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Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism

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Abstract

Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their best-known function is O2 transport. We studied the site of their biosynthesis in the gastropod Concholepas concholepas by using immunological and molecular genetic approaches. We performed immunohistochemical staining of various organs, including the mantle, branchia, and hepatopancreas, and detected C. concholepas hemocyanin (CCH) molecules in circulating and tissue-associated hemocytes by electron microscopy. To characterize the hemocytes, we purified them from hemolymph. We identified three types of granular cells. The most abundant type was a phagocyte-like cell with small cytoplasmic granules. The second type contained large electron-dense granules. The third type had vacuoles containing hemocyanin molecules suggesting that synthesis or catabolism occurred inside these cells. Our failure to detect cch-mRNA in hemocytes by reverse transcription with the polymerase chain reaction (RT-PCR) led us to propose that hemocytes instead played a role in CCH metabolism. This hypothesis was supported by colloidal gold staining showing hemocyanin molecules in electron-dense granules inside hemocytes. RT-PCR analysis, complemented by in situ hybridization analyses with single-stranded antisense RNAs as specific probes, demonstrated the presence of cch-mRNA in the hepatopancreas; this was consistent with the specific hybridization signal and confirmed the hepatopancreas as the site of CCH synthesis. Finally, we investigated the possibility that CCH catabolism in hemocytes was involved in the host immune response and in the generation of secondary metabolites such as antimicrobial peptides and phenoloxidase.

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Acknowledgments

The authors are grateful to Alejandra Fuentes, Andrea González, and Daniel Laporte for their assistance with some of the experiments and for valuable discussions and are also indebted to Dr. Esteban Nova and PhD student Sergio Arancibia for useful discussions during the course of this work. The authors are particularly grateful to Alejandro Munizaga (Servicio de Microscopía Electrónica, Pontificia Universidad Católica de Chile) for his outstanding technical assistance and to Gabriel De Ioannes for preparing the figures.

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Authors and Affiliations

  1. Fundación Ciencia y Tecnología para el Desarrollo, Avenida Alcalde Eduardo Castillo Velasco, 2902, Ñuñoa, Santiago, Chile

    Augusto Manubens, Fabián Salazar, Miguel Del Campo, Jonathan Martínez Pinto & María Inés Becker

  2. Department of Research & Development, BIOSONDA Corporation, Santiago, Chile

    Augusto Manubens & María Inés Becker

  3. Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile

    Denise Haussmann & Jaime Figueroa

  4. Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile

    Laura Huaquín

  5. Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

    Alejandro Venegas

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  1. Augusto Manubens
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  2. Fabián Salazar
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  3. Denise Haussmann
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  4. Jaime Figueroa
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  5. Miguel Del Campo
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  6. Jonathan Martínez Pinto
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  7. Laura Huaquín
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  8. Alejandro Venegas
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  9. María Inés Becker
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Correspondence to María Inés Becker.

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This study was supported in part by Fundación COPEC-PUC SC0014, QC057 and FONDECYT no. 105-0150 grants.

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Manubens, A., Salazar, F., Haussmann, D. et al. Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism. Cell Tissue Res 342, 423–435 (2010). https://doi.org/10.1007/s00441-010-1057-6

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  • DOI: https://doi.org/10.1007/s00441-010-1057-6

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