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.
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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
127 Figure 14: 150 µm confocal projection of whole-body female (PWS2) 72 hours after
128 collection...... 54
129 Figure 15: Confocal images of oocytes with DAPI staining (green cells) in oviducts four weeks
130 after collection at PWS2/June sampling site...... 55
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vi 136 Introduction
137 Most calanoid copepods are income breeders, meaning that they depend on newly ingested food
138 to fuel reproduction (Checkley, 1980; Hirche, 1989). During periods of high food availability,
139 most oocyte developmental stages are present concurrently within the oviducts, which allows
140 females to maximize egg production as broods are spawned at regular intervals (Niehoff, 2007).
141 In contrast, females in the genus Neocalanus are capital breeders. Neither males nor females feed
142 as adults and are characterized by highly reduced mouthparts (Miller, 1988). Females depend
143 entirely on stored energy to fuel reproduction through internal lipid stores (Miller, 1988; Saito
144 and Tsuda, 2000). Furthermore, Neocalanus flemingeri, an ecologically important subarctic
145 calanoid, enters the non-feeding adult stage in the spring before females enter a prolonged period
146 of dormancy termed diapause (Figure 1, Miller and Clemons, 1988; Roncalli et al., 2018). Thus,
147 females depend on energy stores for over half of their lifespan starting with the final molt in
148 May-June and spawning in January-February (Cooney et al., 2001; Miller and Clemons, 1988).
149 The combination of finite resources and the high cost of reproduction raises questions about how
150 these females meter the number of developing eggs to assure their successful maturation and
151 provisioning. A reproductive program with most or all oocyte developmental stages present
152 concurrently would be a disadvantage when dependent on existing energy stores. Therefore, this
153 study focused on the initiation and end of new oocyte production by tracking changes in cell
154 division in the ovary and in lipid content of N. flemingeri starting after the termination of
155 diapause.
156
157 The initiation of the reproductive program post-diapause can be seen at one week after collection
158 through the upregulation of genes relating to germline development in gene expression profiles
1 159 of N. flemingeri (Roncalli et al., 2018). In most calanoid copepods regardless of gonad type, the
160 creation and maturation of oocytes operates similar to a conveyor belt (Hilton, 1931; Niehoff,
161 2007). Early oocytes at the posterior end of the ovary move towards the anterior end of the ovary
162 as oocytes mature (Figure 2, Hilton, 1931; Niehoff, 2007). Development of oocytes starts in the
163 germinative zone or the multiplication zone (Figure 3, Eckelbarger and Blades-Eckelbarger,
164 2005; Hilton, 1931). Hilton (1931) described this zone as the extreme posterior end of the ovary.
165 Oocytes begin as mitotically active germ cells or oogonia that are mitotically dividing in the
166 multiplication zone (Hilton, 1931). Oogonia may undergo multiple mitotic divisions, however
167 after the final mitosis oogonia are considered oocytes (Hilton, 1931).
168
169 Oocytes undergo meiosis 1 but stop in prophase 1, dividing oocytes are found slightly anterior to
170 the oogonia within an area of the ovary called the synapsis zone (Hilton, 1931). Both oogonia
171 and early oocytes described above are classified as within the oocyte developmental stage 0
172 (OS0), the earliest of the five developmental stages (Niehoff, 2007). In Calanus finmarchicus,
173 the most well studied example of a Calanus-type gonad, OS0 oocytes are always present in
174 reproductively active females (Niehoff and Hirche, 1996). Oocytes will stay in prophase 1 until
175 just before eggs are released (Blades-Eckelbarger and Youngbluth, 1984; Eckelbarger and
176 Blades-Eckelbarger, 2005; Niehoff, 2007). Early oocytes begin vitellogenesis, the process of
177 yolk formation (Eckelbarger and Blades-Eckelbarger, 2005; Niehoff, 2007). There are two types
178 of vitellogenesis; vitellogenesis 1 is characterized by the accumulation of yolk from an internal
179 source, while vitellogenesis 2 begins later and accumulates yolk from an extra-ovarian source
180 (Niehoff, 2007). Oocytes in the early stages of vitellogenesis are present in the anterior region of
181 the ovary (Eckelbarger and Blades-Eckelbarger, 2005). Oocytes undergoing vitellogenesis can be
2 182 identified by their increased size (Hilton, 1931; Niehoff and Hirche, 1996). In the late stages of
183 vitellogenesis, just prior to spawning, mature oocytes are located in the oviducts and diverticula
184 (Niehoff and Hirche, 1996; Niehoff, 2007). In the common Calanus-type gonad, stages of oocyte
185 development (OS1-OS4) occur in layers within the diverticula and oviducts with the most mature
186 oocytes in the ventral layer and the least mature oocytes in the dorsal layer (Niehoff, 2007).
187 When a copepod with a Calanus-type gonad spawns, the most mature oocytes are spawned, and
188 the rest of the oocytes layered underneath continue to develop to form the next clutch (Niehoff,
189 2007).
190
191 Neocalanus spp. are unique to calanoids in that egg production is fueled entirely through stored
192 energy (Miller, 1988; Saito and Tsuda, 2000). However, it remains unclear how the process of
193 oogenesis is different in contrast to other calanoid copepods that are income breeders. Currently,
194 the research on oogenesis in N. flemingeri has been limited to egg production studies and
195 characterizing the process of oogenesis through gene expression data (Saito and Tsuda, 2000;
196 Roncalli et al., 2018; Lenz and Roncalli, 2019; Roncalli et al., 2020). The three main objectives
197 of this study were: 1) to define the start and end of cell division in terms of timing and location
198 within the ovary and oviducts of N. flemingeri after the termination of diapause; 2) to evaluate
199 females’ prosome lengths and lipid stores as indicators of reproductive potential; and 3) to
200 estimate lipid utilization of females emerging from diapause and into early oogenesis.
201
202 Methods
203 Sample collection and sorting
3 204 Copepods were collected in Prince William Sound, Alaska in the Summer and Fall of 2019
205 during the Northern Gulf of Alaska Long Term Ecological Research cruises. The June collection
206 date was June 30th, 2019 on the R/V Sikuliaq (cruise number: SKQ201915S;
207 https://nga.lternet.edu/), and the September collection dates were September 12th and 13th, 2019
208 on the R/V Tiglax (cruise number: TGX201909). June females were collected at the sampling
209 site PWS2 (Figure 4, Latitude 60° 32.1'N; Longitude 147° 48.2'W), September females were
210 collected at PWS2 and near the Pleiades Islands (Latitude 60° 16.7'N; Longitude 147° 59.2'W).
211 All copepods were collected using a Multiple Plankton Sampler at an upwards tow speed of 0.5
212 m/sec (MultiNet – 0.5 m2 mouth area; 150 µm mesh nets). In June, experimental copepods were
213 obtained from the MultiNet side net with a non-filtering cod-end and an integrated tow from 725
214 m to the surface. For the September collections, copepods came from the 500-400 m stratum. To
215 confirm all females were in diapause, their posture was checked upon retrieval of the net. Some
216 N. flemingeri had their antennules folded and all had urosomes bent dorsally, which is the typical
217 diapause posture (Lenz and Roncalli, 2019). In addition, a 53 µm CalVET net was towed
218 vertically in the surface 100 m to check that no females were present in surface waters during
219 both PWS2 tows. In June, a few stage C5 N. flemingeri were still present in the upper 100 m but
220 no adult females were seen (Hopcroft, personal communication).
221
222 For sorting, net samples were immediately diluted using filtered seawater taken from depth.
223 Samples were kept cold to prevent heat stress. Females were live sorted, all females selected for
224 the experiments had been mated with as indicated by at least one opaque spermatheca.
225 Experimental females were placed in groups of three into 750 mL Falcon tissue-culture flasks
226 and incubated at a dim light setting in a cold incubator. Incubation temperatures were at or below
4 227 deep-water temperatures in Prince William Sound (temperature settings: 4°C for June and 6°C
228 for September). A total of 186 females in June and 43 in September were sorted into flasks for
229 experimental use. A subset of females was used in cell division experiments; the remaining were
230 imaged for measurements of prosome length and lipid sac area (see below).
231
232 Experimental design
233 5-Ethynyl-2'-deoxyuridine (EdU) is a thymidine analogue that is incorporated into the DNA of a
234 cell during DNA replication. EdU is incorporated into mitotic and meiotic cells during S-phase
235 (Buck et al., 2018). In copepods, oocytes in the ovary undergo S-phase then start meiosis 1, but
236 stop in prophase 1 (Blades-Eckelbarger and Youngbluth, 1984; Eckelbarger and Blades-
237 Eckelbarger, 2005; Niehoff, 2007). The end of meiosis 1 does not occur until just prior to
238 spawning (Blades-Eckelbarger and Youngbluth, 1984; Eckelbarger and Blades-Eckelbarger,
239 2005; Niehoff, 2007). EdU is non-toxic at low concentrations and therefore can be incorporated
240 into an organism while living (Beltz et al., 2011; Benton et al., 2014).
