Progress 10/01/14 to 09/30/17
Outputs
Target Audience:Research Scientists Changes/Problems:We did not anticipate discovery of a second ferritin light chain subunit. We accommodated this finding by characterizing this subunit and evaluating it along with the previously known LCH1 and HCH per objective 2. We do not know the reason for this redundancy of LCH subunits. In mammals, ferritin occurs with 24 subunits and can assume isoforms depending on the number of each subunit present; this variation allows ferritin in some tissues to serve as a rapidly accessible iron reservoir and in other tissues for longer term storage. The only structure of insect ferritin to date showed a molecule of 12 HCH and 12 LCH subunits. In Aedes ferritin serves as both an iron transport protein and an iron storage protein. We speculate that if Aedes ferritin is composed of 12 of each subunit, then perhaps the different light chain subnits allow for differing functions of the protein by permitting subtle changes in molecule struture. What opportunities for training and professional development has the project provided?The professional on the project received training in analysis of data involving large data sets and proteomics. We also trained three undergraduate students in research as part of this project (2014-present)and posters showing various aspects of the work were presented at 8 separate meetings. Two students received national travel awards to attend Experimental Biology; while Conley,Thai and Love each were awarded summer undergraduate research grants by the University of Arizona Honors College. Poster Presentations Conley, Z. R., Geiser, D. L. and Winzerling, J. J. (2013) Visualizing the Effect of Iron on the Localization of Intracellular Ferritin in Mosquito Cells. American Society for Biochemistry and Molecular Biology. Experimental Biology 2013 Meeting, Boston, MA. Student Travel Award Presentation. Geiser, D.L., Li, W., Breci, L.A., and Winzerling, J. J. (2014) Relative quantification of effect of blood meal iron on protein expression in ovaries of Aedes aegypti. First Annual CALS Research Poster Forum, University of Arizona, Tucson, AZ Thai, T., Geiser, D.L. and Winzerling, J. J. (2014) Iron deposition and ferritin accretion in ovaries of yellow fever mosquito, Aedes aegypti. First Annual CALS Research Poster Forum, University of Arizona, Tucson, AZ ]Geiser, D.L., Li, W., Breci, L.A., and Winzerling, J. J (2014) Relative quantification of effect of blood meal iron on protein expression in ovaries of Aedes aegypti. Experimental Biology Meeting 2014, San Diego, California. Thai, T., Geiser, D.L. and Winzerling, J. J. (2014) Iron accumulation in ovaries of Aedes aegypti University of Arizona Honors College Spirit of Inquiry Expo, Tucson, Arizona Geiser, D.L., Li, W., Q.-D. Pham, D., Wysocki,V.H. and Winzerling, J. J. (2015) Shotgun Proteomic Analysis of Protein Expression in Mosquito Ovaries Post Blood Meal. Experimental biology meeting 2015, Boston, Massachusetts. Love, M., Geiser, D. L. and Winzerling, J.J. (2016) Iron Deposition and Ferritin Accretion in the Midgut of the Yellow Fever Mosquito, Aedes aegypti. Experimental Biology Meeting 2016, San Diego, California. Love, M., Geiser, D. L. and Winzerling, J.J. (2016) Iron Deposition and Ferritin Accretion in the Midgut of the Yellow Fever Mosquito, Aedes aegypti. Arizona Imaging And Microanalysis Society (Aims) Annual Meeting, Tucson, Arizona. Love, M., Geiser, D. L. and Winzerling, J.J. (2016) Characterization of an Iron Transport Protein in Aedes aegypti University of Arizona Honors College Spirit Of Inquiry Expo, Tucson, Arizona Love, M., Geiser, D. L. and Winzerling, J.J. Love, M., Geiser, D. L. and Winzerling, J.J. (2016) Iron Deposition and Ferritin Accretion in the Midgut of the Yellow Fever Mosquito, Aedes aegypti. Undergraduate Biology Research Program 27th Annual Conference, Tucson, Arizona Students received training in confocal microscopy, deconvolution microscopy and all other related laboratory techniques. How have the results been disseminated to communities of interest?By publications By research modules for education What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Shotgun proteomic analysis