DOI: 10.5061/dryad.47d7wm3pj
title: Provisioning of vitellogenic follicles continues after green turtles arrive at the nesting beach

description: We analyzed the ovaries of 69 green turtles (Chelonia mydas) preyed upon
 by jaguars (Panthera onca) during the nesting season at Tortuguero, Costa
 Rica. Our findings revealed a bimodal distribution of the diameter of
 vitellogenic follicles. In addition to pre-vitellogenic follicles, ovaries
 contained larger "dominant" follicles that would be ovulated in
 the current nesting season and smaller "non-dominant" follicles
 that were destined for resorption. Small dominant follicles increased in
 diameter by up to 66% during the nesting season, indicating ongoing yolk
 deposition. Analysis of yolk composition showed that small dominant
 follicles had higher percent water content than large dominant follicles.
 Thus, dry matter deposition rather than hydration is responsible for the
 pre-ovulatory increase in diameter of green turtle dominant follicles
 during the nesting season. Furthermore, percentages of lipid, nitrogen
 (N), and phosphorus (P) were constant across green turtle vitellogenic
 follicles, which underscores that the increase in follicle size results
 from provisioning with yolk containing similar proportions of these
 nutrients. Atretic follicles had higher water and lower P percentages than
 dominant follicles, indicating an accelerated resorption of phosphorus
 over lipids and N, which could be due to the importance of this nutrient
 for eggshell production. Finally, the yolk available for resorption in the
 green turtle ovary during the nesting season was not enough to supply the
 energy needed for 83% of the studied females to complete vitellogenesis,
 which means they must either mobilize it from body stores or non-dominant
 follicles or acquire more energy through foraging. These insights into
 follicular dynamics and nutrient provisioning highlight the ongoing
 reproductive investments made by female green turtles at Tortuguero.
 Understanding these dynamics is crucial for devising effective
 conservation strategies aimed at supporting the reproductive health and
 resilience of green turtle populations in their critical nesting habitats.
description: <strong>Study Site</strong> At
 Tortuguero, on the Caribbean coast of Costa Rica (Figure 2), approximately
 100,000 green turtle (<em>Chelonia mydas</em>) nests are
 recorded (Bruno et al. 2020; Restrepo et al. 2023) and over 300 nesting
 green turtles are preyed upon by jaguars (<em>Panthera
 onca</em>) annually (Troëng 2000; Arroyo-Arce and Salom-Pérez 2016).
 Jaguars usually attack a sea turtle through its neck and pull the contents
 out of the body cavity with their paws, often leaving ovaries and oviducts
 undisturbed. Assisted by National Park rangers and
 members of the local community, we conducted necropsies of green turtles
 preyed upon by jaguars at Tortuguero less than 12 hours after the
 predation event. All activities were conducted under research permits
 (SINAC-ACTo-DIR-RES-057-2021, 013-2022, 025-2023).
 Jaguars eating sea turtles in Tortuguero may return to the kill
 for several days afterwards to feed (Arroyo-Arce and Salom-Pérez 2016).
 Therefore, we first identified a predation event from afar by the number
 of black vultures (<em>Coragyps atratus</em>) in the area.
 Upon finding a depredated green turtle, we monitored the site for 10
 minutes using binoculars to see if the jaguar was still present. If the
 jaguar was sighted (Figure 3), we left the area, if not we proceeded to
 conduct a necropsy of the turtle. We collected either one or two ovaries
 from each necropsied turtle and returned to the Tortuguero National Park’s
 “Cuatro Esquinas” headquarters where we proceeded with analyses.
 <strong>Size distribution of ovarian
 follicles</strong> To determine if all follicles
 were at maximum size when females arrived at the nesting beach, from 2021
 to 2023 we used a caliper to measure the maximum diameter of all
 vitellogenic follicles (more yellow and larger than pre-vitellogenic
 follicles). Additionally, we measured all recently formed ovulatory scars,
 which were evident on top of fluid-filled vesicles (see Figure 1). We also
 weighed each ovary to the nearest 500 grams and used a graduated cylinder
 to measure the volume of each ovary by water displacement to the nearest
 50 mL. In 2021, we divided one ovary of four females
 into four segments of similar volume according to the position in the body
 axis (anterior, mid-anterior, mid-posterior, and posterior) and counted
 follicles and scars separately in each segment (Figure S1). Finally, when
 possible, we counted the number of eggs in each oviduct.