241
242 Two to four females were incubated in low concentrations of EdU for three to 24 hours at eight
243 time points in June (Table 1, 0-24, 24-48, 36-60, 72-96 hours and 2, 3, 4, 4.5 weeks), and at nine
244 time points in September (0-3, 0-6, 0-14, 0-24, 24-48, 72-96 hours and 1, 2, 3 weeks) to track the
245 numbers of dividing cells in the ovary from collection (diapause) to early oogenesis based on a
246 previous transcriptomic study (Roncalli et al. 2018). Prior to preservation for confocal
247 microscopy, most females were also examined by light microscopy for any visible
248 morphological changes and imaged for prosome and lipid sac measurements. For some time
249 points, light microscope imaging was not possible as noted in Table 1 (asterisks). Additional
5 250 females, 145 in June and 27 in September, were imaged exclusively by light microscope for lipid
251 sac and prosome measurements. These images provided information on female body size as
252 measured by prosome length and lipid sac size.
253
254 Lipid sac and prosome length imaging
255 Either just prior or after EdU incubations, live females were removed from the tissue flasks and
256 imaged by light microscopy for lipid sac and prosome length measurements. Live females were
257 placed in a chilled embryo dish with a small drop of seawater. All females used for lipid sac and
258 prosome length analysis in June were collected from the PWS2 sampling site, and all females in
259 September were collected from the Pleiades sampling site. Females were imaged on their side at
260 32x magnification (image resolution: 4096 by 3000 pixels) using a Leica MZ16 microscope. A
261 coupling lens was added in September for additional stability. Females were checked for signs of
262 damage. The few copepods that had damaged lipid sacs as indicated by the presence of lipid
263 droplets outside the lipid sac were not included in light microscope imaging. Females with some
264 broken caudal setae and/or antennules were used but were noted to have some damage.
265
266 Using ImageJ, software was calibrated with scale bars based on microscope calibrations. Light
267 images were used to take three measurements: prosome length in mm, area of the lipid sac in
268 mm2, and area of the prosome in mm2 (Figure 5). Prosome length was measured by manually
269 placing a line from the anterior to posterior tip of the prosome. Lipid sac and prosome area were
270 measured by manually outlining the perimeter of each. Area of the lipid sac was measured to
271 estimate total lipid content in mg. Lipid sac area was translated to lipid content in mg using
272 Vogedes et al. (2010) equation: TL=0.197A1.38, where A is lipid sac area and TL is total lipid.
6 273 The Vogedes et al. (2010) equation used previously measured total lipid contents of three
274 Calanus spp. to create an equation that used lipid sac area as a proxy for lipid content. Both area
275 of the lipid sac and prosome were measured to estimate lipid fullness percentage. Lipid fullness:
lipid sac area 276 × 100, incorporates prosome size into lipid measurements since larger copepods prosome sac area
277 can store more lipid than smaller copepods (Miller et al. 2000). Lipid fullness allows for
278 comparisons of lipid content between individuals while minimizing the influence of differences
279 in prosome length, species, and stage on the analysis (Skottene et al. 2019, Schmid et al. 2018).
280 Area, a direct measurement from images, was used in calculations of lipid fullness and lipid
281 content to avoid the assumptions in estimating volume.
282
283 EdU protocol
284 For each experimental time point (Table 1) two to four females were carefully pipetted out of
285 one or more 750 mL flasks and placed in flat bottom well plates with 2 ml of EdU/filtered
286 seawater solution. Females were incubated in the EdU solution for 24 hours except for the first
287 three September time points (0-3, 0-6, 0-14 hours). At the end of incubation, females were
288 removed from EdU, checked for mortality (no mortalities occurred during this step), and fixed in
289 4% paraformaldehyde in Sorensen’s phosphate buffer for 24 hours. Sorensen’s phosphate buffer
290 was used to keep pH neutral while fixing. Staining was done using a ThermoFisher Click-iT EdU
291 Alexa Fluor 594 Imaging Kit (catalog #C10639). Samples were washed in Sorensen’s phosphate
292 buffer three times. Permeabilization of tissue was done by washing copepods three times in 0.5%
293 Triton X-100 in Sorensen’s phosphate buffer. EdU labeled cells were fluorescently tagged with
294 Alexa Fluor 594 dye. Another three washes of Sorensen’s phosphate buffer were done to ensure
295 all of the previous solution was removed. Samples were stored in VECTASHIELD Antifade
7 296 Mounting Medium with DAPI (catalog #H-1200), a counterstain to EdU, at 4C until imaged.
297 Because DAPI in VECTASHIELD frequently did not permeate into the ovary, I diluted
298 VECTASHIELD with DAPI or Hoechst 3342 (catalog #C10639) in phosphate-buffered saline
299 and this solution was used to fully stain the ovary. VECTASHIELD was used as the mounting
300 medium, and a coverslip was placed on top. When June females were confocal imaged, EdU was
301 much brighter than DAPI staining. EdU brightness was decreased by adjusting the concentration
302 of EdU from 1 mg in June to 0.5 mg per copepod in September.
303
304 Confocal imaging and quantification of cell division
305 Samples were imaged using a Leica SP8 X Confocal Laser Scanning microscope with a × 20 or
306 × 63 immersion lens and a white light laser. Glycerol was used as the immersion medium. The
307 405 and 598 laser lines were used for DAPI and EdU, respectively. Samples were imaged by tile
308 scanning through each copepod to locate the entire ovary. Z-stack sections were 1.041 µm apart
309 ensuring no cells were missed due to large imaging gaps. Whole-mount females were imaged
310 until the depth in which resolution was lost due insufficient laser penetration was reached,
311 typically 80-250 µm from the start of the ovary and oviducts. Using Leica’s merge software,
312 individual z-stacks were imaged separately then merged together to form a single z-stack of an
313 ovary larger than the lens’ field of view.
314
315 Cell counts of the ovaries and oviducts were made by projecting each section of a z-stack onto a
316 monitor and outlining each dividing cell onto tracer paper (Benton et al., 2014). Dividing cells
317 were then counted when all slices of a z-stack were analyzed. Any cell that was considered in the
318 ovary or oviducts was counted. The structure of the ovary and oviducts were seen through the
8 319 DNA stain. The ovary and oviducts were identified using previous descriptions of calanoid
320 copepod reproductive structures (Hilton, 1931; Niehoff, 2007; Blades-Eckelbarger and
321 Youngbluth, 1984; Eckelbarger and Blades-Eckelbarger, 2005). Counts were done twice by two
322 separate people then averaged together for more accurate counts.
323
324 Results
325 Description of sampling sites and female collections
326 The PWS2 and Pleiades collection sites are located 16 nautical miles apart in two distinct deep
327 basins separated by a shallowing of the sea floor at points to depths of less than 150 m. PWS2 is
328 located in a deep fjord north of the Knight Island Passage with a max depth of 725 m. The
329 Pleiades sampling site was south of the Knight Island Passage and had a shallower water column
330 depth with a max depth of 580 m.
331
332 Adult females collected in June and September showed no signs of oogenesis: their ovaries were
333 clear, and no oocytes were visible as determined during live sorting and initial imaging (Figure
334 5). While there were no differences in female condition (i.e. signs of stress or damage) between
335 the June and September collections, there was a difference in long-term survival. The survival
336 rate for June experimental females was over >90% during the 32 day experiment. In contrast,
337 survival was lower in September (~60%), most likely due to damage that occurred during the
338 collection in exceptionally high seas.
339
340 Females from the Pleiades sampling site in September had a significantly smaller mean prosome
341 length than copepods from the PWS2 sampling site in June, 3.7 mm and 3.9 mm respectively
9 342 (Figure 6, two sample t-test, p = < .001, t(43) = 4.181). Females had initial total lipid contents
343 that varied greatly; females one day after collection had a range of lipid from 0.31-0.78 mg and
344 0.14-0.58 mg for PWS2/June and Pleiades/September, respectively. Total lipid in mg during the
345 first 24 hours after collection was significantly less in Pleiades/September females (0.36 mg, s.d.
346 = 0.12 mg, n = 13) than in the PWS2/June females (0.53 mg, s.d. = 0.12 mg, n = 21; two sample
347 t-test, p = <.001, t(26) = 3.894). However, lipid content and prosome length are not independent
348 of one another; these two variables were positively correlated (Figure 7, PWS2/June: R2 = 0.46;
349 Pleiades/September: R2 = 0.64). Larger females have a greater body capacity to accumulate lipid
350 stores (Miller et al., 2000). There was not a significant difference in the median initial lipid
351 fullness percentage, measured as lipid fullness 24 hours after collection, between PWS2/June
352 (median = 46 %, n = 21) and Pleiades/September (median = 45 %, n = 13) females (Wilcoxon
353 Rank Sum Test, W = 3381, p = .267). In other words, females from the PWS2/June and the
354 Pleiades/September collections differed in body size and lipid sac content, but not in the relative
355 measure of lipid fullness.
