spherocrystals are deposits of pyrophosphate salt (CaMgP2O7), which traps metal ions.

Elements detected in the spherocrystals of different mollusk families are Ca, Cl, Cu, Fe, K, Mg, Mn, Na, Ni, S, Ti, V, and Zn [START_REF] Nott | Cadmium and the phosphate granules in Littorina littorea[END_REF]Nott et al. 1993;1996;[START_REF] Gibbs | The composition of phosphate granules in the digestive glands of marine prosobranch gastropods: Variation in relation to taxonomy[END_REF]. [START_REF] Nott | The cytology of heavy metal accumulations in the digestive glands of three marine gastropods[END_REF] proposed that metals in cells induce the production of magnesium phosphate in spherocrystals as a source of phosphate ions to bind and detoxify the metals.

After analyzing the elemental composition of spherocrystals from clean and polluted sites, they concluded that spherocrystals of marine gastropods do respond to different levels of metals in the environment. A previous study on S. gigas and S. pugilis highlighted the presence of Al, Ca, Fe, Mg, Mn, P, and Zn in the spherocrystals (Volland et al. 2012). Moreover, analyses of spherocrystals from feces have shown that they still present an important proportion of their trace metal content compared to those analysed from the digestive gland (e.g., 50% for Zn and 33% for Mn) after their passage through the digestive tract [START_REF] Nott | Bioreduction of zinc and manganese along a molluscan food chain[END_REF].

Clearly, these structures represent a detoxification pathway for trace metals in mollusks [START_REF] Marigómez | Cellular and subcellular distribution of metals in molluscs[END_REF].

Exposure to Cu and Zn

No study is available on the levels of Cu and Zn in S. pugilis. Among Strombidae, only S. canarium [START_REF] Said | Arsenic, cadmium and copper in gastropod Strombus canarium in western part of Johor straits[END_REF]Sabri et al. 2014a;2014b) and the queen conch S. gigas were investigated for these metals [START_REF] Rizo | Copper, zinc and lead bioaccumulation in marine snail, Strombus gigas, from Guacanayabo Gulf, Cuba[END_REF][START_REF] Spade | Queen conch (Strombus gigas) testis regresses during the reproductive season at nearshore sites in the Florida keys[END_REF][START_REF] Whitall | Contaminants in queen conch (Strombus gigas) in Vieques, Puerto Rico[END_REF]. Unlike in S. pugilis, the digestive gland of S. gigas is removed before human consumption. The digestive gland of S. gigas shows by far the highest levels of Zn in the organism [START_REF] Spade | Queen conch (Strombus gigas) testis regresses during the reproductive season at nearshore sites in the Florida keys[END_REF]). The digestive gland of S. pugilis represents only 14.4±3.2% of the whole body mass but contains most of the total Zn body burden (87.0±5.9%) in unexposed gastropods. The very high concentration of this metal in the digestive gland already points to a potential role in Zn regulation (and possibly detoxification) through its storage in spherocrystals. Copper is more spread in the body of unexposed individuals but still is more concentrated in the digestive gland. Note that in many mollusks, oxygen is transported by a Cu-based haemocyanin pigment. Each oxygen molecule transported in the blood requires two Cu atoms [START_REF] Van Holde | Hemocyanins[END_REF].

At the end of the exposure period to waterborne metals, both Cu and Zn concentrations increased drastically in the digestive gland. However, only the distribution of Cu between the digestive gland and the rest of the body was significantly modified, with 61.5±16.4% of the Cu being detected in the digestive gland after the exposure to 0.80 µmol L - 1 of CuSO4. Accordingly, storage in this organ is implicated in detoxifying Cu and Zn.

Ultrastructural analysis is required in order to further identify the cell types and structures involved in scavenging these two metals.

The elemental analysis of the spherocrystals from the digestive gland revealed that they naturally contain Zn, which represents 14.2±6.3% of the elements analyzed in spherocrystals. While Zn concentrations increased in the digestive gland during the exposure, the relative proportion of Zn in spherocrystals remained unchanged. The excess Zn might rather be bound to proteins such as metallothioneins [START_REF] Amiard | Metallothioneins in aquatic invertebrates: Their role in metal detoxification and their use as biomarkers[END_REF]. Note that Zn is a transition metal showing properties of class A (affinity for O) and class B (affinity for S) metals [START_REF] Nieboer | The replacement of the nondescript term 'heavy metals' by a biologically and chemically significant classification of metal ions[END_REF]. It has been proposed that spherocrystals scavenge class A metals (K, Na, Li, Ba, Sr, Ca, Mg) more than class B metals (Ag, Cd, Hg), the latter being associated with metallothionein proteins [START_REF] Nieboer | The replacement of the nondescript term 'heavy metals' by a biologically and chemically significant classification of metal ions[END_REF][START_REF] Marigómez | Cellular and subcellular distribution of metals in molluscs[END_REF]. Other structures may be involved in Zn detoxification, including the proteoglycan-rich granules from digestive cells (not analyzed in this study). EDX-ray analyses have reported Zn in similar digestive cell granules in other mollusks [START_REF] Nott | The cytology of heavy metal accumulations in the digestive glands of three marine gastropods[END_REF]. The intracellular symbionts present in the vacuolated cells might also be involved in Zn detoxification because EDX-ray analyses of such structures have revealed the presence of Zn but also Al, Ca, Fe and S [START_REF] Volland | Occurrence of sporozoa-like microorganisms in the digestive gland of various species of Strombidae[END_REF]. Both the digestive cell granules and the symbionts are indeed expelled via the feces [START_REF] Gros | Structural analysis of the digestive gland of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites[END_REF], thus potentially representing an elimination pathway for the excess of metals they contain.

