A Dissenters' View on AppleSnail Immunobiology

Abstract

We stand as dissenters against the acceptance of scientific knowledge that has not been built on empirical data. With this in mind, this review synthesizes selected aspects of the immunobiology of gastropods and of apple snails (Ampullariidae) in particular, from morphological to molecular and "omics" studies. Our trip went through more than two centuries of history and was guided by an evo-devo hypothesis: that the gastropod immune system originally developed in the mesenchymal connective tissue of the reno-pericardial complex, and that in that tissue some cells differentiated into hematopoietically committed progenitor cells that integrate constitutive hemocyte aggregations in the reno-pericardial territory, whether concentrated in the pericardium or the kidney in a species-specific manner. However, some of them may be freed from those aggregations, circulate in the blood, and form distant contingent aggregations anywhere in the body, but always in response to intruders (i.e., pathogens or any other immune challenge). After that, we reviewed the incipient immunology of the Ampullariidae by critically revising the findings in Pomacea canaliculata and Marisa cornuarietis, the only ampullariid species that have been studied in this respect, and we attempted to identify the effectors and the processes in which they are involved. Particularly for P. canaliculata, which is by far the most studied species, we ask which hemocytes are involved, in which tissues or organs are integrated, and what cellular reactions to intruders this species has in common with other animals. Furthermore, we wondered what humoral factors could also integrate its internal defense system. Among the cellular defenses, we give an outstanding position to the generation of hemocyte nodules, which seems to be an important process for these snails, serving the isolation and elimination of intruders. Finally, we discuss hematopoiesis in apple snails. There have been contrasting views about some of these aspects, but we envision a hematopoietic system centered in the constitutive hemocyte islets in the ampullariid kidney.

Keywords: Biomphalaria; Pomacea; hematopoiesis; hemocyanin; hemocyte; nodulation; reno-pericardial complex; rhogocyte.

Figure 1

Circulating hemocyte populations of P. canaliculata and their separation by flow cytometry. (A) Flow cytometry (dot plot of size vs. complexity-granularity) of a representative blood sample indicating the sorted areas where either agranulocytes, granulocytes and hyalinocytes were predominant. (B) Examples of hyalinocytes, agranulocytes and granulocytes in hemolymph smears (hematoxylin-eosin stain). (C) Examples of living hyalinocytes, agranulocytes and granulocytes attached onto a glass slide (phase contrast). GR, agranulocytes; GRA, granulocytes; HYA, hyalinocytes. Scale bars represent 5 μm. From (41).

Figure 2

Circulating hemocytes of P. canaliculata. Panels (A–C), TEM; scale bar represents 1 μm. (A) Hyalinocyte with an eccentric nucleus, numerous SER vesicles, mitochondria and L granules; a few profiles of the RER are also seen. (B) Granulocyte, showing a displaced, bean-shaped nucleus and numerous R granules; some SER vesicles are also seen. (C) Granulocyte in a preparation exposed to E. coli cells showing extensive R granule fusion and a single L granule (arrow). Panels (D–F), laser confocal microscopy, LysoTracker Red-Hoechst 33258 staining; scale bar represents 10 μm. (D) A group of spreading hyalinocytes; small acidic L granules (red) are seen in some of them. (E) A spreading granulocyte (arrowhead) showing numerous rod-shaped, acidic granules; also, there is a group of spreading and round hyalinocytes, which are essentially devoid of acidic granules. (F) Another spreading granulocyte (arrowhead) showing large and merging acidic granules; also, there are two spreading hyalinocytes with no acidic granules. Micrographs from (41).

Figure 3

A circulating agranulocyte of P. canaliculata with a round central nucleus, SER vesicles and RER cisternae, as well as some cytoplasmic granules (under TEM). Its ultrastructural features are compatible with the quiescent cells that have been described in the circulation (32). Scale bars represent 1 μm. Micrograph from (41).

Figure 4

In vitro phagocytosis of fluorescent beads by unsorted hemocytes of P. canaliculata and by sorted circulating hyalinocytes, agranulocytes and granulocytes. (A) Phagocytosis index of unsorted and sorted circulating hemocytes (means ± SEM; N = 6; different letters indicate statistically significant differences, one-way ANOVA, Tukey test). (B) Histograms showing the distribution of phagocytic (red) and non-phagocytic (green) hemocytes. Fluorescence peaks correspond to hemocyte subpopulations with different numbers of phagocytized fluorescent beads. GR, agranulocytes; GRA, granulocytes; HYA, hyalinocytes. From (41).