356
357 Lipid utilization during experimental period
358 Females originated from two different collection months and two different sampling sites. The
359 lipid sac and prosome length data were mutually exclusive with all females from June being
360 collected from PWS2, and all females from September being collected from Pleiades. Females
361 between the two collection months were diapausing for differing lengths of time, roughly a
362 month in June and four months in September. While females would have lost lipid due to
363 metabolic activities between the two collection months, without the understanding of metabolic
364 rates in diapausing N. flemingeri it is difficult to make assumptions about the amount of lipid lost
10 365 between the two collection months. Respiration rates of diapausing N. cristatus were
366 approximately 30% of what active individuals respired (Ikeda et al. 2004). Respirations of some
367 insects have been described lower with Ragland et al. (2009) reporting a 90% depression in
368 respiration rates during diapause in a fly.
369
370 Mean lipid content decreased during the experimental period (Figure 8). This difference was
371 significant during the 32 day PWS2/June incubation, but not the one week Pleiades/September
372 incubation. The estimated lipid loss rate was calculated using a regression analysis. On average
373 females lost an estimated 0.03 mg of lipid per week in the PWS2/June collection. The lipid loss
374 rate of the Pleiades/September collection was higher at 0.07 mg of lipid per week. The lipid loss
375 rate within the first week was higher in both collections (PWS2/June: 0.06 mg of lipid per week;
376 Pleiades/September: 0.07 mg of lipid per week) when compared to the 32 day lipid loss rate, but
377 neither regression analyses of lipid loss in the first week were significant.
378
379 Initiation of oogenesis
380 Females that were incubated in EdU immediately after collection for three hours showed no
381 evidence of cell division within the ovary (Figure 9). All females (n = 9) incubated in EdU for 24
382 hours immediately after collection (0-24 hours after collection) showed evidence for cell division
383 in the posterior end of the ovary regardless of sampling site or collection month. Previous papers
384 have noted an increase in behavioral responsiveness, which is indicative of diapause termination,
385 in Neocalanus spp. one to six hours post-collection (Campbell et al., 2004; Lenz and Roncalli,
386 2019). To capture on a finer resolution when cell division started, additional short incubations
387 were run. One of three females that were given EdU for six hours immediately after collection
11 388 showed presence of cell division within the ovary. Similarly, one of three females that were
389 given EdU for 14 hours after collection showed presence of cell division within the ovary. While
390 a few females may start cell division between 3 and 14 hours after collection, females start cell
391 division consistently between 12 and 24 hours post-collection.
392
393 Location of cell division within the ovary
394 Cell division in the reproductive structures, ovary and oviducts, was predominately situated in
395 the posterior end of the ovary. The posterior end of the ovary was identified by the presence of
396 smaller cells in higher cell densities as well as the variable presence of a rounded outgrowth that
397 Eckelbarger and Blades-Eckelbarger (2005) identified as the germinative zone where oogonia
398 have been observed. The variable presence of the outgrowth was likely due to the limitations of
399 whole mount confocal microscopy, and not variations in ovary structure at least in copepods
400 undergoing early oogenesis. Currently, there is no way through EdU and DAPI staining to
401 definitively differentiate between oogonia mitotically dividing and oocytes starting meiosis 1.
402 However, in this study EdU incorporation occurred in locations that are consistent with cell
403 division of oogonia and oocytes described in the ovaries of C. finmarchicus by Hilton (1931).
404
405 Cell division in the ovary was frequently seen at the extreme posterior edge of the ovary. This
406 location is consistent with the multiplication or germinative zone where oogonia have been
407 previously described, suggesting that oogonia were present and dividing (Eckelbarger and
408 Blades-Eckelbarger, 2005; Hilton, 1931). Confocal images did not always reach a depth where
409 the end of the ovary could be determined. The location of oocytes undergoing meiosis 1 has been
410 described as at or just anterior to the synapsis zone (Hilton, 1931). The synapsis zone does not
12 411 have a well-defined location besides anterior to the multiplication zone. If a germinative zone
412 was seen in confocal images, rounded outgrowth, the synapsis zone was thought to be anterior to
413 the outgrowth where the ovary starts to widen. Cell division was seen in females at this location
414 suggesting that dividing oocytes were also present. Frequently cell division occurred in both the
415 multiplication zone and the synapsis zone concurrently. Occasionally cells were seen dividing in
416 the synapsis zone but not the multiplication zone (Figure 10).
417
418 Peak and end of cell division
419 Cell division ramped up quickly, within three days after collection the number of dividing cells
420 in the ovary had hit a peak in PWS2/June females and was sustained until two weeks after
421 collection (Figure 9). For Pleiades/September copepods the peak in cell division occurred at two
422 weeks. At three weeks after collection, cell division had decreased in females from both
423 collections (Figure 9). Important to note is that week three is the first time point at which
424 morphological changes due to oogenesis can be seen in females through light microscopy,
425 described as a slight graininess or tint in the ovary (Roncalli et al., 2018). Cessation of cell
426 division occurred by 28 days after collection in PWS2/June females. Cell division also did not
427 occur in PWS2/June females 32 days after collection. The absence of cell division at 28 and 32
428 days after collection occurred regardless of the range in prosome length (3.5 – 4.1 mm) and lipid
429 content (0.22 – 0.44 mg) within females.
430
431 Quantification of cell division in ovary and oviducts
432 Number of dividing cells between collections were noticeably different beginning within 24
433 hours after collection (Figure 11). Pleiades/September females had a mean cell count that was
13 434 consistently lower than PWS2/June at all time points sampled in both collection months (Figure
435 12). Mean cell division at each time point was scaled to each collection site’s respective mean
436 peak time point (PWS2/June: 72 hours, 214 cells; September: 14 days, 157 cells) to
437 accommodate for magnitude differences. The temporal pattern of cell division was similar for the
438 two collections as shown in the normalized figure 12. The only deviation from the pattern being
439 at 72 hours when the PWS2/June collection increased in number of dividing cells, but the
440 Pleiades/September collection decreased.
441
442 Females from both sites one and three days after collection were combined to examine if there
443 was a relationship between number of dividing cells in the ovary and oviducts, prosome length,
444 and lipid content. Both prosome length and lipid content were positively correlated with cell
445 division one day after collection, (Figure 13, Spearman’s rank correlation, 휌: 0.82 and 휌: 0.82, n
446 = 5) but negatively correlated three days after collection (Spearman’s rank correlation, 휌: − 0.41
447 and 휌: −0.17, n = 6). Some possible explanations for this negative relationship could be small
448 sample size, external factors like high seas influencing cell division, as well as females being
449 more synchronized just after termination of diapause then showing more variation further into
450 oogenesis. However, the 72 hour time point appeared to be exceptionally low in the
451 Pleiades/September experiment. The lack of relationship between the prosome length, lipid
452 content, and cell division in the ovary could be due to this time point being an outlier.
453
454 Cell division outside of the ovary and oviducts
455 Unexpectedly, cell division was not limited to the ovary and oviducts but was also observed
456 throughout the females. Eleven females were whole-body confocal imaged to elucidate location
14 457 of cell division outside of the reproductive structures; none of these samples had cell division
458 quantified. Of the 11 females that were whole-body confocal imaged, only two females did not
459 have dividing cells clearly present outside of the ovary and oviducts. Cell division outside of the
460 ovary was seen in all locations across the body in females except in the spermatheca (Figure 14).
461 Similar to within the reproductive structures, cell division outside of the ovary was seen in three
462 out of the four females incubated for 24 hours immediately after collection. All females that were
463 imaged from the 72 hour time point showed presence of cell division outside of the ovary (n =
464 3).
465
466 Non-dividing oocytes
467 Oocytes in traditional light microscopy can be identified by their color, clear or tan prior to yolk
468 formation and a golden brown later in development (Blades-Eckelbarger and Youngbluth, 1984).
469 Older oocytes located in the oviducts or the anterior regions of the ovary were easily identifiable
470 by the presence of large nucleus, nucleolus, and condensed chromatin (Figure 15). As expected,
471 EdU was never observed in these older oocytes since oocytes are arrested in meiosis 1. Oocytes
472 grow in size through maturation due to yolk accumulation; in Labidocera aestiva oocytes start at
473 10 µm in their previtellogenic state and end at approximately 90 µm when spawned (Blades-
474 Eckelbarger and Youngbluth, 1984). While DAPI only labelled the nuclei of the oocytes, the
475 nuclei were larger than the nuclei of adjacent somatic cells. Within the nucleus a large nucleolus
476 not stained by DAPI was also present (as previously described for calanoids: Eckelbarger and
477 Blades-Eckelbarger, 2005; Hilton, 1931). Oocytes had a splotchy appearance due to the
478 condensing of chromatin (Eckelbarger and Blades-Eckelbarger, 2005). Older DAPI stained
479 oocytes were first seen in the oviducts at 21 days after collection. At 28 and 32 days, older
15 480 oocytes were common in the in the anterior ovary and oviducts. Oviducts and ovaries expanded
481 across the prosome to accommodate growing oocytes.
