Morphological grounds for the obligate aerial respiration of an aquatic snail: functional and evolutionary perspectives

The freshwater caenogastropod family Ampullariidae is emerging as a model for a variety of studies, among them, the evolution of terrestriality. A common character of the family is that all its members bear a lung while retaining the ancestral gill. This ensures that many ampullariids are able to inhabit poorly oxygenated waters, to bury in the mud during estivation, and to temporarily leave the water, in some species for oviposition. To these characters Pomacea canaliculata (Caenogastropoda, Ampullariidae) adds that is an obligate air-breather. In a recent paper, we showed the gill epithelium of P. canaliculata has a set of characteristics that suggest its role for oxygen uptake may be less significant than its role in ionic/osmotic regulation and immunity. We complement here our morphological investigation on the respiratory organs of P. canaliculata by studying the lung of this species at the anatomical (3D reconstructions of the blood system and nerve supply), histological and ultrastructural levels. The circulation of the gill and the lung are interconnected so that the effluence of blood from the gill goes to the lung where it completes oxygenation. Besides that, we found the lung cavity is lined by a pavement epithelium that encloses an anastomosing network of small blood spaces resting over a fibromuscular layer, which altogether form the respiratory lamina. The pavement cells form a blood-gas barrier that is 80–150 nm thick and thus fulfils the requirements for an efficient gas exchanger. Tufts of ciliary cells, together with some microvillar and secretory cells, are interspersed in the respiratory lamina. Rhogocytes, which have been proposed to partake in metal depuration and in the synthesis of hemocyanin in other gastropods, were found below the respiratory lamina, in close association with the storage cell tissue. In light of these findings, we discuss the functional role of the lung in P. canaliculata and compare it with that of other gastropods. Finally, we point to some similarities in the pattern of the evolution of air dependence in this family.

The preparation of both lung roof and floor samples for TEM involved fixation in 139 Karnovsky's fluid, post-fixation in 1% osmium tetroxide overnight, dehydration in a graded 140 acetone series, and embedding in Spurr's resin. We obtained silver-grey, ultrathin sections, 141 which we mounted on copper grids, stained them with uranyl acetate and lead citrate, and 142 examined them with a Zeiss EM 900 microscope. Besides, we used ~200 nm-thick 143 ultramicrotome sections stained with toluidine blue and mounted with DPX medium (Sigma-144 Aldrich, Cat. #44581) for topographical orientation and examination under light microscopy.

146 Results
147 General organization of the lung 148 The lung of P. canaliculata (Fig. 1A) is a single sac that occupies most of the roof of the mantle 149 cavity. It abuts the gill on its posterior and right margins. An opening in the lung floor, namely 150 the pneumostome, lies anteriorly, near the osphradium and the left mantle edge, and 151 communicates the lung cavity with the mantle cavity. The pneumostome shows two lips (anterior 152 and posterior) and can close by their apposition. Prominent blood sinuses traverse both the lung 153 roof and floor. The muscular layer of the lung floor is formed of muscle strands arranged in two 154 directions, thus is thicker than that of the roof, particularly in the region rostral to the 155 pneumostome. 156 From dorsal to ventral, the lung roof (Fig. 1B) is composed of (1) the outer mantle 157 epithelium, formed by columnar pigment and mucous cells; (2) a single muscle layer; (3) a 158 vascular layer containing the main blood sinuses supplying the lung, which are surrounded by 159 the perivascular storage tissue (Giraud- Billoud et al. 2011); and (4) the respiratory lamina. 160 Almost mirroring that arrangement, the lung floor (Fig. 1C) is composed, from dorsal to ventral, 161 of (1) the respiratory lamina; (2) the vascular and storage tissue layer, which is thicker than that 162 of the roof; (3) a double muscle layer with fibers arranged in perpendicular directions; and (4) 163 the inner mantle epithelium that lines the mantle cavity, and which is formed by columnar 164 ciliated and mucous cells. 165 166 Blood supply and innervation of the lung 167 The 3D reconstruction of the lung and related pallial organs showed the blood supply to the roof 168 and the floor comes from different origins ( Fig. 2; see the interactive 3D model in Fig. S1). The 169 afferent pulmonary vein, which is a prolongation of the afferent branchial vein, runs along the 170 anterior and left margins of the lung, branching repeatedly and supplying the lung roof all along 171 its course. Upon irrigating the lung roof, the blood reaches the efferent pulmobranchial vein, 172 which conveys blood also from the gill leaflets and carries it to the heart auricle (Figs. 2B and 173 2E).

