Table 1.
Hemocyte types according to Cheng [16] and proposed synonymy in architaenioglossan gastropods (families Viviparidae and Ampullariidae).
Fig 1.
Circulating hemocyte types and their separation by flow cytometry.
(A) Examples of hyalinocytes, agranulocytes and granulocytes in hemolymph smears (HE-stain). (B) Examples of living hyalinocytes, agranulocytes and granulocytes attached onto a glass slide (phase contrast). (C) Flow cytometry (dot plot of size vs. complexity-granularity) of a representative hemolymph sample indicating the three areas that were chosen for cell sorting, where the three hemocyte types were predominant. Abbreviations: agr, agranulocytes; gra, granulocytes; hya, hyalinocytes. Scale bars represent 5 μm.
Fig 2.
Circulating hemocytes (LysoTracker Red-Hoechst 33258).
(A) A group of spreading hyalinocytes; small acidic granules (red) are seen in some of them. (B) 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. (C) Another spreading granulocyte (arrowhead) showing large and merging acidic granules; also, there are two spreading hyalinocytes with no acidic granules. Scale bar represents 10 μm.
Fig 3.
(A) Hyalinocyte with an eccentric nucleus, numerous SER vesicles, mitochondria and L granules; a few profiles of the RER are also seen. (B) Agranulocyte with a round central nucleus, SER vesicles and RER cisternae, as well as some mitochondria. (C) Granulocyte, showing a displaced, bean-shaped nucleus and numerous R granules; some SER vesicles are also seen. (D) Pro-granulocyte, showing a bean-shaped nucleus, a Golgi complex and numerous immature R granules of varying electron density; some SER vesicles and extended RER cisternae are also seen. Abbreviations: gol, Golgi complex; Lgr, L granules; mit, mitochondria; n, nucleus; r, immature R granules; Rgr, mature R granules; rer, RER cisternae; ser, SER vesicles. Scale bars represent 1 μm.
Fig 4.
Details of circulating hemocytes (TEM).
(A) Cytoplasm of a hyalinocyte showing numerous mitochondria and some RER and SER profiles. (B) Detail of another hyalinocyte showing an extended RER cisterna, SER vesicles and a membrane-unbound zone with presumptive glycogen granules. (C) Cytoplasm of a hyalinocyte showing numerous SER vesicles, as well as a few L granules and mitochondria. (D) Cytoplasm of a granulocyte showing numerous R granules around a Golgi complex. Abbreviations: gly, presumptive glycogen granules; other abbreviations as in Fig 3. Scale bars represent 1 μm.
Fig 5.
In vitro phagocytosis of E. coli cells by circulating hemocytes (LysoTracker Red-Hoechst 33258).
(A) A group of control hyalinocytes, some of them showing small acidic granules. (B) Hemocytes exposed to E. coli; a phagocyte (upper left) showing a group of internalized red-labeled bacteria, while another phagocyte (lower right) shows a single internalized bacterium. Small acidic granules are not seen in these hemocytes, whether phagocytic or not. Bacteria which are free over and around hemocytes are not labeled (arrows). (C) A group of hemocytes, one of them showing several internalized bacteria in different degrees of digestion. Small acidic granules are not seen in these hyalinocytes. Non internalized bacteria are not labeled by LysoTracker Red (arrows). Scale bar represents 10 μm.
Fig 6.
In vitro microbial phagocytosis by circulating hemocytes (TEM).
(A) Two yeast cells engulfed by a phagocytic hemocyte; a large L granule is attached to one of the phagosomes and may be preceding fusion (arrow). (B) Numerous S. aureus cells engulfed by a hemocyte within seldom interconnected phagosomes. (C) E. coli cells may also be phagocytized in large numbers within complex phagosomes which frequently show more than one compartment (arrows). (D) Granulocyte in a preparation exposed to E. coli cells showing extensive R granule fusion and a single L granule.
Fig 7.
In vitro phagocytosis of fluorescent beads by sorted and unsorted circulating hemocytes.