482
483 Discussion
484 Lipid fullness and comparison between Prince William Sound and Pleiades
485 Females collected both in June at PWS2 and September near the Pleiades were in good condition
486 based on lipid fullness. While Pleiades/September females had less total lipid and less cell
487 division present within the ovary, none of these copepods were what Tsuda et al. (2001)
488 considered as medially stored on the lateral view (<4% lipid fullness). Most copepods from both
489 collections were considered fully stored (>40% lipid fullness) which is the classification of the
490 fullest copepods (Tsuda et al., 2001). The median initial lipid fullness for PWS2/June (median:
491 46%; range: 20-57%) and Pleiades/September (median: 45%; range: 15%-56%) were similar,
492 suggesting that females were largely full of lipid for their respective sizes.
493
494 Diapausing females varied in size with prosome lengths ranging from 3.1 to 4.3 mm at the
495 Pleiades/September sampling site and 3.4 to 4.3 mm at PWS2/June. With a previously described
496 prosome length range of 3.0 to 4.5 mm in N. flemingeri females with a one-year lifespan, both
497 sets of females were within the expected range (Tsuda et al., 2001). While ranges were similar,
498 mean prosome lengths of females collected in September near the Pleiades were significantly
499 shorter than those from PWS2 collected in June. Lipid fullness did not differ between the two
500 collections but given the smaller size and lipid content (mg), females in Pleiades/September were
501 smaller copepods not capable of holding as much total lipid. For example, the fullest copepod in
502 this study that was 3.5 mm long had 0.38 mg of lipid, but the fullest copepod that was 4.0 mm
16 503 long had 0.66 mg of lipid. The sampling sites were only 16 nautical miles apart but with a
504 shallowing of the seafloor in between that effectively separated the two diapausing populations
505 at depth.
506
507 Circulation in Prince William Sound
508 Copepods would have been present in the surface waters during the growing season. Circulation
509 in Prince William Sound has an outflow of water through the north side of Knight Island Passage
510 and out the south end (Niebauer et al., 1994). The Pleiades sampling site was downstream from
511 PWS2. Water circulation likely influenced the local population in the sampling sites prior to the
512 copepods’ migration to depth. Once the movement to depth occurred, females at depth were
513 diapausing at a water temperature of ~6°C at both collection sites. Prince William Sound is
514 unique in that the zooplankton community contains a mix of coastal, middle, and outer-shelf
515 individuals (Cooney, 1986). The variable locations that females could have originated from
516 likely contributed to the range in sizes and conditions. In addition, the significant difference in
517 prosome length between sites suggest that females that accumulated in these two basins may
518 have experienced different spring growth conditions since prosome length and food availability
519 have been correlated in other calanoid copepods (Campbell et al., 2001). While females from the
520 two collection sites were likely not from the same location due to the difference in female size;
521 the circulation patterns in the sound were likely bringing copepods from the same source into the
522 two sampling sites.
523
524 Lipid utilization
17 525 Sampling location and collection month were confounding variables in this study, however the
526 locational difference between the sampling sites is likely the stronger factor in what was
527 influencing females’ size and therefore possibly number of dividing cells. If the collection
528 months were the stronger factor, females from the September collection would have a smaller
529 lipid sac due to lipid being utilized for respiration for a longer amount of time. However, lipid
530 fullness was similar between PWS2/June and Pleiades/September collections suggesting that
531 very little lipid was lost between June and September. With a previous reference to a diapausing
532 respiration rate in N. cristatus that was 30% of the respiration rate of active individuals, N.
533 flemingeri females may likely have a diapausing respiration rate lower than what was described
534 for N. cristatus (Ikeda et al. 2004). Respiration rates of diapausing N. flemingeri may be closer to
535 the depressed metabolic rates of over 90% that was described in a fly (Ragland et al., 2008). In
536 addition, prosome lengths between the two collections were significantly different suggesting
537 that sampling location influenced female size since terminal length is fixed after the molt into
538 adulthood and would not be influenced by diapause duration.
539
540 From net collection to the day of death, N. flemingeri females could approximately live 60-110
541 days depending on how many clutches are released (Roncalli et al., 2018; Saito and Tsuda,
542 2000). Based on the lipid utilization rate from PWS2/June, females from the PWS2/June and
543 Pleiades/September collection were predicted to live for 129 and 88 days, respectively. Both sets
544 of females were relatively full of lipid for their length and were expected to be able to complete
545 the reproductive program.
546
18 547 In a preliminary analysis to better understand possible lipid usage between females of varying
548 lipid contents, PWS2/June and Pleiades/September females were combined into one large
549 dataset. For each time point females were divided in half into two groups: females with more
550 lipid content = fullest 50%, and females with less lipid content = thinnest 50%. When time points
551 had an odd number of data points, the extra data point was added to one category alternating so
552 that the final sample sizes were the same (fullest 50% = 102; thinnest 50% = 102). The mean
553 lipid contents one day after collection were different (fullest 50%: 0.59 mg, s.d.= 0.077 mg, n =
554 17; thinnest 50%: 0.34 mg, s.d.= 0.084 mg), but the projected dates that lipid would run out were
555 similar (fullest 50%: 187 days, y = -0.003x + 0.5607; thinnest 50%: 183 days, y = -0.0019x +
556 0.3471, n = 17) suggesting that females might meter lipid to prevent failure of the reproductive
557 program.
558
559 While this study captured utilization of lipid through early oogenesis, lipid is used throughout the
560 entire reproductive program and likely not at a constant rate. Females 32 days after collection in
561 PWS2/June had on average ~30% less lipid content than females from PWS2/June one day after
562 collection. The lack of a large difference in lipid content at the end of the 32 days suggests that
563 there is a more energy intensive process after early oogenesis. Females may accelerate lipid
564 usage in vitellogenesis 2, a period of rapid yolk accumulation from an extra-ovarian source with
565 a size increase in oocytes (Niehoff, 2007). In N. flemingeri, genes involved in vitellogenesis 2
566 were significantly upregulated at five to seven weeks after collection (Roncalli et al., 2018).
567 Neither experiment reached five weeks, and only one female 4.5 weeks after collection had
568 several rows of small colored oocytes that is typical for oocytes during vitellogenesis 2 (Niehoff
569 and Hirche, 1996). Females at the very end of spawning have been described as having a ghost-
19 570 like body with almost nothing remaining, suggesting that all lipid is utilized during oogenesis
571 (Hopcroft, personal communication).
572
573 Termination of diapause
574 Neocalanus flemingeri females emerged from diapause immediately after collection in late June
575 and September. Thus, they do not appear to have a refractory period. In mosquitos, diapause
576 includes a refractory period in which mosquitos are insensitive to environmental stimuli and will
577 not emerge from diapause even when conditions are favorable (Denlinger and Armbruster,
578 2014). The stress of light, heat, and movement associated with the net collection effectively
579 terminated the state of diapause. Termination of diapause was evidenced by females changing
580 the positioning of the body out of a tent-like posture as well as by the presence of dividing cells
581 in reproductive structures within the first 24 hours after collection. The absence of a refractory
582 period may be a characteristic of diapause in the Calanidae family. Neocalanus plumchrus, C.
583 finmarchicus, and C. helgolandicus all have been described transitioning out of the tent-like
584 positioning and exhibiting escape responses within hours after collection (Campbell et al., 2004;
585 Hirche, 1983).
586
587 By the late June collection date (June 30th, 2019), N. flemingeri have been declining in
588 abundance in the surface waters of Prince William Sound for roughly a month (Cooney et al.,
589 2001). The amount of time N. flemingeri spend mating while at depth before transitioning into
590 diapause is not clear, collections of deep-water copepods in the subarctic requires deep-water
591 sampling which restricts the number of sampling dates that can be obtained. However, since
592 females in this study were collected while diapausing in June, the June collection date was likely
20 593 early in the females’ diapause. At Ocean Station P (~600 nautical miles south of Prince William
594 Sound) the majority of N. flemingeri females were actively spawning in late January, suggesting
595 that the September collection dates (September 12th and 13th, 2019) were towards the middle of
596 diapause (Miller and Clemons, 1988). The two collection months likely cover both the start and
597 middle of when N. flemingeri females are at depth and presumably diapausing. If a refractory
598 period was occurring, one or both of these collection months would have likely captured it.
599
600 In insects a refractory period prevents early termination of diapause. If an organism’s cue to
601 terminate diapause is a warmer temperature, then a period of unseasonably warm weather in
602 winter could cause the organism to awaken when conditions are still unfavorable. In contrast, N.
603 flemingeri spend diapause at depths of 250-2000 m; light does not penetrate to those depths and
604 the water temperature stays nearly constant year-round (Miller and Clemons, 1988). There is no
605 need for a refractory period that protects against external factors if there is little variability in
606 these external factors. While there are many hypotheses about the possible stimuli for the
607 emergence of diapause in calanoid copepods, currently there is no clear conclusion for what
608 terminates diapause (Baumgartner and Tarrant, 2017). In N. plumchrus, it has been proposed that
609 termination from diapause is not stimulated by external factors but by an internal clock that ends
610 diapause after an amount of time has passed (Campbell et al., 2004). In C. finmarchicus it has
611 been suggested that diapause is terminated in response to reaching a lipid threshold (Johnson et
612 al., 2008). A model of C. finmarchicus predicted that if lipid loss triggered termination of
613 diapause, then individuals would end diapause earlier in warmer climates (Pierson et al., 2013).
