Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism.
Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their best-known function is O(2) 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.
Fundación Ciencia y Tecnología para el Desarrollo, Avenida Alcalde Eduardo Castillo Velasco, 2902 Ñuñoa, Santiago, Chile.
Augusto
A
Manubens
Fabián
F
Salazar
Denise
D
Haussmann
Jaime
J
Figueroa
Miguel
M
Del Campo
Jonathan Martínez
JM
Pinto
Laura
L
Huaquín
Alejandro
A
Venegas
María Inés
MI
Becker
D000818:Animals; D005455:Fluorescent Antibody Technique; D049851:Gastropoda / Q000378:metabolism*; D006433:Hemocyanins / Q000096:biosynthesis*; D006434:Hemocytes / Q000378:metabolism*; D043143:Hepatopancreas / Q000378:metabolism*; D017403:In Situ Hybridization; D008854:Microscopy, Electron; D016372:RNA, Antisense; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016372:RNA, Antisense; D006433:Hemocyanins