In cephalopods, exposure of young cuttlefish to waterborne Zn disturbs the metabolism of several metals including Cu (Le [START_REF] Pabic | Trace metal concentrations in post-hatching cuttlefish Sepia officinalis and consequences of dissolved zinc exposure[END_REF]. In the present study, the spherocrystals of unexposed and Zn-exposed individuals show no Cu peak in the EDX-ray spectra: they either lack this element or it is present in amounts below the detection threshold of the technique. In a study focusing on the common cerith, Cerithium vulgatum (Caenogastropoda), similar EDX-ray analysis of digestive gland spherocrystals detected the following elements: Ca, Cl, Co, Cr, Fe, K, Mg, Mn, Na, P, S, and Zn but no Cu [START_REF] Nott | The cytology of heavy metal accumulations in the digestive glands of three marine gastropods[END_REF]. Those authors also analyzed these structures with energy filtered transmission electron microscopy, a more sensitive technique. They reported a small peak for Cu, suggesting that spherocrystals scavenge this element too. After Cu exposure, we detected this metal in the spherocrystals of S. pugilis, where it represented up to 5.7% of the analyzed elements. The presence of Cu in the spherocrystals, together with the increased Cu concentration in the digestive gland, indicate that spherocrystals are involved in the detoxification of this element. EDX-ray analysis, however, is a semi-quantitative analytical technique and cannot determine the absolute amount of Cu detoxified through spherocrystal scavenging.

Conclusion

The digestive gland of mollusks is a key organ involved in the digestion but also in lipid storage, mineral osmoregulation, and metal detoxification [START_REF] Owen | Digestion[END_REF][START_REF] Voltzow | Gastropoda: Prosobranchia[END_REF][START_REF] Porcel | Alterations in the digestive gland and shell of the snail Helix aspersa Müller (Gastropoda, Pulmonata) after prolonged starvation[END_REF][START_REF] Penicaud | Metal bioaccumulation and detoxification processes in cephalopods: A review[END_REF]. Most ecotoxicological studies have investigated the impact of metal exposure on i) the organ metal burden and ii) the subcellular partitioning of metals obtained by differential centrifugation. The resulting fractions contain organelles and other cytoplasmic structures referred to as cellular debris or metal-rich granules [START_REF] Guo | Different responses of abalone Haliotis discus hannai to waterborne and dietary-borne copper and zinc exposure[END_REF].

It is difficult to determine whether the different authors are referring to the same structures.

Other studies have focused on the ultrastructure of the digestive gland, analyzing the elemental composition of metal-rich granules with precise cellular localization [START_REF] Marigómez | Cellular and subcellular distribution of metals in molluscs[END_REF][START_REF] Delakorda | Application of elemental microanalysis to elucidate the role of spherites in the digestive gland of the helicid snail Chilostoma lefeburiana[END_REF]Volland et al. 2012).

Combining ICP analyses with microscopy techniques and elemental analyses of subcellular compartments in targeted cell types enables investigating detoxification processes at two very different scales. This approach is well suited to test hypotheses on the role of specific cell structures in regulating and detoxifying metals. Using this approach for S. pugilis, we demonstrate that spherocrystals help detoxify Cu. The Zn detoxification pathway, on contrast, may involve metallothioneins. The relative contributions of these two detoxification pathways in Strombidae remain to be investigated.

Figure 1 :

 1 Figure 1: Microscopic images of Strombus pugilis digestive gland with ultrastructural details of spherocrystals. a. Histology of a digestive tubule transversal section showing the three cell types. The long columnar digestive cell (DC) with characteristic blue granules (stars). Vacuolated cells (VC) host the sporozoan symbiont (S). Groups of crypt cells (CC) are also visible. b. Semi-thin section of a digestive tubule showing more details of the digestive cell (DC) granules and the spherocrystals (arrowheads). Lipid droplets in vacuolated and digestive cells appear green on this osmificated sample. c. Scanning electron micrograph of a fractured digestive tubule (used for the EDX analysis). Spherocrystals (arrowheads) are readily recognizable. d. TEM detail of two spherocrystals surrounded by rough endoplasmic reticulum (insert) from a single crypt cell. Scale bars: A, 30 µm; B, 40 µm; C, 15 µm; D, 1 µm.

Figure 2 :

 2 Figure 2 : Relative proportion of the elements Al, Ca, Cl, Cu, Fe, K, Mg, P, and Zn in spherocrystals of control individuals and individuals exposed for nine days to 0.80 µmol L -1 of CuSO4 and individuals exposed for nine days to 0.70 µmol L -1 of ZnSO4. Number of analyzed spherocrystals in parentheses. P, Mg, Ca, and Zn are the most abundant elements.

Figure 3 :

 3 Figure 3: EDX-ray analysis showing the relative proportion of K, Fe and Zn in spherocrystals of Strombus pugilis exposed to 0.70 µmol L -1 of ZnSO4. The histograms display the average relative proportion of the respective element in four analyzed cells. The number of spherocrystals (n) analyzed for each cell varied between 3 and 5. While the relative proportion of an element may vary considerably from one cell to another, all spherocrystals analyzed within one cell display similar relative proportions for the considered element (see low standard deviations).

Figure 4 :

 4 Figure 4: Concentrations (mean ± SD; µg g -1 dw) of Cu and Zn and their relative whole body distribution (mean ± SD; %) in the digestive gland and the rest of the body of Strombus pugilis individuals (n=3) from control and exposed groups. a: distribution of Zn in the body. Most of the Zn is located in the digestive gland. b: Zn concentration in the digestive gland and the rest of the body in control and exposed individuals. c: distribution of Cu in the body. In control individuals, most of the Cu is located in the rest of the body. d: Cu concentration in the digestive gland and the rest of the body in control and exposed individuals.