614
615 Description of cell division pattern
21 616 One of the most surprising results of the study was the evidence of cell division in the ovary
617 within the first 24 hours after collection in all females tested. The finer time increments in the
618 Pleiades/September experiments also suggested that cell division in some females starts as early
619 as 6 hours post-collection. These results indicate that the reproductive program in females began
620 within a matter of hours after the diapause stimulus was given (i.e. collection stress). It would be
621 advantageous for females to allocate the most amount of energy as possible to the production of
622 eggs since females will only produce 1-6 clutches before death (Saito and Tsuda, 2000).
623
624 By minimizing the time between the end of diapause and the start of oogenesis, females
625 maximize the resources given to egg production. While oogonia are the precursor to oocytes, in
626 most images with a clearly defined germinative zone, the co-occurrence of EdU incorporation in
627 both locations of oogonia and oocytes suggests that the transition from oogonia to oocyte is
628 likely to be quick. In my experiments, I only observed a few females 24-48 hours after collection
629 with cell division primarily in the synapsis zone (meiosis). The results raise the possibility that
630 females may enter diapause with some oocytes already within the ovary. In C. finmarchicus that
631 have transitioned immediately from stage C5 copepodites to C6 females without diapausing, C5
632 copepodites were found with oocytes within the ovary (Niehoff and Hirche, 1996). However, C.
633 finmarchicus have different life histories that are not easily transferrable to the adult diapausing
634 N. flemingeri. More physiological research on N. flemingeri just prior to the initiation of
635 diapause would need to be done to elucidate this.
636
637 While oocytes and oogonia were thought to be in the ovary based on location, in addition total
638 fecundity of N. flemingeri females and numbers of dividing cells in the ovary would be
22 639 dissimilar if only oogonia or only oocytes were present (Hilton, 1931). Neocalanus flemingeri
640 have been recorded producing a lifetime mean fecundity of 924 (S.E. = +/- 346) and 535 (S.D. =
641 +/- 258) eggs (Saito and Tsuda, 2000; Slater, 2004). Discrepancies between fecundities was
642 likely due to Saito and Tsuda (2000) calculating fecundity with only females who produced four
643 to six clutches. Slater (2004) included all females (n = 35) in her calculations of total fecundity
644 including those who only produces one to four clutches. Maximum fecundity reported by the two
645 studies were both over 1,000 eggs (Saito and Tsuda (2000) = 1398; Slater (2004) = 1036 eggs).
646 On average, females were seen with over 100 dividing cells in the ovary within a 24 hour period.
647 If a female has an estimated 100 new dividing cells every day over the 21 days that cell division
648 was seen (1 – 21 days), she would produce over 2,000 egg cells. Assuming that half of cell
649 division can be attributed to oogonia and the other half to oocytes, a female would produce over
650 1,000 egg cells which is similar to the max recorded fecundity in both studies (Saito and Tsuda,
651 2000; Slater, 2004). Females might also undergo multiple mitotic divisions of oogonia, lowering
652 the final predicted number of egg cells further if these divisions were counted in this study.
653 While the exact location of oogonia and oocytes in the ovary was an estimate, it is likely that
654 both are present and dividing.
655
656 Females were highly synchronized not just in the initiation of oogenesis but throughout early
657 oogenesis. In both collections, cell division in females ramped up quickly and by 14 days after
658 collection were producing their maximum number of oocytes as evidenced by the high
659 magnitude of cell division. Increase in cell division agrees with gene expression data from
660 females one week after collection in which genes related to the cycle of cell division, cyclins,
661 were upregulated (Roncalli et al., 2020). Ramping up of cell division shortly after the start of
23 662 oogenesis would maximize egg production by increasing the duration of time where the ovary is
663 highly productive in forming oocytes. At 21 days post-collection cell division within the ovary
664 decreased in both collections. Innexin-2, a gene required for early germ cell processes, was
665 sharply downregulated at three weeks post-collection in N. flemingeri females (Roncalli et al.,
666 2018). In a clustering analysis of transcripts from N. flemingeri females, copepods from weeks
667 one to three were clustered separately from week zero females (diapausing) and week four
668 females (Roncalli et al., 2020). Both studies suggest that three weeks after collection is the
669 beginning of a significant transition that includes changes in gene expression and the end of
670 oocyte production.
671
672 By four weeks, all signs of cell division in the ovary had ceased. All oogonia and oocytes tracked
673 with EdU would be considered in OS0 (Niehoff, 2007). Neocalanus flemingeri females in
674 contrast with C. finmarchicus no longer had OS0 oocytes present in the ovary in gonad stage 1, a
675 stage described as no oocytes macroscopically seen in oviducts (Niehoff and Hirche, 1996).
676 Calanus finmarchicus which has a Calanus-type gonad, had OS0 oocytes within all gonad stages
677 (Niehoff and Hirche, 1996). The synchronous increase then decrease of new OS0 oocytes
678 combined with the sequential up- and down-regulation of oogenesis genes before the first clutch
679 is released suggests that for N. flemingeri the reproductive program progresses in sequence
680 (Roncalli et al., 2018). Females might be at a disadvantage if they started creating oocytes in the
681 ovary that were unable to complete maturation, potentially reducing their fitness.
682
683 Factors influencing cell division
24 684 Both longer prosome lengths and lipid stores are correlated with egg production in diapausing
685 calanoid copepods (Halvorsen, 2015; Halsband and Hirche, 2001; Runge, 1984). While prosome
686 length and lipid content are interlinked, these two variables are fundamentally different
687 measurements that reflect differences in growth conditions. While N. flemingeri are capable of
688 accumulating lipid as early as the C2 copepodite stage, lipid is mostly a representation of food
689 availability during the C5 copepodite stage (Tsuda et al., 2001). In contrast, prosome length is
690 affected by temperature and food conditions during all copepodite stages (Campbell et al., 2001).
691 In C. finmarchicus prosome lengths were longer during all copepodite stages (C1 to C5) when
692 raised with high food availability at a consistent temperature of 8 ̊C; females on average in the
693 high food environment were 2.8 mm while females in the low food environment were 2.3 mm
694 (Campbell et al., 2001). Temperature also influences body size with copepods in general having
695 smaller overall body sizes in warmer weather due to decreased stage durations (Pershing and
696 Stamieszkin, 2019). Campbell et al. (2001) described the body size of C. finmarchicus to be
697 negatively correlated to temperature.
698
699 Because Pleiades/September females had both lower numbers of dividing cells and less total
700 lipid, it is possible that smaller lipid stores are a factor in cell division due to less energy being
701 available for egg development. A positive relationship between egg production and both
702 prosome length and lipid content has been reported for Calanus hyperboreus, a copepod with the
703 ability to produce eggs over an entire spawning period fueled by internal energy reserves
704 (Halvorsen, 2015; Hirche, 2013). Results for N. flemingeri females one day after collection were
705 consistent with Halvorsen (2015). However, sampling sizes in this study were small (24-48
706 hours; n = 5), and there was no positive correlation at three days. More data is needed to fully
25 707 understand the relationship between cell division, prosome length, and lipid content in N.
708 flemingeri.
709
710 My data suggests that cell division in the ovary in N. flemingeri females is regulated to match
711 resources available to the individual. This is based on several observations. Females with both
712 high and low amounts of lipid content within this study were predicted to have sufficient lipids to
713 complete their reproductive program based on lipid use rates. Correlations were seen between
714 female size and number of dividing cells in the ovary. Both female to female variation in size
715 and number of dividing cells in the ovary were high and consistent with the large coefficient of
716 variation reported in total fecundity of N. flemingeri (Slater, 2004; coefficient of variation =
717 48%). By controlling the number of eggs being produced based on internal energy stores,
718 females would minimize the number of eggs that are unable to be completed or spawned.
719
720 While creation of oocytes is likely metered based on a female’s individual resources; females
721 within a population have reproductive programs that are highly synchronized and independent of
722 female size. Cell division and therefore the production of new oocytes was limited to the first
723 three to four weeks after the termination of diapause. The set end date of new egg production
724 remains the same regardless of lipid or prosome length which could be a mechanism to prevent
725 desynchronization of the population. At Ocean Station P, active spawning of females is
726 concentrated to late January (Miller and Clemons, 1988). The success of N. flemingeri may be
727 dependent on a highly synchronized life cycle. If a mismatch in peak abundance of copepodites
728 and nutrient rich phytoplankton occurs due to copepodites being too early or late, copepodites
729 may suffer food limitations. The first four copepodite stages of N. flemingeri in the northern Gulf
26 730 of Alaska are consolidated into three months, March through May (Liu and Hopcroft, 2006).
