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Separation and behavior in vitro of hemocytes from the moth, Pseudoplusia includens
L L Pech1, D Trudeau, M R Strand
1Department of Entomology, University of Wisconsin-Madison 53706.
Abstract:
Hemocytes collected from larvae of Pseudoplusia includens (Lepidoptera:Noctuidae) were separated by centrifugation on Percoll cushions. The procedure resulted in 95% purity of plasmatocytes and greater than 99% purity of granular and spherule cells. Medium supplemented with chicken serum enhanced cell viability and promoted spreading of plasmatocytes. Cell-free plasma and medium preconditioned by plasmatocytes or granular cells stabilized cells in vitro and also accelerated spreading of plasmatocytes relative to medium supplemented with chicken serum. Oenocytoids were the only morphotype that exhibited endogenous phenoloxidase activity, while granular cells and plasmatocytes were the only cells that endocytosed fluorescent beads in vitro. Granular cells and plasmatocytes ingested fluorescently labelled beads, both in mixed populations of hemocytes and after separation. Plasmatocytes were the only morphotype that encapsulated large foreign targets in vitro following separation. Separated granular cells attached and spread on the surface of foreign targets but never formed a multilayered capsule.
Insights
This study isolated insect hemocytes, revealing plasmatocytes encapsulate foreign targets. Granular cells and plasmatocytes ingest particles, with oenocytoids showing phenoloxidase activity for immune defense.
Area of Science:
- Insect immunology
- Cell biology
- Biochemistry
Background:
- Insect hemocytes are crucial for immune responses.
- Understanding hemocyte function requires pure cell populations.
- Pseudoplusia includens larvae provide a model for studying insect immunity.
Purpose of the Study:
- To isolate and characterize hemocyte subpopulations from Pseudoplusia includens larvae.
- To investigate the in vitro functions of purified hemocytes, including phagocytosis, encapsulation, and enzyme activity.
- To determine the role of different hemocyte types in cellular immunity.
Main Methods:
- Hemocyte isolation using Percoll density gradient centrifugation.
- Assessment of cell purity and viability.
- In vitro functional assays: phagocytosis of fluorescent beads, encapsulation of foreign targets, and phenoloxidase activity measurement.
- Use of supplemented and conditioned media to evaluate cell behavior.
Main Results:
- High purity (>95%) of plasmatocytes, granular cells, and spherule cells was achieved.
- Plasmatocyte spreading and viability were enhanced by chicken serum and conditioned media.
- Oenocytoids exhibited endogenous phenoloxidase activity.
- Granular cells and plasmatocytes demonstrated phagocytic activity.
- Plasmatocytes were capable of encapsulating foreign targets, while granular cells showed attachment and spreading but not encapsulation.
Conclusions:
- Specific hemocyte subpopulations possess distinct immune functions.
- Purified plasmatocytes are key players in the encapsulation response.
- Granular cells and plasmatocytes contribute to phagocytosis.
- Oenocytoids are the primary source of phenoloxidase activity in Pseudoplusia includens hemocytes.