Acknowledgements

This work was supported by a grant from ECOS-NORD 2008-2013 (MO9A2). We thank Manuel Sanchez and Marcela del Río from the CINVESTAV Mérida for their support in conducting the experiments. We also thank Carine Churlaud from the Plateforme Analyses Elémentaires of the LIENSs laboratory for carrying out ICP analyses. The IUF (Institut Universitaire de France) is acknowledged for its support to PB as a Senior Member. Electron microscopy work was performed at the C3MAG laboratory in Guadeloupe.

Conflict of Interest statement:

The authors declare no conflict of interest.


Itziar Garate Lopez
email: itziar.garate-lopez@lmd.jussieu.fr

Sébastien Lebonnois
email: sebastien.lebonnois@lmd.jussieu.fr

Latitudinal Variation of Clouds' Structure Responsible for Venus' Cold Collar

Keywords: Venus atmosphere, cold collar, modelling

Global Climate Models (GCM) are very useful tools to study theoretically the general dynamics and specific phenomena in planetary atmospheres. In the case of Venus, several GCMs succeeded in reproducing the atmosphere's superrotation and the global temperature field. However, the highly variable polar temperature and the permanent cold collar present at 60 • -80 • latitude have not been reproduced satisfactorily yet.

Here we improve the radiative transfer scheme of the Institut Pierre Simon Laplace Venus GCM in order to numerically simulate the polar thermal features in Venus atmosphere. The main difference with the previous model is that we now take into account the latitudinal variation of the cloud structure. Both solar heating rates and infrared cooling rates have been modified to consider the cloud top's altitude decrease toward the poles and the variation in latitude of the different particle

A C C E P T E D M A N U S C R I P T 1 Introduction

In the last two decades, Global Climate Models (GCMs) have turned out to be very useful tools to study the general atmospheric circulation on Venus and the role of different phenomena in the angular momentum budget. Several numerical simulations by different GCMs have already reproduced the main characteristic of the Venus atmosphere: its superrotation (e.g. [START_REF] Yamamoto | The fully developed superrotation simulated by a General Circulation Model of a Venus-like atmosphere[END_REF][START_REF] Lee | Superrotation in a Venus general circulation model[END_REF]Sugimoto et al., 2014a;[START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF]. At about 70 km altitude clouds circle the solid planet at ∼100 m/s, sixty times faster than the rotation of the planet. These simulations support the Gierasch-Rossow-Williams mechanism [START_REF] Gierasch | Meridional circulation and the maintenance of the Venus atmospheric rotation[END_REF][START_REF] Rossow | Large-scale motion in the Venus' stratosphere[END_REF] to explain the maintenance of the superrotation, which holds the latitudinal transport of angular momentum by horizontal planetary-scale waves and by the meridional circulation responsible for the velocity field in the atmosphere. GCMs also confirmed that in the case of Venus, the thermal tides play a key role in transporting the angular momentum vertically [START_REF] Schubert | General circulation and the dynamical state of the Venus atmosphere[END_REF][START_REF] Lebonnois | Superrotation of Venus' atmosphere analysed with a full General Circulation Model[END_REF]. On the other hand, it is well known that there is a wide variety of waves in the main cloud deck [START_REF] Belton | Cloud patterns, waves and convection in the Venus atmosphere[END_REF][START_REF] Rossow | Comments on 'A threedimensional model of dynamical processes in the Venus atmosphere[END_REF][START_REF] Del Genio | Planetary-scale waves and the cyclic nature of cloud top dynamics on Venus[END_REF][START_REF] Peralta | Characterization of mesoscale gravity waves in the upper and lower clouds of Venus from VEX-VIRTIS images[END_REF][START_REF] Piccialli | High latitude gravity waves at the Venus cloud tops as observed by the Venus Monitoring Camera on board Venus Express[END_REF] that may contribute to the transport of angular momentum and, therefore, recent GCMs studies have focused on the analysis of the wave activity present in the Venusian atmosphere (Sugimoto et al., 2014b;[START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF].

A C C E P T E D M A N U S C R I P T

One of the pending questions for the Venus GCMs is the simulation of the 23 atmospheric thermal structure. Although the average temperature field has 24 been reproduced adequately, no model has satisfactorily simulated one of its 25 most puzzling properties: the polar temperature distribution, formed by highly 26 variable vortices and cold air areas that surround the vortices permanently 27 (Zasova et al., 2007;[START_REF] Tellmann | Structure of the Venus neutral atmosphere as observed by the radio science experiment VeRa on Venus Express[END_REF].

28

The temperature in the deep atmosphere is almost constant from equa-29 tor to around 50 • , and slowly decreases polewards [START_REF] Tellmann | Structure of the Venus neutral atmosphere as observed by the radio science experiment VeRa on Venus Express[END_REF]. 30 However, above ∼70 km altitude temperatures increase from equator to pole, 31 in contrast to what is expected from radiative-convective equilibrium, since 32 solar heating is higher at equator than at the poles. This is known as the 33 warm polar mesosphere. At 60 -70 km altitude the temperature field in the 34 60 • -80 • region has a striking feature in both hemispheres, a strong tem-35 perature inversion called the cold collar [START_REF] Taylor | Structure and meteorology of the middle atmosphere of Venus: Infrared remote sounding from the Pioneer Orbiter[END_REF]. It is a collar 36 of cold air circling each pole of Venus. The latitudinally averaged tempera-37 ture between 60 • and 80 • is about 20 K colder than at the equator and 15 K 38 colder than at the poles, i.e. between 80 • and 90 • [START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF]. This 39 temperature difference would cause it to dissipate rapidly, however the cold 40 collar is a permanent structure at the sub-polar latitudes of Venus, implying 41 that it is forced by some unknown mechanism. Enclosed by the cold collar 42 there is a warm polar vortex in each pole of Venus. These are rapidly rotat-43 ing cloud and temperature structures that show highly variable and complex

A C C E P T E D M A N U S C R I P T

morphologies with warm small-scale filaments [START_REF] Piccioni | South-polar features on Venus similar to those near the north pole[END_REF][START_REF] Luz | Venus's southern polar vortex reveals precessing A C C E P T E D M A N U S C R I P T circulation[END_REF],

A C C E P T E D M A N U S C R I P T
atmosphere by comparing radio occultation measurements and GCM results, and concluded that the thermal disturbance associated to the polar vortex could be a neutral barotropic Rossby wave related to barotropic instability.