of proteins expressed in ovaries and eggs of Aedes aegypti at 24 and 72 h post blood meal (PBM) identified ~1490 proteins. Of these, we identified 108 proteins that were detected only at 24 h, and 76 proteins that were detected only at 72 h PBM. The metal-related proteins represented 11% (171/1496) of the female ovary proteome. Of the 171 metal-related proteins we identified, 17 were detected only at 24 h PBM and 7 proteins were detected only at 72 h PBM. We also identified 38 proteins involved in iron/heme metabolism; of these 11 are proteins engaged in the formation of iron sulfur clusters. We also completed a duplicate assay of Tandem Mass Tagged proteins expressed in the ovaries and eggs of Aedes aegypti fed three differnt defined diets: artificial blood meal with iron, artificial blood meal without iron and a porcine blood meal. These data are currently under analysis. Animal ferritin generally consists of 24 heavy and light chain subunits. We previously characterized a heavy chain homologue (HCH) and light chain homologue (LCH1) in Aedes. Unexpectedly, the proteomic analysis identified a second ferritin light chain subunit (LCH2). The gene for this subunit is found on a different chromosome from that of the LCH1 and HCH, and the protein shows 55% identity to the LCH1. To our knowledge a second light chain has been identified in no other species. Studies of this subunit showed that in CCL-125 cells, in contrast to the ferritin LCH1 and HCH subunits, the expression of LCH2 message is unresponsive to iron exposure and the protein is expressed primarily in the absence of iron. Transcripts for all three subunits are present at all life stages. LCH2 protein is present in 4th instar larvae, pupae, males, and sugar-fed females and is not increased with blood feeding despite an increase in message expression. LCH1 shows low expression in eggs,increases with larval stage and is expressed in pupae, males, and females. In contrast to LCH2, LCH1 is expressed in sugar fed females and expression increases following blood feeding by 72 h. HCH is strongly expressed in 4th instar larvae and pupae, is expressed in males, and increases with blood feeding (previous work). In keeping with transcript expression, LCH2 protein is expressed in ovaries and fat body 72 h PBM. It also is detected in midgut at 24 h, but not 72 h PBM. LCH1 and HCH are expressed at very low levels 24 h PBM and expression of both subunits increases in ovaries and fat body by 72 h PBM. We stained ovarian tissues with Prussian blue to visualize the temporal deposition of iron PBM. Staining showed ferric deposits in the ovarian tissues prior to blood feeding localized in the ovarian (OS) and ovariolar (OLS) sheaths. By 24 h PBM, ferric is detected in the secondary follicles (SF), and by 72 h PBM appears primarily in the SF and follicular epithelium (FE). Fluorescent imaging of ferritin in the ovaries showed the HCH sucunit is localized primarily in the OS, OLS, FE, and nurse cells (NC) prior to a blood meal, in the NC and FE 24 h PBM and in the FE only by 72 h PBM. LCH1 is detected in the OS, OLS, and NC prior to blood feeding, with diffuse fluorescent IHC staining in the oocyte (OO). By 24 h PBM, LCH1 is still detected in the OO and NC and present in the FE, SF, and germarium (G) and by 72 h PBM is found FE and SF. LCH2 also is detected in the OS, OLS, and NC, with diffuse fluorescent IHC staining in the OO prior to blood feeding; PBM LCH2 is detected in the FE and SF. Based on current data, we anticipate submitting 4 additional papers to peer-reviewed journals as a result of this project:1) proteomic analysis of the ovarian proteome, 2) TMT studies on iron-responsive proteins, 3) LCH2 ferritin subunit characterization and 4) ferritin and iron location in ovariesand eggs of Aedes aegypti. In addition to these studies although not required by the grant, we evaluated expression of ferritin and the presence of iron in the gut tissues of Aedes. In contast to findings in Drosophila, Aedes aegypti gut tissues showed diffuse staining of iron and ferritin was visualized in discrete puntate vesicles through outthese tissues.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Daphne Q.-D. Pham,Victor H. Perez, Lissette Velasquez, Dharty Bhakta, Erica L. Berzin,Guoli Zhou, and Joy. J. Winzerling. (2014) Mosquito Ribonucleotide Reductase: A site for control. Short Views on Insect Biochemistry and Molecular Biology, (Eds.) R. Chandrasekar, B.K. Tyagi, Z.Z., Gui, G.R., Reeck. International Book Mission, Academic Publisher, Manhattan, USA. Chapter -20, Vol.(1 & 2), pp 449-472. ISBN: 978-1-63315-205-2