 Using R package diptest (Maechler 2024), we used a Hartigan’s dip
 test to assess if the distribution of the diameters of green turtle
 vitellogenic follicles was bimodal. We also used two methods to determine
 the optimal number of clusters for k-means clustering: the elbow method
 (Syakur et al. 2018) and the silhouette method (Kodinariya and Makwana
 2013). We then performed k-mean clustering to further analyze the
 distribution of green turtle ovarian follicles. Based
 on results of the k-mean clustering, we classified follicles larger than
 18 mm as "dominant". Follicles smaller than 18mm in diameter
 were classified as “non-dominant” (see Results). We assumed dominant
 follicles would have been ovulated if the female green turtle had lived,
 and we assumed non-dominant follicles would have been resorbed by the end
 of the nesting season (Hodgen 1982; Etches and Petitte 1990; Fortune et
 al. 1991, 2001; Nielsen et al. 2016; Mayor et al. 2023). As follicles may
 undergo atresia at any stage of development (Price et al. 2016), some
 dominant follicles were probably resorbed by the end of the nesting
 season. The number of clutches remaining in a female
 that would have been laid in that season (clutches left) was calculated by
 counting the number of dominant follicles, whereas the number of clutches
 a female had already laid in the season (clutches laid) was calculated by
 counting the number of recently formed ovulatory scars. These numbers were
 then divided by 110, which is the average clutch size for green turtles in
 this population (Bjorndal and Carr 1989; Bruno 2019). Further, if we had
 only measured and counted follicles in one ovary, we assumed that both
 ovaries contained the same number of scars and follicles and multiplied
 the result by two to obtain the number of clutches laid and clutches left
 per female. We confirmed this assumption (see Results section).
 We classified female green turtles as being in the first or
 second half of their individual nesting season based on the proportions of
 clutches laid and clutches left. Females in the first half of their
 nesting season had fewer clutches laid than clutches left, while those in
 the second half had an equal or greater number of clutches laid compared
 to clutches left. Clutch frequency, defined as the number of clutches
 deposited by a turtle within a nesting season, was estimated by summing
 the number of clutches laid and clutches left.
 <strong>Nutrient analyses</strong>
 To understand what was being added to dominant follicles during
 the green turtle nesting season, in 2022 and 2023 we collected
 approximately 10 large and 10 small dominant follicles and a random sample
 of atretic follicles per female. We dried collected follicles at 60°C in a
 food dehydrator until a stable mass was achieved. We
 exported samples from Costa Rica under CITES permits (2021-CR5515/SJ,
 2022-CR5969, 2023-CR6500) and imported into the United States under CITES
 permits (20US72454/9, 21US72454/9, 23US72454/9). We processed samples in
 the laboratory of the Archie Carr Center for Sea Turtle Research at the
 University of Florida. We used a random number
 generator to select five large (LF) and five small (SF) dominant follicles
 per female. We also used all the dehydrated atretic follicles (AF). We
 ground each of the selected dried follicles to a uniform particle size
 using a mortar and pestle. We subsampled ground follicles and quantified
 lipid content by extraction with hexane in an Ankom system according to
 manufacturer instructions. Another subsample of the same follicles was
 used to quantify nitrogen and phosphorus content via a modified Kjeldahl
 method (Bouchard and Bjorndal 2000). Finally, using the remainder of the
 sample, we quantified organic and mineral content of the yolk by
 combusting the dried samples at 600°C for 8 hours. Throughout this study,
 we express water composition as a percentage of wet mass, while all other
 nutrient compositions are expressed as percentages of dry mass.
 Follicles from 12 females we used for water analyses had to be
 excluded from the nutrient analyses because samples were damaged during
 transport. Further, some of the females did not have atretic follicles,
 and sample mass of some follicles was not sufficient for all nutrient
 analyses. Therefore, number of females and follicles used per analysis
 varied; sample size and results of each analysis are in Tables 1 and
 2. <strong>Nutrient investment and
 resorption</strong> We measured the initial
 diameter of each vitellogenic follicle in the ovaries we studied, and we
 measured dry weight only for the follicles we used for nutrient analyses.
 Therefore, to understand how much yolk and energy had been invested and
 would be further invested into green turtle ovarian follicles during the
 nesting season, we first used a linear regression (Figure S2) to predict
 dry weight (g) of each follicle in the studied ovaries:
  The dry weight of each dominant follicle (predicted
 or measured) was taken as the yolk investment already made by the female.
 To estimate the additional yolk (in grams) that would be invested in each
 follicle during the nesting season, we subtracted the dry weight of each
 dominant follicle from the weight of the largest dominant follicle in the
 same ovary. The dry weight of all non-dominant follicles was considered as
 the yolk available for resorption by the female. We
 then calculated the total grams of yolk each female had deposited and
 would have deposited and resorbed throughout the nesting season. If
 follicles were measured and counted in both ovaries, we summed the yolk
 amounts from each ovary. If only one ovary was measured, we multiplied the
 yolk sum by two to estimate the total yolk investment. Finally, using the
 average values of lipid, nitrogen, and phosphorus per gram of dry yolk
 specific to each female, we determined the total grams of each nutrient
 that would be deposited into and resorbed from green turtle ovarian
 follicles during the nesting season at Tortuguero.
