Fig 1.
Classification of Deroceras leave and Ambigolimax valentianus.
(A) D. laeve and (B) A. valentianus adult individuals. The radula of D. laeve (C-H) and A. valentianus (I-N) was analyzed by scanning electron microscopy. (O) The sequence of a fragment of cytochrome oxidase 1 (Cox1) was analyzed by multiple alignment with the corresponding sequences of individuals of fourteen stylommatophoran species including D. laeve and A. valentianus. Green and blue arrows indicate lab-reared D. laeve and A. valentianus, respectively, from this work.
Fig 2.
Growth and reproduction of D. laeve and A. valentianus.
Growth was measured by weighing individuals each week (A, C), survival rate was determined by counting the members of isolated clutches over time. (B, D), degrowth of D. laeve was determined by starvation of animals for eight weeks and feeding ad libitum for the subsequent four weeks (E), and the hatching rate was measured for isolated groups of eggs of both species (F). For (A), n = 200 animals. For (B), n = 4325 animals. For (C), n = 200 animals. For (D), n = 679 animals. For (E), n = 10 animals. For (F), D. laeve, n = 438 clutches; A. valentianus, n = 84 clutches.
Fig 3.
Histology and anatomy of D. laeve.
Whole specimens were sectioned and stained with hematoxylin-eosin on the horizontal (A, A’), transversal (B, B’), or sagittal planes (C, C’). Each series was digitized, assembled in mosaic and layered into a digital stack (D). Manual tracing of the external contour of each section and segmentation of the major internal organs were used to build a 3D model (D, D’). Abbreviations in Table 1.
Fig 4.
Histology and anatomy of A. valentianus.
Whole specimens were sectioned and stained with hematoxylin-eosin on the horizontal (A, A’), transversal (B, B’), or sagittal planes (C, C’). Each series was digitized, assembled in mosaic and layered into a digital stack (D). Manual tracing of the external contour of each section and segmentation of the major internal organs were used to build a 3D model (D, D’). Abbreviations in Table 1.
Fig 5.
Screenshots of the different sections of SlugAtlas (https://slugatlas.lavis.unam.mx) exemplify the various functionalities of the resource. (A) Landing page. (B) Second page with the buttons to access the 3D visualizer and the 2D histology collections. (C) Example of histological section of D. laeve and (D) a magnified view of a region of that section. (E) Example of 3D model view of the whole body and (F) of the circumesophageal nerve ring of D. laeve.
Table 1.
Abbreviations used in this work.
Fig 6.
Body wall of D. laeve and A. valentianus.
(A, C, I, and K) Dorsal or (B, J) ventral body wall in longitudinal sections. (D and L) Lateral fold of the foot in transverse section. (E and M) Epidermis of the mantle in transverse section (E is a semithin section). (F and F’) Semithin longitudinal sections of the foot. (G-H) Semithin sections of the sec in horizontal plane. (N) Muscle bundles of the subepidermal connective (sec) in horizontal plane. (O) Muscular mesh in the subepidermal connective of the mantle in the horizontal plane. Scale bar in B applies to A, B, F, F’, I, J, and O; bar in L applies to C-E, G, H, and K-N. Abbreviations in Table 1.
Fig 7.
Foot and suprapedal gland of D. laeve and A. valentianus.
(A and H) 3D models of the whole body (brown) and the suprapedal gland (blue). (B and I) Transverse sections of the foot in anterior and (C and J) in posterior regions (arrows indicate suprapedal gland). (D and K) Transverse sections of the suprapedal gland in anterior and (E and L) in posterior regions. (F and M) Horizontal sections on the posterior and (G and N) anterior regions of the suprapedal gland. Asterisks indicate the lumen of the suprapedal gland, dotted lines indicate the anatomical midline, and arrows in D-M indicate secretory streaks of the suprapedal gland. Scale bar in J applies to B, C, and I. Bar in L applies to D, E, and K. Bar in N applies to F, G, and M. Abbreviations in Table 1.
Fig 8.
Muscle bundles of D. laeve visualized with phalloidin staining.
(A-E) Lateral pedal fold in a transverse section showing (A) tissue texture obtained by DIC optics, (B) phalloidin staining, (C) Hoechst 33342 (nuclei), (D) 3-channel projection, and (E) 3D rendering. (F) Horizontal section showing the free muscle stained with Phalloidin (arrow) with Hoechst 33342 counterstain. Abbreviations in Table 1.
Fig 9.
Tentacles and Semper’s organ of D. laeve and A. valentianus.
(A) Diagram of posterior tentacle in everted and (B) in retracted configuration. (C) Horizontal semithin section of the right posterior tentacle of D. laeve. (D) Diagram of anterior tentacle in retracted configuration. (E-F) Posterior and (G-H) anterior tentacle and Semper’s organ (So) of D. laeve. Inset in (G), Semper’s organ in a more lateral position. (I) Posterior and (J-K) anterior tentacle of A. valentianus. Values of scale bar: E (400 μm); F-H and J (100 μm); C and E (300 μm); inset in G (300 μm); and K (80 μm). Diagram in (A), modified from [33]. Image in (I) was assembled in mosaic manually from several micrographs of the same section in Gimp. Abbreviations in Table 1.
Fig 10.
Nervous system of D. laeve and A. valentianus.
(A) 3D render of D. laeve and (B) of A. valentianus. (C) 3D model of the circumesophageal nerve ring of D. laeve showing the ganglia in different colors, and in D and E, the cellular cortical regions in dark color and the neuropil in light color. (F-G) Sagittal sections of D. laeve and (H) transverse section with Kluver-Barrera stain. (I-J) Sagittal sections of A. valentianus. (K) 3D-printed model of the nervous system of D. laeve for educational purposes. Scale bar in I applies to F. Bar in J applies to G. Abbreviations in Table 1.