731 Neocalanus plumchrus appear in the surface waters after N. flemingeri have descended, late N.
732 flemingeri individuals would mean increased competition with N. plumchrus; it is beneficial to
733 N. flemingeri to be highly synchronized more than other Neocalanus spp. (Saito and Tsuda,
734 2000).
735
736 Cell division outside of the ovary and oviducts
737 Somatic cell division in adult stage copepods from the cyclopoid and calanoid family has been
738 described to be uncommon or non-existent (Rasch and Wyngaard, 2008). The exception to this is
739 a minority of somatic cells that undergo endopolyploidy which duplicates DNA, but no mitosis
740 occurs (Rasch and Wyngaard, 2008). However, the statement that no somatic cell division is
741 present originates from literature that analyzed chromosomes and DNA content with no
742 experimental studies that clearly demonstrated a lack of somatic cell division (Lécher et al.,
743 1995). Copepoda do undergo chromatin-diminution which is the loss of DNA during
744 development, but it is not clear if chromatin-diminution would impact an adult copepod (Lécher
745 et al., 1995). To our knowledge, this is the first study using EdU on copepods. Because of a lack
746 of research in this area, it is difficult to identify the origin of dividing cells outside of the ovary
747 and oviducts. However, DNA replication is present across the body.
748
749 Environmental context
750 From 1970 to 2006, waters of an inshore station in the Northern Gulf of Alaska increased in
751 year-round temperature by more than 0.8 ̊C within the surface 250 m (Royer and Grosch, 2006).
752 Starting in late 2013, a mass of warm water known as “The Blob” was present off the Gulf of
27 753 Alaska (Kintisch, 2015). From 2013 through 2019 the water temperatures in the same inshore
754 station have been atypically warm both at the surface and below 200 m (Danielson et al. 2020).
755 Warming waters associated with climate change have been hypothesized to influence not just N.
756 flemingeri but many other diapausing copepods by negatively impacting reproductive success
757 and decreasing diapause length (Baumgartner and Tarrant, 2017; Pierson et al., 2013).
758
759 Body size of a diapausing copepod is dependent on temperature and food availability (Campbell
760 et al., 2001). In general, the same species of copepod will have a smaller body size in warmer
761 temperatures due to decreased life stage durations (Pershing and Stamieszkin, 2019). A smaller
762 overall body size in N. flemingeri would mean smaller lipid sacs, which could translate to less
763 energy being available to fuel oogenesis. In this study, there was some evidence that females
764 with smaller lipid sacs and prosome lengths had smaller numbers of dividing oogonia and
765 oocytes, but this needs to be confirmed with larger sample sizes.
766
767 Warmer temperature could affect the quantity and the quality of the copepod’s food. On a global
768 scale, increasing temperatures tend to shift phytoplankton assemblages towards smaller size
769 classes (Mousing et al., 2014). When available, N. flemingeri preferentially feed on plankton
770 larger than 20 μm (Dagg et al., 2006). During the spring bloom in Prince William Sound, 80% of
771 chlorophyll is contributed by phytoplankton larger than 20 μm (Ward, 1997). If phytoplankton
772 assemblages during the spring bloom shift towards smaller size classes or if there is a trophic
773 mismatch between large phytoplankton and N. flemingeri copepodites during the growth period,
774 females’ energy reserves could be smaller, which could reduce total fecundity.
775
28 776 Diapausing metabolic rates of copepods are temperature sensitive which suggests that diapause
777 duration could be impacted by warming waters (Pierson et al., 2013). Over the next several
778 decades, it has been projected that C. finmarchicus might decrease the duration of diapause by
779 over a month if an endogenous lipid-mediated timer is the cue to end diapause (Pierson et al.
780 2013). Calanus finmarchicus must terminate diapause earlier in the year when phytoplankton
781 abundances might be non-optimal (Pierson et al. 2013). Without any similar studies focused on
782 N. flemingeri there are no predictions for how the duration of diapause might change with global
783 warming in this species. While this study cannot predict how diapause duration will change in N.
784 flemingeri, my results demonstrate that females within the same year cohort are highly
785 synchronized independent of size or lipid content. Thus, N. flemingeri females may not utilize an
786 endogenous lipid cue since variations in female size and lipid reserves would be expected to
787 desynchronize the population. It is unclear if the timing and duration of cell division in the ovary
788 (24 hours - 3 weeks) will remain the same if the diapause duration is shortened, but it seems
789 likely that females will stay synchronized within their year cohort since females will have
790 experienced similar diapause temperatures.
791
792 Diapause in N. flemingeri remains understudied due to the species’ relatively recent discovery
793 (1988), and difficulty in collection compared with terrestrial arthropods. Diapausing individuals
794 require nets and ship infrastructure that can handle deep water sampling. In the Gulf of Alaska,
795 the one-year life cycle of N. flemingeri designates that stage specific studies are limited to
796 specific times of year. However, with the advent of large research vessels like the R/V Sikuliaq
797 with laboratory infrastructure, time sensitive cellular and molecular studies in combination with
798 shipboard experiments on diapausing copepods have been made more possible in recent years.
29 799 This study combined both a molecular and physiological approach via cell division staining and
800 female size measurements to better understand the process of oogenesis within N. flemingeri. An
801 experimental design using the integration of multiple measurements on the same live individual
802 provided complementary forms of context to a physiological process.
803
804 Diapause and oogenesis in capital breeder copepods is a subject rich in potential future studies.
805 While described in C. hyperboreus, a better understanding of the correlation between egg
806 production, lipid content, and prosome length in N. flemingeri is required to better understand the
807 relationship between female size and reproductive potential. In addition, an estimate of the
808 minimum threshold of lipid content needed for successful completion of diapause and egg
809 production would be useful in predicting reproductive success in females. With a previous
810 reference to lipid size and prosome length being representative of different life stages, teasing
811 apart which aspect of female size is more influential over reproductive potential is essential to
812 understanding which developmental stages might be the most vulnerable to climate change. For
813 predictions to be made about change in diapause duration as waters warm, studies are needed
814 focusing on metabolic rates of diapausing N. flemingeri individuals at different temperatures as
815 well as empirical evidence of the mechanisms that trigger the termination of diapause. Lastly, a
816 future extension of this research would be to continue EdU experiments on N. flemingeri females
817 from subsequent years and to collect females closer to their in situ start of oogenesis to examine
818 if the patterns of cell division are consistent with what was found in 2019.
819
820 References
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822 Marine Copepods. Annu. Rev. Mar. Sci. 9, 387-411.
823
824 Benton, J. L., Kery, R., Li, J., Noonin, C., Söderhäll, I. and Beltz, B. S. (2014). Cells from
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828 Beltz, B. S., Zhang, Y., Benton, J. L. and Sandeman, D. C. (2011). Adult neurogenesis in the
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830 Neuroscience. 34, 870-883.
831
832 Blades-Eckelbarger, P. I. and Youngbluth, M. J. (1984). The Ultrastructure of Oogenesis and
833 Yolk Formation in Labidocera aestiva (Copepoda: Calanoida). J Morphol. 179, 33-46.
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835 Buck, S. B., Bradford, J., Gee, K. R., Agnew, B. J., Clarke, S. T. and Salic, A. (2018).
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839
840 Campbell, R. G., Wagner, M. M., Teegarden, G. J., Bourdeau, C. A. and Durbin, E. G.
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842 laboratory. MEPS. 221, 161-183.
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31 844 Campbell, R. W., Boutillier, P. and Dower, J. F. (2004). Ecophysiology of overwintering in
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846 cycle. MEPS. 280, 211-226.
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848 Checkley, D. M. (1980) The egg production of a marine planktonic copepod in relation to its
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851 Cooney, R. T. (1986). The seasonal occurrence of Neocalanus cristatus, Neocalanus plumchrus,
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859 Coyle, K.O. and Pinchuk, A.I. (2003). Annual cycle of zooplankton abundance, biomass and
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863 Dagg, M. J., Liu, H., Thomas, A. C. (2006). Effects of mesoscale phytoplankton variability on
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873 Eckelbarger, K. J. and Blades-Eckelbarger, P. I. (2005). Oogenesis in calanoid copepods.
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876 Halsband, C. and Hirche, H. J. (2001). Reproductive cycles of dominant calanoid copepods in
877 the North Sea. MEPS. 209, 219-229.
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879 Halvorsen, E. (2015). Significance of lipid storage levels for reproductive output in the Arctic
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882 Hilton I. F. (1931). The Oogensis of Calanus finmarchicus. The Quarterly Journal of
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885 Hirche, H. J. (1983). Overwintering of Calanus finmarchicus and Calanus helgolandicus.
886 MEPS. 11, 281-290.
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888 Hirche, H. J. (1989). Egg production of the Arctic copepod Calanus glacialis: laboratory
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33 890
891 Hirche, H. J. (2013). Long-term experiments on lifespan, reproductive activity and timing of
892 reproductive activity and timing of reproduction in the Arctic copepod Calanus
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894
895 Ikeda, T., Sano, F., Yamaguchi, A. (2004). Metabolism and body composition of a copepod
896 (Neocalanus cristatus: Crustacea) from the bathypelagic zone of the Oyashio region,
897 western subarctic Pacific. Marine Biology. 145, 1181-1190.