 <strong>Statistical analyses</strong>
 We used R program version 4.4.0 (R Core Team 2024) for all
 statistical analyses, and we generated plots with package ggplot2 (Wickham
 2016). We checked if data were parametric by using a Shapiro-Wilks test in
 base R to assess normal distribution and a Levene’s test or Bartlett’s
 test in package car (Weisberg 2019) to assess data
 homoscedasticity. We used a Welch’s T-test to
 investigate whether the number of dominant follicles varied significantly
 according to the stage of the nesting cycle each female green turtle was
 in (first or second half). We used a Wilcox test in package rstatix
 (Kassambra 2023) to test if the number of non-dominant follicles differed
 significantly between the two stages of the nesting cycle.
 We investigated whether nutrient content of yolk was different
 among follicle types (fixed effect) while blocking for the effect of
 female id (random effect) in a complete block design. Because of the much
 smaller sample sizes of AF, we first ran these models using all follicle
 types (AF, SF, and LF) and then again using only dominant follicles (SF
 and LF). For these analyses, when the data did not fit the assumptions for
 parametric statistics, we used a Durbin test in package PMCMRplus (Pohlert
 2023). In case of a significant result (p &lt; 0.05) in the analyses
 among the three groups, we used a DurbinAllPairs test to understand the
 variation between groups. When the data fit the assumptions for parametric
 statistics, we used a general linear mixed effects model (LMM) in the lme4
 package (Bates et al. 2015). In case of a significant result (p &lt;
 0.05) in the analyses among the three groups, we used a TukeyHSD test with
 a Bonferroni correction to understand the variation between
 groups.
description: # Provisioning of vitellogenic follicles continues after green turtles
 arrive at the nesting beach
 [https://doi.org/10.5061/dryad.47d7wm3pj](https://doi.org/10.5061/dryad.47d7wm3pj) ## Description of the data and file structure Methodology included in the relevant article. ### Files and variables #### **File: RBruno\_9524.R** **Description:** R script used to process the rdata.xlsx dataset for publication #### File: rdata.xlsx **Description:** Analyses of ovaries and oviducts of green turtles (*Chelonia mydas*) preyed upon by jaguars (*Panthera onca*) at Tortuguero National Park, Costa Rica. The .xlsx contains 5 tabs and 39 variables, which are described below. Blank cells are missing values. ##### Variables * “water” tab: * tid: Individual ID. * ftype: Follicle type. LF = Large dominant, SF = Small Dominant, AF = Atretic Follicle. * water: % water composition per follicle ((wet mass – dry mass)/wet mass) x 100. * “follcount” tab: * id: Individual ID. * type: Follicle type. F = Vitellogenic follicle, S = Ovulatory scar, AF = Atretic Follicle. * size: Maximum follicle/scar diameter (mm). * ova: Ovary to which that follicle/scar belonged (rt = right, lf = left). * ovab: Whether we measured only one or both ovaries for that turtle. * “females” tab * id: Individual ID. * ccl: Curved carapace length of the individual (cm). * rtovavol: Volume of right ovary to the nearest 50 mL. * lfovavol: Volume of left ovary to the nearest 50 mL. * rtovawt: Weight of right ovary to the nearest 0.5 kg. * lfovawt: Weight of left ovary to the nearest 0.5 kg. * rtovi: Number of eggs found in the right oviduct. * lfovi: Number of eggs found in the left oviduct. * “yolkdata” tab * tid: Individual ID. * ftype: Follicle type. F = Vitellogenic follicle, S = Ovulatory scar, AF = Atretic Follicle. * fsize: Maximum follicle/scar diameter (mm). * initial fwt: Follicle wet mass (g) * dry fwt: Follicle dry mass (g). * samplenp: Weight of sample used in the analyses of N and P (g). * Ng: N per gram of dry yolk (g). * Pg: P per gram of dry yolk (g). * sampleomdm: Weight of sample used in the organic matter analyses (g). * ashg: Minerals per g of dry yolk (g). * omatg: Organic matter per g of dry yolk (g). * samplelip: Weight of sample used in the lipid analyses (g) * lipidg: Lipids per g of dry yolk (g). * “segment” tab * id: Individual ID. * FA: Maximum diameter (mm) of follicles in the anterior segment of the ovary. * FB: Maximum diameter (mm) of follicles in the mid-anterior segment of the ovary. * FC: Maximum diameter (mm) of follicles in the mid-posterior segment of the ovary. * FD: Maximum diameter (mm) of follicles in the posterior segment of the ovary. * SA: Maximum diameter (mm) of ovulatory scars in the anterior segment of the ovary. * SB: Maximum diameter (mm) of ovulatory scars in the mid-anterior segment of the ovary. * SC: Maximum diameter (mm) of ovulatory scars in the mid-posterior segment of the ovary. * SD: Maximum diameter (mm) of ovulatory scars in the posterior segment of the ovary. Empty cells in the dataset indicate data that was not available due to several factors. For instance, samples from 12 females used for water analyses were excluded from nutrient analyses due to damage during transport. Additionally, some females did not have atretic follicles, making these data not applicable. In other cases, the sample mass of certain follicles was insufficient for all nutrient analyses. ## Code/software rdata.xlsx can be visualized using Excel and the relevant code can be run using R Program version 4.4.0. ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * Collected by author