Fig 11.
Buccal organ of D. laeve and A. valentianus.
Sagittal sections are shown. (A) Medial region of buccal organ. (B) Dorsal lip with jaw and cuticle. (C) Medial region of buccal organ. (D) More lateral region than in C. (E) Dorsal lip with jaw and cuticle. (F) Esophagus. Scale bar in A applies to C and D. Bar in B applies to E and F. Abbreviations in Table 1.
Fig 12.
Digestive system of D. laeve and A. valentianus.
(A-M) D. laeve. (A) 3D model of digestive system (digestive tube in cyan and digestive gland in green). (B, C, and K) Epithelium of the anterior region of the stomach (B is a Kluver-Barrera stain and K is silver stained). (D) Posterior region of the stomach. (E) Descending intestine. (F) Ascending intestine. (G) Ascending intestine near the anus. (H) Salivary gland. (I and J) Region where the digestive gland drains into the intestine. (L and M) Digestive gland (M is a semithin section). (N-W) A. valentianus. (N) 3D model of digestive system (digestive tube in cyan and digestive gland in green). (O) Epithelium of the anterior region of the stomach. (P) Posterior region of the stomach. (Q-R) Descending intestine. (S) Ascending intestine. (T) Ascending intestine near the anus. (U) Salivary gland. (V) Region where the digestive gland drains into the intestine. (W) Digestive gland. Arrows in B-G and O-T indicate the digestive tube epithelium. Scale bar in G applies to B-H, K-M, and O-U and W. Bar in J applies to I and V. Abbreviations in Table 1.
Fig 13.
Heart of D. laeve and A. valentianus.
(A-F) D. laeve. (A) 3D model of the whole body (brown) and the heart (pink). (B-D) Sections of the heart of a juvenile D. laeve and (E and F) of an older individual (semithin sections stained with toluidine blue). (G-L) A. valentianus. (G) 3D model of the whole body (brown) and the heart (pink). (H-J) Sections of the heart of a juvenile and (K-L) an older individual. Arrow in C and K-L indicates the atrioventricular valve, in D and J it indicates squamous cells of the myocardium. Scale bar in C applies to D-E. Bar in L applies to I-J. Abbreviations in Table 1.
Fig 14.
Kidney and lung of D. laeve and A. valentianus.
(A-J) D. laeve. (A) 3D model of the whole body (brown), lung (blue), and kidney (orange). (B-C and H-J) Transverse sections of the lung and kidney. (D-G) Horizontal semithin sections. (H-J) Close interaction of the ureter with the airway in D. laeve. Arrow in F indicates renocytes bursting into the renal lumen releasing their concretions, in G it indicates the villi in the lung epithelium, and in I-J the merging of the ureter with the airway. (K-N) A. valentianus. (K) 3D model of the whole body (brown), lung (blue), and kidney (orange). (L-N) Transverse sections of the lung and kidney. (C and E) Close interaction of ureter with airway and their merging (arrow in N). Scale bar in B applies to C and D. Bar in N applies to E-J and M. Abbreviations in Table 1.
Fig 15.
Reproductive system of D. laeve and A. valentianus.
(A-B’) Diagrams of both species show the topology of the different components but not their proportions. The topology differs between the two species and between the two ages analyzed of A. valentianus. White curved lines indicate the lumen of the different ducts that constitute the system and show their connection patterns. Modified from [38]. (C-M) Reproductive system of D. laeve. (C) 3D model showing the reproductive system in red. (D) ovotestis. (E) Albumen gland. (F) Albumen gland with silver stain. (G) Hermaphroditic duct and albumen gland. (H) Nidamental gland. (I) Spermoviduct. (J) Oviduct and vas deferens. (K) Penial complex, bursa copulatrix/spermatheca, and spermoviduct. (L) Penial gland, penial complex, peduncle of bursa copulatrix, vas deferens, and oviduct. (M) genital atrium. Scale bar in F applies to D, E, G, and J. Bar in M applies to H, I, K, and L. Abbreviations in Table 1.
Fig 16.
Reproductive system of A. valentianus.
(A) 3D model showing the reproductive system in red. (B-I) Micrographs of a juvenile individual and (I-O) an older individual. (B) Ovotestis (predominantly spermatogenic). (C) Hermaphroditic duct. (D) Spermoviduct/prostate gland. (E) Spermoviduct/prostate gland. (F) Spermoviduct/prostate gland. (G) Spermoviduct and penis, (H) Penial complex. (I) Ovotestis (hermaphrodite). (J) Albumen gland. (K) Nidamental gland and spermoviduct/prostate gland. (L) Spermoviduct and bursa copulatrix. (M) Spermoviduct. (N) Oviduct and bursa copulatrix. (O) Genital atrium and gonopore. Values of scale bar: B and I (100 μm); C-G, J, and M-O (200 μm); H, K, and L (400 μm). Abbreviations in Table 1.
Fig 17.
Regeneration of the tail in D. laeve.
Sagittal sections after amputation: (A and B) stained with hematoxylin and eosin and (C-F) labeled with F-ara-EdU (red) counerstained with Hoechst 33342 (blue) and Sitox (green). Non-amputated control (Ctrl), 6 hours (6h) and days post-amputation (2d, 7d, and 14d) are shown in each panel. Rectangles in each panel indicate the magnified region shown below in each inset. Arrows indicate the location of the blastema. White arrowheads in B indicate fibroblastoid cells (f), muscle cells (m), and round cells resembling hemocytes (h). Dorsal (D), ventral (V), posterior (P), and anterior (A) orientations are indicated in D. Scale bar in B also applies to A and bar in D applies to C-F. Abbreviations in Table 1.