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899 Johnson, C. L., Leising, A. W., Runge, J. A., Head, E. J. H., Pepin, P., Ploudre, S., Durbin,
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902
903 Kintisch, E. (2015). ‘The Blob’ invades Pacific, flummoxing climate experts. Science. 348, 17-
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906 Lécher, P., Defaye, D. and Noel, P. (1995). Chromosomes and nuclear DNA of Crustacea.
907 Invertebrate Reproduction and Development. 27, 85-114.
908
909 Lenz, P. H. and Roncalli, V. (2019). Diapause within the Context of Life-History Strategies in
910 Calanid Copepods (Calanoida: Crustacea). The Biological Bulletin. 237, 170-179.
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34 912 Liu, H. and Hopcroft, R. R. (2006). Growth and development of Neocalanus
913 flemingeri/plumchrus in the northern Gulf of Alaska: validation of the artificial-cohort
914 method in cold waters. J Plankton Res. 28, 87-101.
915
916 Miller, C. B. (1988). Neocalanus flemingeri, a new species of Calanidae (Copepoda: Calanoida)
917 from the subarctic Pacific Ocean, with a comparative redescription of Neocalanus
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919
920 Miller, C. B. and Clemons, M. A. (1988). Revised life history analysis for large grazing
921 copepods in the subarctic Pacific Ocean. Progress in Oceanography. 20, 293-313.
922
923 Miller, C. B., Crain, J. A. and Morgan, C. A. (2000). Oil storage variability in Calanus
924 finmarchicus. ICES Journal of Marine Science. 57, 1786-1799.
925
926 Mousing, E. A., Ellegaard, M., Richardson, K. (2014). Global patterns in phytoplankton
927 community size structure-evidence for a direct temperature effect. MEPS. 497, 25-38.
928
929 Niebauer, H. J., Royer, T. C. and Weingartner, T. J. (1994). Circulation of Prince William
930 Sound, Alaska. Journal of Geophysical Research. 99, 14113-14126.
931
932 Niehoff, B. (2007). Life history strategies in zooplankton communities: The significance of
933 female gonad morphology and maturation types for the reproductive biology of marine
934 calanoid copepods. Progress in Oceanography. 74, 1-47.
35 935
936 Niehoff, B. and Hirche, H. J. (1996). Oogenesis and gonad maturation in the copepod Calanus
937 finmarchicus and the prediction of egg production from preserved samples. Polar Biology.
938 16, 601-612.
939
940 Pershing, A. J. and Stamieszkin, K. (2020). The North Atlantic Ecosystem, from Plankton to
941 Whales. Annual Review of Marine Science. 12, 339-359.
942
943 Pierson, J. L., Batchelder, H., Saumweber, W., Leising, A., Runge, J. (2013). The impact of
944 increasing temperatures on dormancy duration in Calanus finmarchicus. Journal of
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946
947 Ragland, G. J., Fuller, J., Feder, J. L., Hahn, D. A. (2009). Biphasic metabolic rate trajectory
948 of pupal diapause termination and post-diapause development in a tephritid fly. Journal of
949 Insect Physiology. 55, 344-350.
950
951 Rasch E. M. and Wyngaard G. A. (2008). Endopolyploidy in Cyclopoid Copepods. Journal of
952 Crustacean Biology. 28, 412-416.
953
954 Roncalli, V., Sommer, S. A., Cieslak, M. C., Clarke, C., Hopcroft, R. R., and Lenz, P. H.
955 (2018). Physiological characterization of the emergence from diapause: A transcriptomics
956 approach. Scientific Reports. 8, 12577.
957
36 958 Roncalli, V., Cieslak, M. C., Hopcroft, R. R. and Lenz, P. H. (2020). Capital Breeding in a
959 Diapausing Copepod: A Transcriptomics Analysis. Front. Mar. Sci. 7.
960
961 Royer, T. C., Grosch, C. E. (2006). Ocean warming and freshening in the northern Gulf of
962 Alaska. Geophysical Research Letters. 33.
963
964 Runge, J. A. (1984). Egg production of the marine, planktonic copepod, Calanus pacificus
965 Brodsky: laboratory observations. J. Exp. Mar. Biol. Ecol. 74, 53-66.
966
967 Saito, S. and Tsuda, A. (2000). Egg production and early development of the subarctic
968 copepods Neocalanus cristatus, N. plumchrus, and N. flemingeri. Deep-Sea Research 1. 47,
969 2141-2158.
970
971 Schmid, M. S., Maps, F. and Fortier, L. (2018). Lipid load triggers migratin to diapause in
972 Arctic Calanus copepods-insights from underwater imaging. J Plankton Res. 40, 311-325.
973
974 Skottene, E., Tarrant, A. M., Olsen, A. J., Altin, D., Østensen, M. A., Hansen, B. H.,
975 Choquet, M., Jenssen, B. M. and Olsen, R. E. (2019). The 훽-oxidation pathway is
976 downregulated during diapause termination in Calanus copepods. Scientific Reports. 9,
977 16686.
978
979 Slater, L. (2004). Development, Growth, and Egg Production of Centropages abdominalis and
980 Neocalanus flemingeri from the Eastern Subarctic Pacific (Unpublished master’s thesis).
37 981 University of Alaska Fairbanks. Fairbanks, Alaska.
982
983 Tsuda, A., Saito, H. and Kasai, H. (2001). Life history strategies of subarctic copepods
984 Neocalanus flemingeri and N. plumchrus, especially concerning lipid accumulation patterns.
985 Plankton Biol. and Ecol. 48, 52-58.
986
987 Tsuda, A., Saito, H. and Kasai, H. (2001). Geographical Variation of Body Size of Neocalanus
988 cristatus, N. plumchrus, and N. flemingeri in the Subarctic Pacific and Its Marginal Sea:
989 Implication for the Origin of Large Form of N. flemingeri in the Oyashio Area. Journal of
990 Oceanography. 57, 341-352
991
992 Vogedes, D., Varpe, Ø., Søreide, J. E., Graeve, M., Berge, J. and Falk-Peterson, S. (2010).
993 Lipid sac area as a proxy for individual lipid content of arctic calanoid copepods. J Plankton
994 Res. 32, 1471-1477.
995
996 Ward, A. (1997). A temporal study of the phytoplankton spring bloom in Prince William Sound,
997 Alaska (Unpublished master’s thesis). University of Alaska Fairbanks. Fairbanks, Alaska.
998
999
1000
1001
1002
1003
38 1004 List of tables
1005
1006 Table 1: Summary of Neocalanus flemingeri experiments completed in the Summer (PWS2,
1007 June collection) and Fall (PWS2 and Pleiades, September collections) of 2019. Start time and
1008 duration of EdU incubations are listed in first two columns. Experimental start time is given
1009 relative to collection, starting within two hours after net retrieval. EdU incubations were 24
1010 hours, except for the first set of PWS2/September experiments (first three rows). Values indicate
1011 number of individuals incubated in EdU and light imaged prior to preservation (EdU columns) or
1012 just imaged through a light microscope (light imaging only columns). In some cases, females
1013 incubated in EdU could not be light imaged before preservation and these are indicated by an
1014 asterisk (*).
1015
1016
1017
1018
1019
1020
39 1021 List of charts, graphs, figures, illustrations, plates, maps
1022
1023 Figure 1. Diagram of life cycle of N. flemingeri in the Gulf of Alaska. Cartoon drawing of
1024 nauplii, copepodites, and adults (non-feeding) shown when these stages are found in the water
1025 column (figure taken from Lenz with permission). Months are listed on the bottom starting with
1026 November, and vertical depths are labeled in diagram. Life cycle based on descriptions in Miller
1027 and Clemons (1988), Coyle and Pinchuk (2003), and Liu and Hopcroft (2006).
1028
1029
1030
1031
1032
1033
40 1034
1035 Figure 2: Diagram of location and structure of ovary and oviducts in N. flemingeri. General
1036 path of egg maturation in a Calanus-type gonad shown in blue arrows as described in Niehoff
1037 (2007). Maturing oocytes begin in the posterior end of the ovary and move anteriorly through the
1038 ovary. Oocytes accumulating yolk are found across both anterior and posterior oviducts (also
1039 referred to as diverticula) with the most mature oocytes present ventrally in the oviducts. When
1040 spawning occurs, oocytes move towards the posterior oviduct and are released through the
1041 gonopore after fertilization. Blue dotted lines are a schematic drawing of the oviducts and
1042 diverticula in a Calanus-type gonad modified from Niehoff (2007). Red rectangle indicates
1043 approximate confocal imaging location and grey outline shows ovary and anterior oviducts as
1044 seen in the confocal. Diagram shows a female with no developing oocytes. Ovary and oviducts
1045 expand as egg cells develop.
1046
1047
1048
41 1049
1050 Figure 3: Modified diagram of a section through thorax of C. finmarchicus showing ovary
1051 and oviducts from Hilton (1931). Arrows point to locations where cells are dividing;
1052 mitotically dividing oogonia (m: multiplication zone), and oocytes beginning prophase of
1053 meiosis 1 (s: synapsis zone).
