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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 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.
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pone.0123964.g006: 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.

Mentions: Hemocytes were able to phagocytize yeast cells in spite of their large size, resulting in a marked distortion of the phagocyte. A microgranular material of low electron density fills the phagosome space surrounding the yeast cell (Fig 6A). Membrane-bound L granules, which were a common feature of unexposed hyalinocytes, were only rarely seen in phagocytic cells, which would indicate that phagocytosis is accompanied either by degranulation (exocytosis) or by fusion of L granules to phagosomes (Fig 6A). Phagocytic hemocytes are most likely to be hyalinocytes, but granulocytes may also be phagocytic (see below) though they may not be recognizable after loss of their characteristic R granules.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123964.g006: 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.
Mentions: Hemocytes were able to phagocytize yeast cells in spite of their large size, resulting in a marked distortion of the phagocyte. A microgranular material of low electron density fills the phagosome space surrounding the yeast cell (Fig 6A). Membrane-bound L granules, which were a common feature of unexposed hyalinocytes, were only rarely seen in phagocytic cells, which would indicate that phagocytosis is accompanied either by degranulation (exocytosis) or by fusion of L granules to phagosomes (Fig 6A). Phagocytic hemocytes are most likely to be hyalinocytes, but granulocytes may also be phagocytic (see below) though they may not be recognizable after loss of their characteristic R granules.

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