Figure 5

Constitutive hemocyte islets (panels (A, B), paraffin and hematoxylin-eosin; panel (C), Spurr resin and toluidine blue). (A) Section of the dorsal renal epithelium, perpendicular to the outer surface of the organ. The pigmented outer mantle epithelium separates the kidney from the extrapallial space. Renal hemocyte islets are seen as elongated basophilic masses between the cortical renal crypts, while they appear transversally sectioned in the region overlying the renal chamber. (B) Section of a hemocyte islet in the space between two epithelial crypts, at higher magnification. (C) Section through a medium-sized hemocyte islet which is retained in place by basal extensions of the renal epithelial cells. (D) Diagram of the same section shown in (C), highlighting a hemocyte islet, together with some smaller aggregations; the entire set is retained in place by cytoplasmic extensions (asterisks) of renal epithelial cells (light brown); the space for the flowing blood plasma is shown in pale blue. Hyalinocytes are shown in dark blue while granulocytes are shown in pink. Scale bar represents 20 µm. ECN, epithelial cell nuclei; EPS, extrapallial space; PPE, pigmented pallial epithelium; RCC, renal cell concretion; RCH, renal chamber; RCL, renal crypt lumen; RCN, renal cell nucleus; RHI, renal hemocyte islet; URC, urinary concretions. From (29, 41).

Figure 6

Constitutive hemocyte islets (panels A-C, under TEM). (A) Numerous hyalinocytes and two tangential sections of granulocytes at the core of a renal islet. Non-membrane bound areas with presumptive glycogen clumps appear in most cells and are larger than those in circulating hemocytes. (B) Detail of a hyalinocyte from the preceding panel, showing a Golgi stack, areas of presumptive glycogen clumps, SER vesicles and mitochondria. (C) A granulocyte in a renal islet, showing SER vesicles, some RER profiles and numerous R granules of different sizes (some of them appear merging); a single L granule is also seen. Microgranular material and membrane remnants are found in the intercellular space.AGR, agranulocyte; GLY, presumptive glycogen granules; GOL, Golgi stack; GRA, granulocyte; HYA, hyalinocyte; LGR, L granule; RGR, R granule. Scale bars in (A, B) panels represent 5 μm, while those in other panels represent 1 μm. From (41).

Figure 7

Nodular hemocyte reactions after an immune challenge (hematoxylin-eosin stain). (A) Field view of the dorsal kidney epithelium showing enlarged islets and nodules 96 h after yeast inoculation. (B, C) Detail of hemocyte nodules in another treated snail showing acellular cavities, apoptotic bodies, lipofuscin-like deposits, some granulocytes, and a delimiting band of flattened cells (black arrowheads). The nodule is pushing against the cavity of a crypt in (B), while is in contact with a blood sinus in (C). (D) A nodule showing a core and a cortex; the core appears to be in a more advanced stage of regression; bands of flattened cells separate the core from the cortex, and the cortex from the surrounding tissues and a blood sinus. Scale bars represent: (A) 200 µm; (B–D) 50 µm. IBS, intercryptal blood sinus; RCE, renal cryptal epithelium; RCL, renal cryptal lumen; RHI, renal hemocyte islet. From (29, 41).

Figure 8

Circulating hemocytes of P. canaliculata in culture. (A) Dynamics of an early aggregate formation by time-lapse video-microscopy. Time elapsed after seeding (in minutes) is shown in the upper-left corner of each selected frame. Arrowheads indicate two cells together which are approximating the larger aggregate and finally merge with it (8–20 min). Open arrowheads show an elongated cell which rapidly joins the aggregate (18–20 min). Open arrows point to a cell which detaches from the aggregate and leaves the observation field (50–55 min). Also, a smaller cell aggregate appears in the lower right corner approximating the larger aggregate and finally joining it (18–31 min). Asterisks indicate two cells which appear attached to the substrate and did not move during all the period of observation (0–57 min). Scale bar represents 20 µm. (B) Hemocyte aggregates formed in culture and merging into large floating masses (phase contrast, micrographs taken 96 h after seeding). Scale bar represents 200 µm (all panels). From (15).

Figure 9

Hemocyte aggregates in vitro. (A) Large nodule mostly covered by filopodia/lamellipodia emitting cells, 96 h after seeding; scanning electron microscopy; scale bar represents 20 µm. (B) Detail of the smooth external aspect of a nodule, with some filopodia/lamellipodia emitting cells on it, 96 h after seeding; panels (A, B) are scanning electron microscopy micrographies; scale bar represents 10 µm. Panels (C–H) Floating hemocyte aggregates in culture, 72 h after seeding (DAPI and propidium iodide staining; laser confocal microscopy). (C) Hemocyte nodule grown in vitro showing the peripheral cell zone, the intermediate lacunar zone, and the inner core (DAPI emission, blue). (D) Same hemocyte aggregate lacking propidium iodide staining (red emission), indicating that all cells were viable. (E) Merging of micrographs (C, D). (F). Another aggregate showing the same basic organization (DAPI emission, blue). (G) Same aggregate showing abundant cells marked with propidium iodide (red emission), indicating that most cells in this aggregate were not viable. (H) Merging of micrographs (F, G). Scale bar for panels (C–H) represents 100 µm. IC, inner core; LA, lacuna; PZ, peripheral zone. From (15).

