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Differential partitioning of maternal fatty acid and phospholipid in neonate mosquito larvae

Georgia C. Atella^* and Mohammed Shahabuddin

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
^* Present address: Universidade Federal do Rio de Janeiro, UFRJ, Centro de Ciências da Saúde, Instituto de Ciências Biomédicas, Departamento de Bioquímica Médica, Ilha da Ciudade Universitária, Rio de Janeiro, CEP 21941-590, Brazil

View larger version (80K): [in a new window]   Fig. 1. Uptake and distribution of lipophorin, fatty acid and phosphatidylethanolamine by the ovary and oocyte of the mosquito Anopheles gambiae strain G3. (A) Uptake of 32P-labeled lipophorin by different stages (Christophers, 1911) of the developing ovary. (B) Localization of FITC-labeled maternal lipophorin in developing oocytes. Scale bar, 25 µm. (C) Phosphatidylethanolamine analog labeled with Texas Red (Texas Red-PE, excitation 582 nm/emission 601 nm) on the polar head. (D) Fatty acid analog labeled with BODIPY (BODIPY-FA, excitation 505 nm/emission 515 nm) on the acyl chain. (E) Localization of the fatty acid (BODIPY-FA) in A. gambiae oocytes. Scale bar, 15 µm. (F) Localization of phospholipid (Texas Red-PE) in the same oocyte as in E. (G) Merged panels E and F demonstrate that fatty acids and phosphatidylethanolamine colocalized in the same vesicles.

View larger version (69K): [in a new window]   Fig. 2. Maternal lipid localization in neonate mosquito larvae. Purified lipophorin was double labeled with Texas Red-PE and BODIPY-FA before injecting into vitellogenic females; eggs were collected and kept in water at 28°C until larval hatching. The fluorescence associated with newborn larvae was examined within 30 min after hatching. (A) Localization of BODIPY-FA in A. gambiae larvae. (B) Localization of Texas Red-PE in the same newborn larvae as shown in A. (C) Merged image of A and B shows differential localization of BODIPY-FA and Texas Red-PE. (D) Phase-contrast image of the same larvae as shown in C. (E) All neonate larvae hatched from the same batch of eggs, showing identical pattern of differential partitioning of fatty acid (BODIPY-FA) and phosphatidylethanolamine (Texas Red-PE). (F) Phase-contrast image of the larvae shown in E. (G) Maternal lipid distribution in the larvae of A. aegypti mosquitoes. (H) Phase-contrast image of larvae shown in G. Scale bar, 200 µm.

View larger version (18K): [in a new window]   Fig. 3. Intact imported phosphatidylethanolamine in neonate A. gambiae larvae. Texas Red-PE-loaded lipophorin was injected into vitellogenic females. Extracted phospholipids from larvae were fractionated by thin-layer chromatography. Fluorescence from the lipid analog was visualized by illuminating the plate with UV light. Lane UL, unlabeled phospholipids extracted from unlabeled neonate larvae; lane L, labeled phospholipids extracted from neonate larvae hatched from eggs laid by the females injected with Texas Red-PE; lane S, standard containing commercial Texas Red-PE that was injected into the mosquitoes.

View larger version (56K): [in a new window]   Fig. 4. Distribution of phospholipids and fatty acids in neonate and embryonic larvae. (A) BODIPY-labeled phosphatidylcholine analog (BODIPY-PC). (B) Location of phosphatidylcholine analog (BODIPY-PC) in newly hatched A. gambiae larvae. (C) Location of phosphatidylethanolamine analog (Texas Red-PE) in the same larvae. (D) Phase-contrast image of the larvae shown in B and C. Scale bar, 200µm. (E) Phase-contrast image of approximately 30-h-old embryonic A. gambiae larvae. Scale bar, 150µm. (F) Fluorescent image of the same larvae in E, showing the distribution of BODIPY-labeled maternal fatty acid (BODIPY-FA). (G) Image of the same embryonic larvae showing the distribution of the Texas Red-labeled maternal phosphatidylethanolamine (Texas Red-PE). (H) Diagram of a neonate A. gambiae larvae depicting the major accumulation sites of the maternal phospholipids (red) and fatty acids (green).