These three works obtained a temporally evolving polar region and some horizontal structure within the vortex. However, the average latitude-altitude distributions of temperature do not exactly reproduce the observed cold collar structure. In observation-based maps (Zasova et al., 2007;[START_REF] Tellmann | Structure of the Venus neutral atmosphere as observed by the radio science experiment VeRa on Venus Express[END_REF][START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF] the cold collar is an isolated region and is located slightly lower than in the maps of [START_REF] Yamamoto | Dynamics of polar vortices at cloud top and base on Venus inferred from a general circulation model: Case of a strong diurnal thermal tide[END_REF] and [START_REF] Ando | The puzzling Venusian polar atmospheric structure reproduced by a general circulation model[END_REF][START_REF] Ando | Vertical structure of the axi-asymmetric temperature disturbance in the Venusian polar atmosphere: comparison between radio occultation measurements and GCM results[END_REF]. In addition, the average temperature values obtained by [START_REF] Ando | The puzzling Venusian polar atmospheric structure reproduced by a general circulation model[END_REF] are not warm enough compared to observations. On the other hand, [START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF] used the IPSL (Institut Pierre Simon Laplace) Venus GCM, that has a full radiative transfer module, to model the Venus atmosphere paying a special attention to polar regions. Their zonally and temporally averaged latitudinal profiles showed a small cold collar signature at the cloud top (∼70 km), located slightly higher than observed.

Due to its axi-asymmetric distribution, the obtained cold collar looked subtle when zonally averaged but it was more evident in the polar temperature fields.

They related the shape of the polar temperature distribution (cold collar and warm vortex) to the combination of thermal tide and high-frequency wave activity but the temperature contrasts obtained inside the polar regions (∼10 K) were weaker than those presented by [START_REF] Ando | The puzzling Venusian polar atmospheric structure reproduced by a general circulation model[END_REF][START_REF] Ando | Vertical structure of the axi-asymmetric temperature disturbance in the Venusian polar atmosphere: comparison between radio occultation measurements and GCM results[END_REF]) (∼20-25 K) and

A C C E P T E D M A N U S C R I P T
were far from the observed ∼30 K on average (Garate-Lopez et al., 2015).

89

In order to perform an in-depth study of the Venus polar atmosphere and The general characteristics of the IPSL Venus GCM have been described in 103 detail in [START_REF] Lebonnois | Superrotation of Venus' atmosphere analysed with a full General Circulation Model[END_REF] and [START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF]. Therefore, only 104 a short summary is given here. The model is based on the LMDZ latitude-105 longitude grid finite-difference dynamical core [START_REF] Hourdin | The LMDZ4 general circulation model: climate performance and sensitivity to parameterized physics with emphasis on tropical convection[END_REF]. Since 106 the goal of the current study is the polar regions, it must be noted that this

A C C E P T E D M A N U S C R I P T
dynamical core includes a longitudinal polar filter: this Fourier filter is applied at latitudes higher than 60 • , removing high frequencies and limiting the effective resolution to that at 60 • latitude [START_REF] Jablonowski | The pros and cons of diffusion, filters and fixers in Atmospheric General Circulation Models[END_REF]. Some of the most important features of the model are: the boundary layer scheme used, based on [START_REF] Mellor | Development of a turbulent closure model for geophysical fluid problems[END_REF]; the implementation of topography, with hybrid vertical coordinates (50 vertical levels, from surface to roughly 95 km altitude); the temperature dependence of the specific heat C p (T ), which affects the definition of the potential temperature (as detailed in [START_REF] Lebonnois | Superrotation of Venus' atmosphere analysed with a full General Circulation Model[END_REF]; and the horizontal resolution currently used, 96 longitudes x 96 latitudes, i.e. 3.75

• x 1.875 • .
The previous version of the model [START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF] reproduced consistently the average zonal wind and temperature fields, but the obtained equatorial jet was too intense compared to mid-latitude jets and the winds below the clouds were too slow compared to measurements made by Venera and Pioneer Venus probes [START_REF] Schubert | General circulation and the dynamical state of the Venus atmosphere[END_REF][START_REF] Gierasch | The general circulation of the Venus atmosphere: an assessment[END_REF]. On the other hand, the modeled average temperature structure, although generally consistent with observations, resulted in colder temperatures in the deep atmosphere and at surface compared to observed profiles (Seiff et al., 1985), and in slightly higher temperatures above the clouds [START_REF] Tellmann | Structure of the Venus neutral atmosphere as observed by the radio science experiment VeRa on Venus Express[END_REF][START_REF] Migliorini | Investigation of air temperature on the nightside of Venus derived from VIRTIS-H on board Venus-Express[END_REF][START_REF] Grassi | The Venus nighttime atmosphere as observed by VIRTIS-M instrument. Average fields from the complete infrared data set[END_REF]. The latitudinal profiles showed a small cold collar signature at ∼70 km and the polar temperature distribution at ∼67 km displayed some inner structure. However, in the average

A C C E P T E D M A N U S C R I P T
altitude-latitude temperature map the cold collar was not an isolated feature 130 as in observation-based maps [START_REF] Tellmann | Structure of the Venus neutral atmosphere as observed by the radio science experiment VeRa on Venus Express[END_REF][START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF] and 131 the contrast between cold collar and warm vortex was weaker than observed, 132 about 10 K in the model vs ∼30 K in temperature retrievals from VIRTIS in-133 strument (Garate-Lopez et al., 2015). The latitudinal structure of the clouds used for control by comparing it to the varcld simulation in the discussion of the formation of the cold collar, but this simulation and its results have no physical sense and should not be considered alone. In the latter case (varcld simulation) a stable state of the atmosphere was obtained after roughly 60 Vd of simulation, so it is the simulation that represents the current IPSL Venus GCM model. The different inputs used in each stage and simulations are summarized in Table 1.