(A) Dot plot of cell size vs. complexity-granularity of unsorted circulating hemocytes exposed to fluorescent beads. In this and in panels C, E and G, red dots indicate phagocytic hemocytes associated to fluorescent beads, while green dots indicate non phagocytic ones. (B) Histograms of the sample shown in A: in this and in panels D, F and H, red and green lines show the distribution of phagocytic and non-phagocytic hemocytes, respectively. The red histogram indicates the existence of 4–5 hemocyte populations associated to different amounts of fluorescent beads. (C) Dot plot of size vs. complexity-granularity of sorted hyalinocytes exposed to fluorescent beads. (D) Histograms of the hyalinocyte sample shown in C: the red histogram indicates the existence of 3–4 hyalinocytes populations associated to different amounts of fluorescent beads. (E) Dot plot of size vs. complexity-granularity of sorted agranulocytes exposed to fluorescent beads. (F) Histograms of the agranulocyte sample shown in E: the red histogram indicates the existence of 2–3 agranulocyte populations associated to different amounts of fluorescent beads. (G) Dot plot of size vs. complexity-granularity of sorted granulocytes exposed to fluorescent beads. (H) Histograms of the granulocyte sample shown in E: the red histogram indicates the existence of 2–3 granulocyte populations associated to different amounts of fluorescent beads. (I) Phagocytosis index of unsorted circulating hemocytes and of sorted hyalinocytes, agranulocytes and granulocytes (means ± SE; N = 6; different letters indicate statistically significant differences, one-way ANOVA, Tukey test). (J) Phase contrast micrographs of sorted phagocytic hemocytes. Abbreviations: agr, agranulocytes; gra, granulocytes; hya, hyalinocytes. Scale bar represents 10 μm.
Fig 8.
Renal hemocyte islets (HE-stain).
(A) Kidney section perpendicular to the pallial surface of the organ. The mantle separates the kidney from the extrapallial space and is covered by the pigmented pallial epithelium. 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 mostly composed of hyalinocytes and delimited by the renal epithelium. Abbreviations: eps, extrapallial space; ppe, pigmented pallial epithelium; rcc, renal cell concretion; rch, renal chamber; rcn, renal cell nucleus, rhi, renal hemocyte islet.
Fig 9.
(A) An agranulocyte and two hyalinocytes appear loosely attached to the surface region of a renal islet. (B) Detail of the larger hyalinocyte on the preceding panel, showing R granules, SER vesicles and numerous mitochondria. The intercellular space contains a microgranular material and membrane remnants. (C) Numerous hyalinocytes and two tangential sections of granulocytes at the core of a renal islet. Membrane-unbound areas with presumptive glycogen granules appear in most cells and are larger than those in circulating hemocytes. (D) Detail of a cell from the preceding panel, showing a Golgi stack, areas of presumptive glycogen granules, SER vesicles, R granules and mitochondria. The intercellular space is also occupied by a microgranular material and membrane remnants. (E) Detail of the Golgi stack and an area of presumptive glycogen granules; numerous SER vesicles and free ribosomes are also seen. (F) 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. Again, a microgranular material and membrane remnants are found in the intercellular space. Abbreviations: agr, agranulocyte; gly, presumptive glycogen granules; gol, Golgi stack; gra, granulocyte; hya, hyalinocyte; Lgr, L granule; Rgr, R granule. Scale bars in A and B panels represent 5 μm, while those in other panels represent 1 μm.
Fig 10.
In vivo microbial phagocytosis by renal hemocytes (TEM).
(A) Partly digested yeast cell engulfed by a phagocytic hyalinocyte; a small multivesicular body is seen close to the phagosome; (B) Several S. aureus cells engulfed by a hyalinocyte within individual phagosomes; multivesicular bodies of different sizes are also seen; arrow indicates a basal interdigitation of a renal epithelial cell, surrounded by the basal membrane (arrow); (C) E. coli cells in different stages of digestion are contained within complex phagosomes; the basal membrane of the renal epithelium is indicated by arrows. Abbreviation: mvb, multivesicular body. Scale bar represents 1 μm.
Fig 11.
In vitro phagocytosis of fluorescent beads by dispersed renal hemocytes.
(A) Dot plot of cell size vs. complexity-granularity of dispersed renal hemocytes exposed to fluorescent beads. Red dots indicate phagocytic hemocytes associated to fluorescent beads, while green dots indicate non phagocytic ones. (B) Histograms of the sample shown in A: the red and green lines show the distribution of phagocytic and non-phagocytic hemocytes, respectively. The red histogram indicates the existence of 3–4 hemocyte populations associated to different amounts of fluorescent beads. (C) Phase contrast micrographs of sorted hemocytes exposed to fluorescent beads: those in the upper row are not phagocytic while those in the lower row show internalized bead/s. Scale bar represents 10 μm.
Table 2.
Properties of circulating hemocyte types in Pomacea canaliculata.