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A new intracellular pathway of haem detoxification in the midgut of the cattle tick Boophilus microplus: aggregation inside a specialized organelle, the hemosome

Flavio Alves Lara1, Ulysses Lins2, Gabriela Paiva-Silva1, Igor C. Almeida3, Cláudia M. Braga4, Flávio C. Miguens5, Pedro L. Oliveira1 and Marílvia Dansa-Petretski5,*

1 Departamento de Bioquímica Médica, ICB, Universidade Federal do Rio de Janeiro, Brazil
2 Departamento de Microbiologia Geral, IMPPG, Universidade Federal do Rio de Janeiro, Brazil
3 Departamento de Parasitologia, ICB, Universidade de São Paulo, Brazil
4 Divisão de Química, Petrobrás/CENPES, Rio de Janeiro, Brazil
5 Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Brazil



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  		Fig. 1. Haem peroxidase activity in the tick midgut epithelium during blood digestion. Midgut sections were stained for 3-3-diaminobenzidine (DAB) oxidation, and observed by differential interference contrast (DIC) microscopy. (A) First day after blood meal (ABM); arrowheads show contact between digest cells (DC) and the basement membrane (BL). Scale bar, 35 µm. (B) Third day ABM; arrowheads show the diminished contact between digest cells and basement membrane. Scale bar, 35 µm. (C) Day 15 ABM. Scale bar, 60 µm. (D) Day 20 ABM. Scale bar, 60 µm. (E) Day 5 ABM, negative control without H2O2; arrowheads show detachment of digest cells. Scale bar, 40 µm. BC, basophilic cell.

 


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  		Fig. 2. Digest cells on the third day ABM, isolated and observed by differential interference contrast (DIC) microscopy. (A) Digest cells separated from the midgut were observed without a coverslip. Arrowheads show the site of attachment to the gut epithelium. Scale bar, 55 µm. (B) Digest cell observed with a coverslip, at higher magnification. Hemosomes are concentrated in the perinuclear area (arrowheads). Asterisks indicate digestive vesicles. Scale bar, 5 µm. NU, nucleus.

 


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  		Fig. 3. Hemosome ultrastructure and formation. Transmission electron microscopy of the cytoplasm region of digest cells. (A) Mature hemosome. The arrowhead indicates a lipid bilayer membrane. Scale bar, 150 nm. (B) Growing hemosome. Scale bar, 150 nm. (C) Detail of B. The hemosome is formed by association of homogeneous sub-particles (arrowheads), possibly migrating towards the core (asterisk). Scale bar, 50 nm.

 


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  		Fig. 4. Hemosome composition as determined by high performance liquid chromatography (HPLC) and electrospray-ionisation mass spectrometry (ESI-MS). (A) Transmission electron microscopy of hemosome cores after isolation through a Percoll gradient and washing with deionised water. Scale bar, 1.0 µm. (B) Reverse-phase HPLC fractionation of the hemosome content, monitored at 220 nm. Arrow, a peak with a retention time similar to a haem standard; inset, the light absorption spectrum of the same region. (C–F) Positive-ion mode electrospray-ionisation mass spectrometry (ESI-MS) profile of the intact haem fraction (C) and tandem ESI-MS (ESI-MS/MS) spectra of its daughter ions (D–F). (G–I) ESI-MS/MS spectra of standard haem. (D,G) Daughter ions of m/z 616.4. (E,H) ESI-MS/MS spectra of the daughter ion m/z 557.4. (F,I) Tandem ESI-MS spectra of the daughter ion m/z 498.5. (J) Structure of the iron-protoporphyrin IX and the proposed assignment for its observed daughter ions.

 


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  		Fig. 5. Fourier Transform infrared (FTIR) spectrum of the hemosome. Hemosomes were isolated as described in Materials and methods. Shown are FTIR spectra of the hemosome core (broken line) and haem standard (solid line). Arrow, haem carboxylate peak at 1704 cm–1. Asterisks, peaks at 1652 cm–1, 1546 cm–1, 1392 cm–1 and 1279 cm–1 that can be designated as non-haem components of hemosome.

 


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  		Fig. 6. Elemental mapping of hemosome development. Cytoplasm of digest cells was observed by energy-filtering transmission electron microscopy. Each row of panels shows a section from cells on different days ABM (4, 8 and 20 ABM, from top to bottom). (Left) A reference image obtained at 250 eV, which depicts a structure-sensitive contrast. Nitrogen (middle) and iron (right) distributions are shown. DV, digestive vesicles; LU, lumen. Scale bar, 5 µm.

 




© The Company of Biologists Ltd 2003