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Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney.

Cueto JA, Rodriguez C, Vega IA, Castro-Vazquez A - PLoS ONE (2015)

Bottom Line: Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes.Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads.A role for the kidney as an immune barrier is proposed for this snail.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Fisiología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina; Instituto de Histología y Embriología "Dr. Mario H. Burgos", Consejo Nacional de Investigaciones Científicas y Técnicas, Mendoza, Argentina.

ABSTRACT
Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.

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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.
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pone.0123964.g005: 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.

Mentions: Most control hemocytes (i.e., not exposed to bacteria) showed small and round acidic granules (corresponding to L granules, Fig 5A), while cells bearing rod-like granules (corresponding to R granules) were occasionally seen (not shown in Fig 5) and they were not phagocytizing cells. Hemocytes exposed to an E. coli suspension behaved differently whether they had internalized bacteria or not: those without internalized bacteria had lost all acidic granules while those with internalized bacteria showed intensely labeled phagocytic vesicles with the shape of the phagocytized bacterium in different stages of digestion (Fig 5B and 5C). Also, unlabeled vesicles sometimes surround the labeled vesicles (Fig 5B), which may be correlated with the phagosomes with more than one compartment that are seen under TEM (see next section). No additional acidic compartments (such as those corresponding to L granules seen in control hemocytes, Fig 5A) are seen in phagocytic hemocytes, which correlates well with the lack of granules observed in phagocytic hemocytes under TEM (next section). Most non-phagocytic hemocytes also appeared devoid of acidic compartments in preparations exposed to bacteria.


Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney.

Cueto JA, Rodriguez C, Vega IA, Castro-Vazquez A - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4404100&req=5

pone.0123964.g005: 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.
Mentions: Most control hemocytes (i.e., not exposed to bacteria) showed small and round acidic granules (corresponding to L granules, Fig 5A), while cells bearing rod-like granules (corresponding to R granules) were occasionally seen (not shown in Fig 5) and they were not phagocytizing cells. Hemocytes exposed to an E. coli suspension behaved differently whether they had internalized bacteria or not: those without internalized bacteria had lost all acidic granules while those with internalized bacteria showed intensely labeled phagocytic vesicles with the shape of the phagocytized bacterium in different stages of digestion (Fig 5B and 5C). Also, unlabeled vesicles sometimes surround the labeled vesicles (Fig 5B), which may be correlated with the phagosomes with more than one compartment that are seen under TEM (see next section). No additional acidic compartments (such as those corresponding to L granules seen in control hemocytes, Fig 5A) are seen in phagocytic hemocytes, which correlates well with the lack of granules observed in phagocytic hemocytes under TEM (next section). Most non-phagocytic hemocytes also appeared devoid of acidic compartments in preparations exposed to bacteria.

Bottom Line: Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes.Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads.A role for the kidney as an immune barrier is proposed for this snail.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Fisiología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina; Instituto de Histología y Embriología "Dr. Mario H. Burgos", Consejo Nacional de Investigaciones Científicas y Técnicas, Mendoza, Argentina.

ABSTRACT
Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.

Show MeSH
Related in: MedlinePlus