TABLE 1

Improved radiative transfer

Previously, the radiative transfer included solar heating rate profiles as a function of solar zenith angle taken from look-up tables based on [START_REF] Crisp | Radiative forcing of the Venus mesosphere. I -Solar fluxes and heating rates[END_REF], and used the infrared net-exchange rate matrix formulation [START_REF] Eymet | Net-exchange parameterization of the thermal infrared radiative transfer in Venus' atmosphere[END_REF] with horizontally uniform opacity sources (gas and clouds).

For the current study, the main modification in the radiative transfer is related to the cloud model. Our computations now take into account the more recent cloud model described by [START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF][START_REF] Haus | Radiative heating and cooling in the middle and lower atmosphere of Venus and responses to atmospheric and spectroscopic parameter variations[END_REF], which is based on the most recent observations performed by the Venus-Express spacecraft. In particular, this model describes latitudinal variations of the cloud structure.

According to [START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF], the cloud top altitude (defined as the altitude where the optical depth equals the unity at 1 µm) decreases slowly from

A C C E P T E D M A N U S C R I P T
about 71 km at the equator to about 70 km at 50 • , but then it quickly drops poleward reaching about 61 km over both poles. Authors also found a strong latitude dependence of cloud opacity, which is transposed into the latitudinal behavior of cloud mode factors, scaling the abundance of the different modes compared to the equatorial vertical distribution. Retrieved mode 1 and 2 factors gradually decrease poleward of 30 • showing a small local minimum at 55 • . The mode 3 factor is nearly constant from equator to ∼ 15 • , decreases between 15 • and 30 • , remains almost constant up to 55 • , but then strongly increases poleward [START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF]. This new cloud model and some tuning (see details below) were implemented both in the solar heating and infrared cooling rates' computations.

Solar heating

For the short-wavelength part of the radiative transfer scheme, the principle used is basically the same as previously. Look-up tables are used, that contain the vertical profile of the solar heating rate as a function of solar zenith angle.

The model described in [START_REF] Haus | Radiative heating and cooling in the middle and lower atmosphere of Venus and responses to atmospheric and spectroscopic parameter variations[END_REF] is now used to produce these tables.

In addition, since the latitudinal structure of the cloud is now taken into account, the tables vary with latitude, in bins of 5 • . It can be noted that in this cloud model, the unknown ultraviolet absorber is modeled independently from the cloud particle modes, regardless of the absorber's chemical composition.

The vertical profile of the solar heating plays a crucial role in the temper-

A C C E P T E D M A N U S C R I P T
ature profile, as shown e.g. by [START_REF] Lebonnois | Analysis of the radiative budget of Venus atmosphere based on infrared Net Exchange Rate formalism[END_REF]. However, it is poorly constrained by available data. A series of tests were performed with the new solar heating rates (Lebonnois and Schubert, 2017, Methods and Supplementary Materials). Below approximately the cloud base, these solar heating rates are smaller than the ones that were used previously [START_REF] Crisp | Radiative forcing of the Venus mesosphere. I -Solar fluxes and heating rates[END_REF], and this resulted in deep atmospheric temperature profiles significantly colder than observed. The composition of the lower haze particles, located between the cloud base (48 km) and 30 km and observed by in-situ probe instruments [START_REF] Knollenberg | The clouds of Venus: A A C C E P T E D M A N U S C R I P T synthesis report[END_REF], is not established, so their optical properties are not well constrained. This sub-cloud haze is not taken into account in the [START_REF] Haus | Radiative heating and cooling in the middle and lower atmosphere of Venus and responses to atmospheric and spectroscopic parameter variations[END_REF] model, except for the tail of the sulfuric acid cloud mode vertical distributions. The absorption of the solar flux in this region is therefore subject to uncertainty. To better reproduce the temperature structure in the middle cloud and below, we decided to increase the solar heating rates in this region, multiplying the solar heating rates provided by the [START_REF] Haus | Radiative heating and cooling in the middle and lower atmosphere of Venus and responses to atmospheric and spectroscopic parameter variations[END_REF] model by a factor of 3. This tuning brings the values in the same range as other models [START_REF] Crisp | Radiative forcing of the Venus mesosphere. I -Solar fluxes and heating rates[END_REF][START_REF] Lee | A Discrete Ordinate, Multiple Scattering, Radiative Transfer Model of the Venus Atmosphere from 0.1 to 260µm[END_REF], and allows the temperature profile to reach observed values.

Infrared cooling

The long-wavelength part of the radiative transfer scheme is based on the net-exchange rate (NER) formalism [START_REF] Eymet | Net-exchange parameterization of the thermal infrared radiative transfer in Venus' atmosphere[END_REF]. The cloud model an additional continuum of 1.3 × 10 -6 cm -1 amagat -2 is taken into account in 231 the 30 -48 km region, and 4 × 10 -7 cm -1 amagat -2 in the 16 -30 km region, 232 for a best fit of the VIRA (Seiff et al., 1985) and VeGa-2 [START_REF] Linkin | Thermal structure of the Venus atmosphere according to measurements with the Vega-2 lander[END_REF] 233 Zasova et al., 2006) temperature profiles (see Lebonnois and Schubert, 2017, 234 Methods and Supplementary Information).