Figure 10

Rhogocytes in the lung (under TEM). (A) A rhogocyte and the cytoplasmic extension of another one, showing the extracellular lacunae with the characteristic “slit apparatus”; they are surrounded by a lamina densa and extracellular matrix where muscle fibers and storage cells are embedded. Most components of the slit apparatus (i.e., bars and slits) appear sectioned transversally, but the two dashed boxes show some longitudinally sectioned. Scale bar represents 2 µm. (B) Detail of the extracellular lacunae of the same rhogocyte. Arrowheads indicate the extracellular matrix that surrounds the rhogocyte. Scale bar represents 1 µm. (C) Rough endoplasmic reticulum with interspersed glycogen clumps; profiles of the smooth endoplasmic reticulum are also seen in the vicinity of the extracellular lacunae. (D) Wide cisternae of the rough endoplasmic reticulum, and a large nucleolus within an oval nucleus; cytoplasmic extensions found in the vicinity (probably from other rhogocytes) contain rough endoplasmic reticulum and extracellular lacunae. (E) A cytoplasmic region showing a wide cistern of the smooth endoplasmic reticulum as wells as multiple tubuli, some of which connect with extracellular lacunae. (F) Close-up of a connection of the smooth endoplasmic reticulum with an extracellular lacuna; the slit apparatus opens to a region of packed collagen fibers. BAR, bar of the slit apparatus; COL, collagen matrix; EDG, electron-dense globule; EXL, extracellular lacunae; GCP, glial cell process; GLY, glycogen; MIT, mitochondria; MSC, muscle cell; NUC, nucleus; NUL, nucleolus; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; SLT, slit of the slit apparatus. Scale bars represent: (A) 2 µm; (B–D) 1 µm; (E) 500 nm; (F) 200 nm. From (112).

Figure 11

Renal blood sinuses, hemocytes, and hemocyanin polymers under TEM. (A) Blood sinus containing hemocyanin polymers and an agranulocyte lying beside a projection of a renal epithelial cell. Parts of other hemocytes and of the epithelial extensions show dark clumps and non-membrane bound areas, which are likely glycogen deposits. (B) A hyalinocyte showing eccentric nucleus, profiles of the SER, and numerous mitochondria. Intimate contacts between different hemocytes and thin epithelial projections are seen (arrowheads), but there are no intercellular junctions. (C) A hemocyanin filled blood sinus containing a granulocyte with an eccentric nucleus and electron-dense R-granules. (D) Detail of hemocyanin polymers. Scale bars represent: (A–C) 1 µm; (D) 100 nm. BLA, basal lamina; BSI, blood sinus; CEN, centriole; grn, R-granules; MIT, mitochondrion; NUC, cell nucleus; REP, renal epithelium; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum. From (29).

Figure 12

Location of rhogocytes in the lung (illustration credit: Guido I. Prieto). The main structures are indicated in the thumbnail in the right-upper corner. The diagram depicts a portion of the respiratory lamina (with a ciliary tuft and some blood sinuses), the fibromuscular layer that underlies it (the collagen matrix of the fibromuscular tissue has not been drawn for clarity) and the upper part of the storage tissue layer. Rhogocytes mainly occur in contact with storage cells, but also with the perpendicular blood sinuses that feed the respiratory lamina and, less frequently, are found in the proximity of the basal part of the ciliary tufts (not shown in the diagram). Rhogocytes are encased in a thin lamina of extracellular matrix (dashed lines). 1, pulmonary cavity; 2, pavement cells; 3, ciliary tuft; 4, blood sinuses of the respiratory lamina; 5, hemocyte; 6, fibromuscular tissue; 7, endothelial-like cell; 8, radial sinus; 9, rhogocyte; 10, storage cell; ADH, adherent junction; BLA, basal lamina; CIL, cilia; EXL, extracellular lacunae; EXM, extracellular matrix; ICS, intercellular space; MSC, muscle cells; MUG, mucin granules; MVI, microvilli; NEU, neurite bundle; PSE, pseudopodia; PTM, particulate material; SEG, secretory globules; SEP, septate junctions; STP, slit apparatus; VES, vesicles of storage cells. From (109).