Characterization of Cell Division in the Tissues of the Calanoid

Characterization of Cell Division in the Tissues of the Calanoid

1 CHARACTERIZATION OF CELL DIVISION IN THE TISSUES OF THE CALANOID 2 COPEPOD, NEOCALANUS FLEMINGERI FROM DIAPAUSE THROUGH EARLY 3 OOGENESIS 4 5 A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF 6 HAWAI'I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 7 DEGREE OF 8 9 MASTER OF SCIENCE 10 IN 11 MARINE BIOLOGY 12 DECEMBER 2020 13 14 By 15 Kira J. Monell 16 17 Thesis Committee: 18 Petra Lenz, Chairperson 19 Erica Goetze 20 Megan Porter 21 22 Keywords: 5-Ethynyl-2´-deoxyuridine, lipid, oogenesis, Neocalanus flemingeri, Gulf of Alaska, 23 zooplankton 24 Acknowledgements 25 First and foremost, I would like state my sincere appreciation for my advisor Petra Lenz whose 26 seemingly encyclopedic knowledge of copepod literature, ability to ask the hard questions, and 27 most importantly compassion and empathy has made me a better scientist. She has tasted my 28 cooking and knows that I should probably stay in science. This research was supported by the 29 National Science Foundation Grant: OCE-1459235, OCE-1459826, and OCE-1756767. This 30 thesis would not be in existence if not for Tina Carvalho, thank you for being an expert teacher 31 in confocal microscopy and somehow always knowing what obscure copepod imaging issue I 32 was dealing with. An extremely special thanks to Russell Hopcroft whose mastery in invertebrate 33 taxonomy made this research possible. Thanks to the crew of the R/V Sikuliaq and R/V Tiglax 34 for help with collection of specimens. Thanks to Caitlin Smoot and Emily Stidham for additional 35 shipboard help. I am grateful to Daniel Hartline for his expert advice on 5-ethynyl-2'- 36 deoxyuridine. Many thanks to Vittoria Roncalli for invaluable help both shipboard and in the lab. 37 Thanks to Myly Le for undergoing the task of counting cells. Thanks to Marilyn Dunlap in the 38 Biological Electron Microscope Facility at UH Manoa. Thanks to Megan Porter and Erica 39 Goetze for providing advice and being wonderful thesis committee members. Thanks to Lauren 40 Block for listening to my long monologues about copepod oogenesis. Thanks to everyone at the 41 Békésy Laboratory of Neurobiology for your support and constant candy supply. Lastly, I want 42 to thank my family and lovely friends from: my cohort, the NGA LTER program, and life; your 43 support and cheerleading has made my graduate degree a joy to have undergone. 44 45 46 ii 47 Abstract 48 Unlike most calanoid copepods, females of the diapausing copepod, Neocalanus flemingeri 49 (Miller, 1988) fuel oogenesis entirely through stored energy. Due to the reliance on stored 50 energy, N. flemingeri females manage their internal lipid stores to maximize oocyte production 51 which differs from the reproductive program of most calanoids that couple food availability with 52 oogenesis. In this study, both lipid content and cell division within the reproductive structures in 53 females were examined as diapause was terminated and oogenesis began. In June and September 54 2019, diapausing females were collected from depth in Prince William Sound, Alaska. 55 Incubation experiments in 5-Ethynyl-2´-deoxyuridine (EdU) were conducted to quantify and 56 pinpoint the location of cell division within the body from diapause through early oogenesis. 57 Imaging of EdU-treated females using confocal microscopy revealed evidence of cell division in 58 the ovary within 24 hours after collection. Both oogonia and oocytes incorporated EdU based on 59 the location of cells in the posterior end of the ovary. Dividing cells in the ovary peaked in 60 number at 72 hours, remained high over two weeks, and decreased thereafter with no staining 61 detected at four weeks after collection. Thus, the production of new oocytes stopped two to four 62 weeks before females release their first clutch of eggs. The pattern of cell division in the ovary 63 parallels the up- and down-regulation of early germline development genes reported in an earlier 64 transcriptomics study. These results suggest that oogenesis is sequential in N. flemingeri which 65 synchronizes egg maturation, unlike other calanoid copepods where most oocyte stages are 66 observed concurrently within the ovary. The magnitude of cell division in the ovary of individual 67 females were compared with their respective total lipid contents and prosome lengths. Numbers 68 of dividing cells in the ovaries were positively correlated with both prosome length and lipid 69 content, suggesting that total fecundity is higher in copepods with longer prosome lengths and iii 70 more lipid. In this study, duration of the period of active cell division appeared to be similar in 71 all females independent of prosome size or lipid content. Understanding the internal 72 physiological process of reproduction in lipid-rich copepods like N. flemingeri is an important 73 step in knowing how and to what magnitude egg production can be affected by climate change. 