285

The coldest region shows a rotation around the pole with a period of about 286 5.85 Earth days in the model, that agrees with the 5 -10 Earth days period 287 found by [START_REF] Luz | Venus's southern polar vortex reveals precessing A C C E P T E D M A N U S C R I P T circulation[END_REF] for the drift of the rotation center of the vor-288 tex around the pole. However, the modeled inner core does not show a clear 289 rotation around the pole, so it is difficult to compare its motion to the one 290 observed for the warm vortex [START_REF] Luz | Venus's southern polar vortex reveals precessing A C C E P T E D M A N U S C R I P T circulation[END_REF][START_REF] Garate-Lopez | A chaotic long-lived vortex at the southern pole of Venus[END_REF].

Average wind field

Considering the latitudinal structure of the clouds has also improved the zonally and temporally averaged zonal wind field. The modeled equatorial jet is now less intense than the mid-latitude jets (Figure 4) as cloud-tracking measurements suggest [START_REF] Hueso | Six years of Venus winds at the upper cloud level from UV, visible and near infrared observations from VIRTIS on Venus Express[END_REF]. The modeled mid-latitude jets are still located too close to the poles (and, therefore, the latitudinal wind gradient is still high poleward of 70 • ) but their altitude has gone down slightly, while the equatorial jet has ascended a bit.

FIG 4

In the previous model [START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF] winds at 40 -60 km were approximately half of the observed values, but the currently modeled winds at this altitude range are on average ∼ 20 m/s (about 75%) faster and are now in good agreement with the Venera and Pioneer Venus probes' profiles (see Figure 5). According to a new wave activity analysis that is still on-going work and will be presented in a subsequent publication, this improvement of the averaged zonal wind could be due to a new baroclinic activity found between 40 and 60 km altitude and that transports heat and angular momentum equatorwards. Nevertheless, the velocity of the zonal winds below 40 km altitude has

A C C E P T E D M A N U S C R I P T

decreased showing winds significantly slower than observed, which was not the case previously. The large-scale gravity waves present in the deep atmosphere in the previous simulation [START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF] are no longer obtained, which probably explains this worsening in the zonal wind below 40 km.

FIG 5 4 Cold Collar Analysis

To analyze in detail the influence of the latitudinal structure of the clouds in the formation of the cold collar, the simulation with the latitudinal variations of the cloud structure (varcld simulation) is compared to a control simulation in which a uniform cloud structure is maintained in the IR cooling computation (unifcld simulation). Note that the simulations differ in the second stage of the implementation (see Section 2.1), i.e. in the last 100 Vd, so the solar heating rates take into account the latitudinal variations in both simulations (same look-up table). The unifcld simulation has no physical sense since the cloud model is different between the solar heating and IR cooling treatments. But its results are very similar to the previous simulation presented by [START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF], so we think that the comparison between unifcld and varcld simulations is useful to identify which are the key elements in the formation of the cold collar.

The cold collar is formed in the varcld simulation but not in the unifcld

A C C E P T E D M A N U S C R I P T

simulation, which indicates that the generation of this characteristic feature is 337 not affected by latitudinal variations of the clouds in the solar heating rates.

338

It is the influence that the cloud structure has in the IR cooling rates that 339 forms the cold collar.

340

In the unifcld simulation (Figure 6a), there is a cold region around 10 3 Pa 341 and 65 • that extends downward and poleward, creating a warmer region close 342 to the pole and above that altitude level, but this cold feature is not exactly 343 as the one observed (e.g. [START_REF] Tellmann | Structure of the Venus neutral atmosphere as observed by the radio science experiment VeRa on Venus Express[END_REF][START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF], it is not 344 completely surrounded by warmer air. This structure, as well as the global 345 thermal structure in the unifcld simulation, is very similar to the structure 346 obtained in previous works and presented there as the cold collar (Ando et al., 347 2016(Ando et al., 347 , 2017;;[START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF]. The relation between this structure and 348 the thermal tides in the polar atmosphere was demonstrated by Ando et al.

349

(2016) and it should be similar in the unifcld simulation presented here. During tens of Venusian days the polar atmosphere evolves in such a way that after 60 Vd it reaches a new equilibrium, although the average temperature at the cold collar oscillates slightly afterwards. Figure 7b shows that the stronger and weaker cooling regions in the varcld simulation are still present 40 Vd after reaching the equilibrium, at 100 Vd. This is important because it means that the atmosphere at the newly formed cold collar will be constantly cooling by means of IR radiation in a quite stronger way than the atmosphere above it (referred to as the warm mesospheric pole hereon). As long as this new equilibrium endures, the cold collar will remain colder than its surroundings due to this stronger IR cooling.

Figure 8 displays the vertical profile of the IR energy exchange between different atmospheric levels and space for both unifcld and varcld simulations at the end of simulation (100 Vd) and at 75 • latitude (cold collar region). The energy loss to space from the upper clouds (∼50 -70 km altitude) is more localized in altitude in the varcld simulation than in the unifcld simulation. This, as well as the decay rate of the cooling with altitude above the clouds, is

A C C E P T E D M A N U S C R I P T
inherited from the cloud model by [START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF]. At high-latitude (higher than 60 • ) the retrieval of the cloud top altitude required the estimation of another quantity, the cloud upper altitude boundary, above which no cloud particles are considered. This cloud upper altitude boundary (located slightly above the cloud top defined at 1 µm) strongly affects the highest peak in the cooling rates, intensifying it. This peak corresponds mainly to the cooling rates in the 6-12 µm range and it is associated to the uppermost layer of cloud particles in the model. The lowest peak in the vertical profiles of Figure 8 corresponds mostly to the cooling at 20-30 µm and is associated to the base of the upper clouds, while the cooling above the clouds occurs mainly in the aisles of the 15 µm band [START_REF] Lebonnois | Analysis of the radiative budget of Venus atmosphere based on infrared Net Exchange Rate formalism[END_REF].