1054
1055
1056
1057
42 1058
1059 Figure 4: Map of Prince William Sound, Alaska with sampling sites for N. flemingeri
1060 collections. Samples were collected at PWS2 (June and September: Latitude 60° 32.1'N;
1061 Longitude 147° 48.2'W) and Pleiades (September only: Latitude 60° 16.7'N; Longitude 147°
1062 59.2'W) sampling sites. Latitude lines (light blue) are 0.5 degrees.
1063
1064
1065
1066
1067
1068
1069
43 1070
1071 Figure 5: Light microscope images of N. flemingeri females. (A) Female collected from
1072 PWS2 in Prince William Sound, Alaska (Latitude 60° 32.1'N; Longitude 147° 48.2'W); light
1073 microscope images were used to measure prosome length (red dashed line), lipid sac area (blue
1074 dashed line contour), and prosome area (green dashed line contour) with ImageJ. Lipid sac and
1075 prosome area were used in total lipid content in mg and lipid fullness percentage calculations.
1076 Pictured female: prosome length = 4 mm, prosome area = 4 mm2, lipid area = 2 mm2, computed
1077 lipid content = 0.51 mg. (B) Female collected from Pleiades (Latitude 60° 16.7'N; Longitude
1078 147° 59.2'W); arrows point to indicators used in sorting. Clear ovary; no signs of oogenesis,
1079 opaque spermathecae: mating has occurred. Pictured female: prosome length = 3.8 mm, lipid
1080 content = 0.34 mg. Images were taken three days after collection. Microscope magnification:
1081 × 32, scale bars are 1000 µm.
1082
1083
1084
1085
1086
44 1087
1088 Figure 6: Histograms showing the distribution in prosome length in mm (A) and initial
1089 lipid fullness percentage (B) between sampling sites. (A) Prosome length was directly
1090 measured from light microscope images of experimental females (PWS2: grey bars, n = 168;
1091 Pleiades: black bars, n = 36; Table 1). Pleiades females (mean: 3.7 mm, s.d. = 0.29 mm) were
1092 significantly smaller than PWS2 females (3.9 mm, s.d. = 0.21 mm; two sample t-test, p < .001,
lipid sac area 1093 t(43) = 4.181). (B) Median initial lipid fullness ( × 100) was calculated using prosome sac area
1094 measurements from light microscope images. Initial lipid fullness used females one day after
1095 collection from PWS2 (n = 21) and Pleiades (n = 13). Initial lipid fullness was slightly lower at
1096 Pleiades (median = 45 %, IQR = 8%) compared with PWS2 (median = 46 %, IQR = 8%), but the
1097 difference was not significant (Wilcoxon Rank Sum Test, W = 3381, p = 0.267).
45 1098
1099 Figure 7: Scatterplot between prosome length in mm and lipid content in mg for
1100 PWS2/June (grey triangles, n = 168) and Pleiades/September (black circles, n = 36)
1101 collections. Female prosome length was measured directly from light microscope images. Lipid
1102 content was calculated from measured lipid sac area using Vogedes et al. (2010) equation:
1103 TL=0.197A1.38 where A is measured lipid sac area and TL is total lipid content in mg. Trendlines
1104 are linear regression lines, statistics are reported in figure (grey box: PWS2, black box: Pleiades).
1105
1106
46 1107 1108 Figure 8: Females’ lipid contents at different times post-collection. (A) Scatterplot of females
1109 from the PWS2/June collection over the 32 day experimental timeline. Blue line shows a fitted
1110 regression line. (B) Boxplot of lipid content of females both from Pleiades/September (black)
1111 and PWS2/June over the first seven days post-collection.
1112
47 1113
1114 Figure 9: Maximum Intensity Projections (MIP) of ovaries of females incubated in EdU
1115 showing a time series from immediately after collection to four weeks post-collection.
48 1116 Images were created using a MIP of merged confocal z-stacks where the brightest voxels over a
1117 specified depth are consolidated into one image. All females were collected from the PWS2
1118 sampling site. Red: EdU stained cells, blue: DAPI stained cells. In images, cephalosome is to the
1119 left; urosome is to the right. White outline used to emphasize ovary; ov: ovary, od: oviduct. (A)
1120 Female was incubated in EdU for three hours directly after collection; Image is a 75 µm
1121 projection, number of dividing cells = 0. Arrows point to the approximate locations of the
1122 multiplication/germinative zone (dividing oogonia) (right arrows) and the synapsis zone
1123 (dividing oocytes) (left arrows). (B) Female was incubated in EdU for 24 hours directly after
1124 collection. Image is a 104 µm projection, number of dividing cells = 70. (C) Female was
1125 incubated in EdU for 24 hours at three days after net collection. Image is a 63 µm projection;
1126 number of dividing cells = 299. **: image doubled due to tile merge artifact. (D) Female was
1127 incubated in EdU for 24 hours at three weeks after net collection. Image is a 36 µm projection;
1128 number of dividing cells = 138. (E) Female was incubated in EdU for 24 hours at four weeks
1129 after collection. Image is a 10 µm projection; number of dividing cells = 0. Dotted line indicates
1130 shape of ovary not viewable in MIP image. Images were taken at × 20 magnification, scale bars
1131 are 100 µm.
1132
1133
49 1134
1135 Figure 10: MIP of merged confocal z-stacks of ovaries 24-48 hours after collection at PWS2
1136 (A) (June) and Pleiades (B) sampling sites. (A) Image is a 15 µm projection; number of
1137 dividing cells = 111. (B) Image is a 24 µm projection; number of dividing cells = 52. Red: EdU
1138 stained cells, blue: DAPI stained cells. Arrows point to the approximate locations of the
1139 multiplication/germinative zone (dividing oogonia) (right arrows) and the synapsis zone
1140 (dividing oocytes) (left arrows); ov: ovary, c: cuticle segment. In images, cephalosome is to the
1141 left; urosome is to the right. Images were taken at × 20 magnification, scale bars are 100 µm.
1142
1143
50 1144
1145 Figure 11: MIP of merged confocal z-stacks of ovaries at 0-24 hours after collection in
1146 PWS2 (A) (June) and Pleiades (B) sampling sites. (A) Image is a 104 µm projection, number
1147 of dividing cells = 70. (B) Image is a 53 µm projection, number of dividing cells = 27. Red: EdU
1148 stained cells, blue: DAPI stained cells. Images were taken at × 20 magnification; ov: ovary, od:
1149 oviduct. In images, cephalosome is to the left; urosome is to the right. Scale bars are 100 µm.
1150
1151
1152
1153
1154
1155
1156
1157
1158
1159
51 1160
1161 Figure 12: Description of EdU incorporation in dividing cells within reproductive
1162 structures at different times post-collection. (A) Each data point represents mean number of
1163 dividing cells in the ovary and oviducts of two to six females at each time point. EdU-stained
1164 cells were counted using confocal z-stacks. Error bars are standard deviation. (B) Each data point
1165 represents normalized cell division computed as the mean number of EdU-stained cells at a time
1166 point divided by that experiment’s mean number of cell division at its peak time point
1167 (PWS2/June: 72 hours, 214.2 cells, dashed line; Pleiades/September: 14 days, 157.2 cells, solid
1168 line). Note difference in x-axis between A and B; B only shows time points shared between late
1169 June and September. (A, B) Pleiades data includes also includes females collected from PWS2 in
1170 September (Table 1). (C) Diagram summarizing pattern of EdU incorporation in dividing within
1171 the ovary of N. flemingeri post-collection with important time points listed in bar along bottom.
52 1172
1173 Figure 13: Scatterplots comparing cell division in the ovary and oviducts with lipid content
1174 and prosome length at one day after collection. Females with both cell counts (confocal
1175 imaging) and body measurements (light microscope imaging) taken were used. Females were
1176 from both the PWS2/June and the Pleiades/September sampling sites. All correlations are
1177 Spearman’s rank correlations.
1178
1179
1180
1181
53 1182
1183 Figure 14: 150 µm confocal projection of whole-body female (PWS2) 72 hours after
1184 collection. Prosome length = 3.7 mm, lipid content = 0.41 mg, female is the same copepod from
1185 Figure 9c. White: EdU stained cells. Image was taken at × 10 magnification, z-stack step size
1186 was 4 µm. Scale bar was 1000 µm.
1187
1188
1189
1190
1191
1192
1193
1194
1195
1196
1197
1198
54 1199
1200 Figure 15: Confocal images of oocytes with DAPI staining (green cells) in oviducts four
1201 weeks from PWS2/June collection. Green false color used to highlight details. (A) Oocytes
1202 with condensed chromatin in the nucleus and the presence of a large nucleolus; Nu: nucleolus, N:
1203 nucleus, arrow: cell boundary. (B) Oocytes lined in paired oviducts. Arrows point to cell walls of
1204 the paired oviducts. Images were taken at × 63 magnification, scale bars are 10 µm.
1205
55