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First published online June 16, 2005
Journal of Experimental Biology 208, 2497-2502 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01664

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Laminin and the malaria parasite's journey through the mosquito midgut

Romanico B. G. Arrighi^1,*, Gareth Lycett^1,2,*, Vassiliki Mahairaki^1,3, Inga Siden-Kiamos¹ and Christos Louis^1,3,

¹ Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology – Hellas, Vassilika Vouton, PO Box 1527, 71110 Heraklion, Crete, Greece
² European Molecular Biology Laboratory, Meyerhofstr. 1, 69000 Heidelberg, Germany
³ Department of Biology, University of Crete, 71110 Heraklion, Crete, Greece

View larger version (73K): [in a new window]   Fig. 1. Association of laminin and developing Plasmodium berghei in the mosquito midgut. Mosquitoes were given a blood meal containing P. berghei parasites. At different time points, the midguts were dissected and immunostained using the antibodies described in detail in Materials and methods. (A,B) A parasite is seen reaching the basal lamina 20–24 h after blood feeding. Green, SOAP protein; red, laminin; insets in B show details of the parasite staining showing only the red or green colour channel. Scale bars: 2 mm in A, 1 mm in B. (C) A young oocyst localized close to the muscle layer surrounding the midgut 3 days after the blood meal; green, P28 protein; red, laminin. Insets in C: details of the parasite staining showing only the red or green colour channel. Scale bar: 10 mm. (D) 10 days after the blood meal laminin (green) is seen concentrated around the larger oocyst. Bar: 10 mm. (E) 15 days after blood feeding, a thin coat of laminin (green) is seen on a mature oocyst protruding through the muscle layer shortly before rupturing. Scale bar: 10 mm. (F) A midgut 15 days after the blood meal. Individual sporozoites can be seen (green: CSP) along the muscles (red: laminin) while an almost mature oocyst is seen on the right; blue T-PRO stained nuclei of immature oocysts or epithelial cells. Scale bar: 100 mm. (G) Higher magnification of the midgut shown in F. Individual sporozoites can be seen (green: CSP) along the muscles (red: laminin). Scale bar: 10 mm.

View larger version (19K): [in a new window]   Fig. 2. (A) Schematic representation of the Anopheles gambiae LANB2 gene. The coding area is represented by boxes corresponding to the domains of the protein (grey: domain V, containing EGF repeats and shown previously to interact with P25 and P28). The black line indicates non-coding segments, while the small map below shows the extent of the segment used to obtain the dsRNA used in the experiments spanning the nucleotides indicated. (B) Effects of dsRNA injections. The bars indicate the mean number of oocysts found in the midguts of mosquitoes that had previously been injected with dsRNA directed against the GFP gene as a control (a) or the LANB2 gene (b). Error bars represent standard error of the mean (N=85 and N=79, respectively). The asterisk indicates a significant difference in oocyst burden (P<0.0001). See Results for details. (C) Estimates of the LANB2 gene mRNA copy number expressed in percent of the S7 gene mRNA copies after injection of dsRNA determined by quantitative real-time PCR. a, sugar-fed mosquitoes injected with dsRNA against GFP; b, sugar-fed mosquitoes injected with dsRNA against LANB2; c, blood-fed mosquitoes injected with dsRNA against GFP; d, bloodfed mosquitoes injected with dsRNA against LANB2. Error bars represent the standard error of the mean (N=3). See Materials and methods for experimental details.

View larger version (35K): [in a new window]   Fig. 3. LANB2 mRNA accumulation in the midgut. Semi-quantitative PCR was performed to determine LANB2 mRNA accumulation in isolated midguts (Lam) using S7 mRNA as a standard (S7). 1, sugarfed mosquitoes injected with dsRNA against GFP; 2, sugar-fed mosquitoes injected with dsRNA against LANB2; 3, blood-fed mosquitoes injected with dsRNA against GFP; 4, blood-fed mosquitoes injected with dsRNA against LANB2. See Materials and methods for experimental details.