FIG 8

The position of the cold collar with respect to the cloud top (defined at 1 µm) and the cloud upper boundary is better seen in Figure 7. It is located just in between, coinciding with the highest peak of the cooling rates in the vertical profile. This means that the opacity at 1 µm equals unity within the upper clouds, but that there is a region with cloud particles above it where the cold collar is formed. The cold collar is, therefore, located at the uppermost layer of the clouds. Its position is, however, cloud model dependent since it is strongly linked to the cloud upper boundary defined by [START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF].

The induced modifications in the temperature structure affects the circu- 9b), where dynamics heats the atmosphere 454 (Figure 9a). Above the clouds, in the warm mesospheric pole, the polar heat-455 ing due to dynamics occurs by means of vertical transport (Figure 9d), but 456 it is mitigated by horizontal transport (Figure 9c). On the other hand, in the 457 cold collar and inner warm core areas, horizontal transport mostly heats the 458 area (although a region of dynamical cooling is appreciated between the main 459 two heating regions). This heating is mitigated by vertical heating transport. in both unifcld and varcld simulations (in Pa/s units, so negative values mean upwelling motion). Although the main differences take place at the clouds and levels below, an altered subsidence structure that extends down to 50 • latitude is seen in the varcld simulation, responsible of the vertical heating seen in Figure 9d. Ascending air is found in the cold collar between 60 • and 70 • latitudes and at about 10 4 Pa altitude for the varcld simulation which is associated with the dynamical cooling seen in Figure 9d. A reversal of the vertical circulation in the 70 • -90 • polar region from unifcld to varcld simulations is also clearly seen.

FIG 10

Hence, according to the results of the new IPSL Venus GCM, the cold collar feature observed in Venus' polar atmosphere is formed by the infrared cooling region generated by the decrease of the cloud top altitude poleward and the latitudinal variation of the particles modes. Since the radiative forcing cools the area while the dynamics heats it, resulting in a region characteristically cold, it seems that the driver of the cold collar is the radiative transfer rather than dynamics. However, the presence of the cold collar strongly influences the dynamics, thus affecting the polar circulation of Venus. Even though the cloud distribution, radiative transfer, and dynamics form a coupled system in reality and, therefore, any change in one of the mentioned aspects will bring along a change in the other aspects, this coupling is not yet present in our GCM and we can not characterize it in detail.

A C C E P T E D M A N U S C R I P T

The temporal evolution observed in the modeled polar temperature maps at 7×10 3 Pa altitude (not shown here) presents a cold collar that is displaced from the pole and rotates around it. The obtained rotation period of 5.85 Earth days agrees with the period found by [START_REF] Luz | Venus's southern polar vortex reveals precessing A C C E P T E D M A N U S C R I P T circulation[END_REF] for the drift of the rotation center of the vortex around the pole. This result, together with the structure of the cold feature in the average temperature field, indicates that the current IPSL Venus GCM models satisfactorily the subpolar latitudes. However, the current polar latitudes' treatment is not ideally suited and, therefore, the modeled inner core does not show neither a clear rotation around the pole nor detailed horizontal structure within it, as the observed polar vortex does.

Conclusions

The previous IPSL Venus GCM [START_REF] Lebonnois | Wave analysis in the atmosphere of Venus below 100-km altitude, simulated by the LMD Venus GCM[END_REF] The main result, however, is the appearance of a feature in the averaged altitude-latitude temperature map that resembles the subpolar cold collar observed in Venus's atmosphere. The atmosphere between 60 • and 85 • shows a thermal minimum at 2 × 10 4 -4 × 10 3 Pa (∼ 62 -66 km) altitude, being ∼20 K colder than the equator and about 10 K colder than the pole. The coldest region shows values about 220 K, in good agreement with observed values, but the inner warm core shows an average value of 230 K that is colder than the observed values. The structure simulated within the inner core is smoother than that obtained by [START_REF] Yamamoto | Dynamics of polar vortices at cloud top and base on Venus inferred from a general circulation model: Case of a strong diurnal thermal tide[END_REF] and [START_REF] Ando | The puzzling Venusian polar atmospheric structure reproduced by a general circulation model[END_REF][START_REF] Ando | Vertical structure of the axi-asymmetric temperature disturbance in the Venusian polar atmosphere: comparison between radio occultation measurements and GCM results[END_REF], but the altitude level where the cold collar is reproduced and the modeled temperature values are more consistent with observations. According to our analysis the cloud's latitudinal structure is an essential element in the formation of the cold collar via the IR cooling rates. A strong cooling region at ∼ 75 Once the atmosphere reaches a new thermal equilibrium, the radiative cooling is compensated by heating due to dynamics. Consequently, the dynamical heating is stronger at the cold collar area than in the warm mesospheric pole (85 • and 4 × 10 2 Pa). Our analysis points to vertical transport (associated to a subsidence motion that extends down to 50 • ) as the main source of dynamical heating in the warm mesospheric pole and to horizontal transport at the cold collar.

Since the radiative forcing cools the area while the dynamics heats it, resulting in a region characteristically cold, it seems that the driver of the cold collar is the radiative transfer rather than dynamics.