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In turn, the lung floor receives blood from several origins and, for clarity, will be treated 175 separately as the right (Fig. 2C) and the left afferent systems (Fig. 2D). Connection of the gill 176 and lung circulation occurs through the right afferent sinuses. The ventral afferent pulmonary 177 vein is the main right afferent and runs parallel to the already mentioned efferent pulmobranchial 178 vein. It receives blood from the gill leaflets and divides within the right half of the lung floor into 179 numerous right afferent sinuses (Figs. 2C and 2F). All the latter will end at the ventral efferent 180 pulmonary vein, which follows a diagonal path within the lung floor. This large vein arises as a 181 branch of the efferent pulmobranchial vein, and finally joins it again close to its entrance into the PeerJ reviewing PDF | (2020:03:46818:2:0:NEW 16 Dec 2020) Manuscript to be reviewed 227 phagocytosis of a particulate material of variable size and appearance that occurs in the sinus 228 blood.

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Muscle and collagen fibers appear sectioned both transversally and longitudinally in the 230 fibromuscular layer (Figs. 7E and 7F). Neurite bundles with vesicles of moderate to high electron 231 density and glial cells with granules of high electron density (Fig. 7F) occur in this fibromuscular 232 layer, frequently in the proximity of the ciliary tufts, but no evidence of intraepithelial 233 innervation (as that seen in the gill, Rodriguez et al. 2019) could be discerned in the lung.

234
It should be noted that this layer of fibromuscular tissue separates the respiratory lamina 235 from the large blood sinuses that supply the lung. Radial sinuses originating from them traverse 236 the fibromuscular layer conveying blood to the sinus of the respiratory lamina (Figs. 1B and 1C). 237 These radial sinuses are of varying length, but they are much narrower than the main sinuses, and 238 in certain occasions, there are direct connections between the main blood sinuses and the blood 239 spaces of the respiratory lamina (Fig. 1A). We found sparse endothelial-like cells lining the 240 radial sinuses (Fig. 6).  Manuscript to be reviewed 274 glycogen clumps (Fig. 10A), as well as some well-delimited cytoplasmic globules, with varying 275 sizes and electron densities (Figs. 10A, 10C and 10D), also occurred in the rhogocytes of the 276 examined P. canaliculata individuals.

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A summary of the lung structures in P. canaliculata and their proposed functions is 278 presented in Table 1. . The majority of lung-bearing gastropods, however, are 284 comprised within the Stylommatophora (superorder Eupulmonata) and the "basommatophora" 285 (superorder Hygrophila)(Bouchet et al. 2017), in which the pallial cavity has acquired the role of 286 an air-breathing organ and is partly closed by extensions of the mantle edge. In these cases, the 287 lung and the pallial cavity are homologous structures (Ruthensteiner 1997) and the pneumostome 288 opens to the exterior (Fig. 11A).

289
In the particular case of the Ampullariidae, however, the lung is a flattened sac exhibiting 290 distinct roof and floor regions, and a pneumostome that opens through the floor, connecting the 291 lung cavity with the mantle cavity (

299
To our knowledge, and before the current study, the detailed histology of these 300 presumptive gas exchanging areas had being adequately studied in just one tropical slug (Maina 301 1989; Polytoxon robustum, Urocyclidae, as Trichotoxon copleyi, a junior synonym) and one 302 ampullariid snail (Lutfy & Demian 1965; Marisa cornuarietis). The study in P. robustum was 303 made using SEM and TEM, and the inner surface of the lung (the presumptive gas exchanger) 304 appears covered by approximately parallel ridges, which appear hollow in TEM sections and that 305 Maina (1989) called 'capillaries'. They are lined externally by pavement cells, much similar to 306 those lining the blood respiratory sinuses in P. canaliculata. Also, the blood sinuses contain a 307 particulate material among the hemocytes, similar to that seen in P. canaliculata. We interpret 308 they are cell debris that the slow blood flow accumulates in the sinuses.

309
Instead, the study in M. cornuarietis (Lutfy & Demian 1965) was made exclusively using 310 light microscopy of paraffin sections, which we think adds merit to that outstanding paper, in 311 which they accurately described the blood sinuses of the respiratory lamina but, unfortunately, 312 they mistakenly equated the storage cells of the lung wall with blood lacunae. In fact, the 313 cytoplasmic contents of these large cells may be extracted during histological processing, and the 314 remaining cytoplasmic rim may give the false appearance of the wall of blood sinuses. Another 315 important limitation of that paper is its strictly descriptive character and, consequently, the fact 316 that the authors did not advance any functional interpretation. In both studied Ampullariidae 317 (Lutfy & Demian 1965; and this paper), the vascularized area involves all the inner surface of the 318 lung cavity. Manuscript to be reviewed 320 The lung floor, the gill, and the adaptation of Pomacea canaliculata to air 321 breathing 322 The lung roof and the lung floor receive blood from partly different origins. The lung roof 323 receives blood from right afferents (Fig. 2E), while the lung floor receives blood from right 324 afferents that have already traversed the gill and from left afferents that come directly (Fig. 2F). 325 Deoxygenated blood from the visceral hump reaches the afferent branchial vein through the 326 right pallial and rectal sinuses. That vein not only supplies the gill but, after leaving it, this vein 327 is termed the dorsal afferent pulmonary vein (Andrews 1965) and distributes blood to all of the 328 lung roof and to the anterior and right side of the lung floor. Deoxygenated blood conveyed by 329 blood sinuses from the mantle edge also reaches the lung roof through the dorsal afferent 330 pulmonary vein. After oxygenation in the respiratory lamina of the lung roof, the blood is 331 drained with that coming from the gill through a common efferent route, i.e., the efferent 332 pulmobranchial vein ( Fig. 2A). Instead, the lung floor receives oxygenated blood from the gill 333 leaflets through the ventral afferent pulmonary vein (Fig. 2B, right sinuses). It also receives 334 deoxygenated blood from the left side of the body, through several slender left sinuses and from 335 a large sinus that collects blood from the siphon and the left mantle border (Fig. 2B, left sinuses).