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Target audience: Research scientific community Changes/Problems:No major problems were encountered. We are expanding the work to include ferritin assessment in the midgut tissues; see the above report. What opportunities for training and professional development has the project provided?Confocal microscopy training for students Opportunity to present at poster sessions of local and national meetings How have the results been disseminated to communities of interest?By current publications. What do you plan to do during the next reporting period to accomplish the goals?Complete the TMT studies and complete midgut analysis for ferritin. Publish a proteomics paper on the ovarian proteome in blood fed mosquitoes (control). Publish a paper on the second LCH2 ferritin subunit.
Impacts
What was accomplished under these goals?
Objective 1: We have completed the first analysis of a duplicate assay of Tandem Mass Tagged (TMT) proteins expressed in Aedes aegypti ovaries fed three different defined diets: artificial blood meal with iron (e.g., hemoglobin added) (ABM +Fe), artificial blood meal without iron (e.g., hemoglobin removed) (ABM -Fe), and a porcine blood meal (BM). A second analysis is underway with the same samples to show reproducibility of the duplicate experiment. Objective 2: During oogenesis in Ae. aegypti, we observed the accumulation of iron overtime post blood meal (PBM) and the differential localization of iron at 0, 24, and 72 h PBM using Prussian blue staining to detect ferric iron. The changes observed in iron location overtime PBM are consistent with the changes in the ovarian follicle anatomy during oogenesis. This suggests that iron may play a role in the process of oogenesis. We hypothesize that female mosquitoes retain iron in the ovaries from earlier developmental stages, the iron accumulates over time after the first blood meal, and the location of iron accretion in the ovaries shifts during oogenesis. We visualized the presence of ferritin, the major iron storage and transport protein in Ae. aegypti, in the ovaries using confocal microscopy. Immunohistochemistry imaging of the three subunits that assemble the ferritin molecule, heavy chain homologue (HCH) and two light chain homologues (LCHs), showed that these subunits and ferric iron are found in similar regions of the Ae. aegypti ovarian tissues prior to and after a blood meal. Based on the data presented here, we hypothesize that ferritin and iron play a role in oocyte development. The coalescence of ferritin and iron in mosquito ovaries supports our previous findings that iron from the blood meal is absorbed, loaded into ferritin in the midgut, secreted into the hemolymph from the midgut, and then detected in the ovaries PBM [1]. In the future, we intend to study iron accretion and ferritin expression in Ae. aegypti midguts PBM to further clarify the role of dietary iron in the midgut-ovary axis during oogenesis. This will enhance our understanding of where iron and ferritin are localized within these tissues to potentially interfere with iron metabolism and thereby reduce the numbers of vectors and disease transmission.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
International Glossina Genome Initiative. (2014) Genome sequence of the tsetse fly (Glossina morsitans): vector of African trypanosomiasis. Science. 344(6182), 380-386. doi: 10.1126/science.1249656.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Geiser, D. L., Conley, Z. R., Elliott, J. L., Mayo, J. J., Winzerling, J. J. (2015) Characterization of Anopheles gambiae (African Malaria Mosquito) Ferritin and the Effect of Iron on Intracellular Localization in Mosquito Cells. J Insect Sci. 15. pii: 68. doi: 10.1093/jisesa/iev049.
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