74 This is the first study that tracked cell division in post-diapause N. flemingeri. This capital 75 breeder meters its energy sources by varying the number of dividing cells and limiting cell 76 division in the ovary to the first three to four weeks post-diapause. As waters continue to warm, 77 predictions of shorter diapause lengths, and both smaller lipid reserves and prosome lengths have 78 been hypothesized. Negative impacts on egg production in this species could lead to a decrease 79 in population numbers and thus a decrease in a vital food source for many birds and fishes. 80 81 82 83 84 85 86 87 88 89 90 91 92 iv 93 Contents 94 Acknowledgements........................................................................................................................ii 95 Abstract.........................................................................................................................................iii 96 Introduction....................................................................................................................................1 97 Methods...........................................................................................................................................3 98 Results.............................................................................................................................................9 99 Discussion.....................................................................................................................................16 100 References.....................................................................................................................................30 101 List of tables..................................................................................................................................39 102 Table 1: Summary of Neocalanus flemingeri experiments completed in the Summer (PWS2, 103 June collection) and Fall (PWS2 and Pleiades, September collections) of 2019..........................39 104 List of charts, graphs, figures, illustrations, plates, maps........................................................40 105 Figure 1. Diagram of life cycle of N. flemingeri in the Gulf of Alaska.........................................40 106 Figure 2: Diagram of location and structure of ovary and oviducts in N. flemingeri....................41 107 Figure 3: Modified diagram of a section through thorax of Calanus finmarchicus showing ovary 108 and oviducts from Hilton (1931)....................................................................................................42 109 Figure 4: Map of Prince William Sound, Alaska with sampling sites for N. flemingeri collections 110 ........................................................................................................................................................43 111 Figure 5: Light microscope images of N. flemingeri females........................................................44 112 Figure 6: Histograms showing the distribution in prosome length in mm and initial lipid fullness 113 percentage between sampling sites................................................................................................45 114 Figure 7: Scatterplot between prosome length in mm and lipid content in mg for PWS2/June 115 (grey triangles, n = 168) and Pleiades/September (black circles, n=36).......................................46 v 116 Figure 8: Females’ lipid contents at different times post-collection..............................................47 117 Figure 9: Maximum Intensity Projections (MIP) of ovaries of females incubated in EdU showing 118 a time series from immediately after collection to four weeks post-collection.............................48 119 Figure 10: MIP of merged confocal z-stacks of ovaries 24-48 hours after collection at PWS2 (A) 120 (June) and Pleiades (B) sampling sites..........................................................................................50 121 Figure 11: MIP of merged confocal z-stacks of ovaries at 0-24 hours after collection in PWS2 122 (A) (June) and Pleiades (B) sampling sites....................................................................................51 123 Figure 12: Description of EdU incorporation in dividing cells within reproductive structures at 124 different times post-collection.......................................................................................................52 125 Figure 13: Scatterplots comparing cell division in the ovary and oviducts with lipid content and 126 prosome length at one day after collection....................................................................................53

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