336
When the animal is submerged and the gill leaflets are deployed, oxygenated blood may 337 reach the auricle directly, through the efferent pulmobranchial vein. However, when the animal 338 is outside of the water, the gill leaflets collapse and stick together, thus drastically reducing both 339 the circulation through the leaflet sinuses and the available surface for gas exchange. Then, the 340 blood would shunt through the basal leaflet sinuses (Rodriguez et al. 2019) converging into the 341 ventral afferent pulmonary vein, which will distribute blood in the right side of the lung floor. 342 Also, even when breathing in water, blood conveyed by the rectal and right pallial sinuses may 343 traverse the gill leaflets, also reaching the lung floor through the ventral afferent pulmonary vein.   360 In general, a blood-gas barrier must have both a thin and an extensive surface to facilitate gas 361 diffusion, but it should be sturdy enough to withstand the rough work of respiratory movements 362 and currents. Comparative studies have stressed that this is achieved by structures that combine a 363 "soft" and a "hard part", the former to allow gas-exchange, and the latter to keep the soft part in 364 place, and in functional conditions (Maina & West 2005).
In P. canaliculata, the thin gas exchange membrane (80-150 nm width, Fig. 7D) needs to 366 be anchored to a "hard part" (Maina & West 2005), which in this case is the fibromuscular layer 367 (Figs. 7A-7D). This is achieved by the intercellular junctions that bind the pavement cells to the 368 epithelial cells of the ciliary tufts, which are themselves deeply rooted in the fibromuscular layer 369 at frequent intervals (Fig. 4A), and thereby may be acting as rivets that keep the epithelial and 370 connective parts together (Fig. 4A). We have also occasionally observed some fibroblast cells in 371 direct contact with the basal part of pavement cells, and that may be an additional structural 372 component that needs to be explored further.

373
However, other important functions of the ciliary tufts should be those related to their 374 secretions and to the ability of their cilia to spread them over the surface of pavement cells. 375 Possible roles would be the agglutination of dust particles that have entered with air, and that 376 would be expelled by the ciliary rows of the pneumostome (Fig. 5), as well as the lubrication of 377 the delicate respiratory lamina during breathing movements. Besides that, a phospholipidic 378 surfactant, found in the lungs or tracheoles of a wide variety of animals (Orgeig et al. 2007), has 379 been reported in the lung of the garden snail Cornu aspersum (Stylommatophora, Helicidae) 380 (Daniels et al. 1999). This should encourage the search for compounds with a similar function in 381 the ciliary tufts' secretions in P. canaliculata.

382
Taken together, our histological and ultrastructural findings on the respiratory organs may 383 constitute the morphological basis to explain why P. canaliculata is an obligate air-breather . 398 503 pneumostome, the osphradium, and each of the gill leaflets through the leaflet nerves (Fig. 3). 504 The accessory visceral ganglion gives off several branches that innervate the lung floor and a 505 single branch that runs all the way through the branchial base, namely the branchial base nerve 506 (Fig. 3C). 507 The shared innervation as well as the interconnections between the supraesophageal and 508 the accessory visceral ganglia might also provide the neural mechanisms for the behavioral 509 shifts between branchial and pulmonary breathing, which is coupled with the deployment of the Manuscript to be reviewed 547 6. We described rhogocytes, for the first time in an ampullariid, and discussed their association 548 with storage tissue in other organs. Roles in heavy metals detoxification and hemocyanin 549 production have been proposed for these cells.              gill; lcv, lung cavity; mth, mouth; omm, ommatophore; opc, operculum; osp, osphradium; pca, pallial cavity; pfo, pallial fold; pne, pneumostome; pod, foot; rnl, right nuchal lobe; shl, shell; ten, tentacle.