90

  scheme are described in Section 2. Then, Section 3 shows the average temper-95

134

  was not taken into account in this previous IPSL model, though Lebonnois 135 et al. (2016) noted that it could play a role in the shape and strength of the 136 cold collar. 137 Due to the development of different tools necessary to implement the lati-138 tudinal variation of the cloud structure in the solar heating rates and in the 139 infrared cooling rates, the improvements in the radiative transfer were done 140 in two stages; (1) implementation of the cloud structure in the solar heating 141 rates, plus some tuning, and (2) implementation of the cloud structure in the 142 IR cooling rates (these improvements and tuning are described in the next 143 section). The simulation started from an already superrotating atmosphere 144 obtained from previous simulations (Lebonnois et al., 2016) and it was then 145 run for 200 Venus days (Vd, 117 Earth days) with the partially-improved ra-146 diative transfer, the one obtained after the first stage of the improvements. 147 Two simulations were then pursued for another 100 Vd: one with latitudinally 148 uniform clouds in the IR cooling computation (referred to as unifcld hereon) 149 and another one with latitudinal variations of the cloud structure for study-150 ing the cold collar (varcld hereon). The former case, unifcld simulation, is A C C E P T E D M A N U S C R I P T

  A C C E P T E D M A N U S C R I P T(density profile of each mode, n i (z)) used in the previous computation of the 214 net-exchange rate matrices was fromZasova et al. (2007). In this work, we 215 now use the cloud model described in[START_REF] Haus | Atmospheric thermal structure and cloud features in the southern hemisphere of Venus as retrieved from VIR-TIS/VEX radiation measurements[END_REF]. In order to take 216 into account the latitudinal variations of this model, matrices are computed 217 for five latitudinal bins: 0 • -50 • , 50 • -60 • , 60 • -70 • , 70 • -80 • and 80 • -90 • . 218 When running the GCM, the matrices are interpolated between the central 219 latitudes of each bin. 220 The gas opacities are computed first for high-resolution spectra, taking into 221 account updated spectral dataset, Voigt line shape profiles, and a truncation at 222 200 cm -1 . Then correlated-k coefficients are computed and used in the NER 223 matrix calculation. In addition, CO 2 and H 2 O collision-induced absorption 224 are taken into account, as detailed in Lebonnois et al. (2015). However, as 225 mentioned in Lebonnois et al. (2015) and Lebonnois and Schubert (2017), it 226 is difficult to have consistent temperature profiles in the middle and lower 227 cloud and in the atmosphere below without including some small additional 228 continuum to close the windows located in the [3 -7] µm range, through which 229 energy is exchanged between the cloud base and the layers just below. Here, 230

FIG 1 250

 1 FIG 1

Figure 3

 3 FIG 2

291Figure 3

 3 Figure 3 in Lebonnois et al. (2016) displayed four temperature fields at

  FIG 6

  FIG 9

460

  Studying the variation of the circulation, particularly of the zonal and 461 meridional winds, is not trivial due to the high variability present in the po-462 lar regions. This study of the horizontal distribution of the dynamical polar 463 atmosphere in the varcld simulation will be presented in a subsequent paper 464 together with the new wave activity. However, the variation of vertical wind is 465 interesting and noteworthy. Figure 10 shows the vertical wind velocity fields A C C E P T E D M A N U S C R I P T

  has been modified in order to improve the radiative transfer, update the cloud model, and take into account the latitudinal variation of the clouds (by means of cloud top altitude and different modes' abundances) in the solar heating profiles and infrared cooling computation. The new cloud model used in the current version is described by Haus et al. (2014) and shows that the cloud top altitude decreases towards the pole in Venus's atmosphere and that the different particle modes' abundances change from equator to pole. The zonally and temporally averaged zonal wind and temperature fields A C C E P T E D M A N U S C R I P T have been improved showing now a better agreement with the observations. Particularly, the equatorial jet's intensity has decreased compared to the midlatitude jets' intensity, the zonal wind velocities between 40 and 60 km altitude are faster than previously, and the averaged temperature values below (resp. above) the clouds are now warmer (resp. colder), thus closer to the VIRA reference profile.

  • and 10 4 Pa is developed as soon as the new cloud structure is implemented in the IR computations and it remains cooling strongly after a new equilibrium is reached and the cold collar is formed. A region of A C C E P T E D M A N U S C R I P T weak IR cooling is also developed above the polar clouds just where the warm mesospheric pole is observed. The position of the cold collar is cloud model dependent since it is strongly linked to the cloud upper altitude boundary (level above which no particles are considered) defined by Haus et al. (2014) at high latitudes. The IR cooling peak is located right below this cloud upper boundary, above the cloud top altitude. This means that the cold collar is located at the uppermost layer of the clouds, in a region with cloud particles but above the level where the opacity equals one at 1 µm.

Fig. 1 .

 1 Fig.1. Horizontally and temporally averaged temperature profile for the previous (red) and current (green) IPSL Venus GCM models at the end of simulation (300 Vd) compared to VIRA reference profile (black). Data is globally averaged over all longitudes and latitudes in each altitude, and over 2 Vd in time.

Table 1

 1 Chosen inputs for the simulations. H15 b L16 c updated Z07 d H13 e H14 f L16 c updated g Haus et al. (2015), with additional tuning in lower haze area (see text) c Lebonnois et al. (2016) d Zasova et al. (2007) e Haus et al. (2013), clouds taken as uniform with latitude f Haus et al. (2014), clouds taken as variable with latitude g see text

	Solar heating Correlated-k set	Cloud model (IR)	Continuum (3-7µm)
	C86 a Initial state ×		×	×	×
	0-200 Vd	×	×	×	×
	200-300 Vd:
	-unifcld	×	×	×	×
	-varcld a Crisp (1986)	×	×	×	×

b

Seiff, A., Schofield, J. T., Kliore, A. J., et al., 1985. Model of the structure of the atmosphere of Venus from surface to 100 km altitude. Adv. Space Res. 5 (11), 3-58. Sugimoto, N., Takagi, M., Matsuda, Y., 2014a