The effect of respiratory gases and incubation temperature on early stage embryonic development in sea turtles

David Terrington Booth; Alexander Archibald-Binge; Colin James Limpus

doi:10.1371/journal.pone.0233580

. 2020 Dec 2;15(12):e0233580. doi: 10.1371/journal.pone.0233580

The effect of respiratory gases and incubation temperature on early stage embryonic development in sea turtles

David Terrington Booth ^1,^*, Alexander Archibald-Binge ¹, Colin James Limpus ²

Editor: Frank Melzner³

PMCID: PMC7710074 PMID: 33264278

Abstract

Sea turtle embryos at high-density nesting beaches experience relative high rates of early stage embryo death. One hypothesis to explain this high mortality rate is that there is an increased probability that newly constructed nests are located close to maturing clutches whose metabolising embryos cause low oxygen levels, high carbon dioxide levels, and high temperatures. Although these altered environmental conditions are well tolerated by mature embryos, early stage embryos, i.e. embryos in eggs that have only been incubating for less than a week, may not be as tolerant leading to an increase in their mortality. To test this hypothesis, we incubated newly laid sea turtle eggs over a range of temperatures in different combinations of oxygen and carbon dioxide concentrations and assessed embryo development and death rates. We found that gas mixtures of decreased oxygen and increased carbon dioxide, similar to those found in natural sea turtle nests containing mature embryos, slowed embryonic development but did not influence the mortality rate of early stage embryos. We found incubation temperature had no effect on early embryo mortality but growth rate at 27°C and 34°C was slower than at 30°C and 33°C. Our findings indicate that low oxygen and high carbon dioxide partial pressures are not the cause of the high early stage embryo mortality observed at high-density sea turtle nesting beaches, but there is evidence suggesting high incubation temperatures, particularly above 34°C are harmful. Any management strategies that can increase the spacing between nests or other strategies such as shading or irrigation that reduce sand temperature are likely to increase hatching success at high-density nesting beaches.

Introduction

The oxygen limitation hypothesis predicts that decreased oxygen availability in the environment and / or limitations in internal oxygen transport can limit aerobic metabolic processes at the intracellular level and thus limit cellular metabolism [1]. In ectothermic animals this potential limitation on cellular process may be exacerbated by high temperatures because high temperatures within the viable temperature range accelerate biochemical reactions and thus oxygen demand, a phenomenon termed the oxygen-capacity-limited thermal tolerance hypothesis [2]. For example, lethal temperature decreased from 44°C to 40°C as oxygen decreased from 21% to 10% in a viviparous lizard [3].

Sea turtle embryos experience varying oxygen and carbon dioxide partial pressures and incubation temperatures during their development in the female reproductive tract and natural nests [4, 5]. Embryos begin their development inside the oviduct of the female where the oxygen partial pressure is very low, less than 1 kPa and embryo development arrests at the mid to late gastrula stage due to lack of oxygen [4]. The carbon dioxide partial pressure is probably also considerable higher than in air but this has not been measured. Once the eggs are laid, they are exposed to higher oxygen partial pressures so that the oxygen limitation is lifted and the embryos break developmental arrest about 12 hours after laying and recommence development [4]. However, once developmental arrest is broken, if embryos are re-exposed to extremely low oxygen partial pressures they die of asphyxiation [6].

Typically, during the early stages of incubation, sea turtle embryos experience oxygen and carbon dioxide partial pressures close to those of the atmosphere above the sand [7, 8] and temperatures in the range 28–30°C and these are considered to be optimal conditions for embryonic development. However, during the second half of incubation, as the embryos grow rapidly in size, the combined metabolism of the entire clutch causes the oxygen partial pressure within the nest to decrease and the carbon dioxide partial pressure to increase [7, 8]. In green turtle (Chelonian mydas) nests, during peak metabolism, the oxygen partial pressure can fall to 10 kPa and carbon dioxide partial pressure climb to 8 kPa [7], while in loggerhead turtle (Caretta caretta) nests the equivalent values are 15–17 kPa and 3–5 kPa, respectively [7, 8]. In natural nests oxygen and carbon dioxide always change as mirror images of each other, i.e., as oxygen is consumed by the embryo it releases carbon dioxide so that as oxygen decreases within the nest, carbon dioxide increases [7, 8]. The combined metabolism of embryos also generates considerable heat which causes the nest temperature to rise between 2–5°C above the surrounding sand temperature which is typically 28°C to 32°C during the final stages of incubation so maximum nests temperatures can exceed 36°C [9, 10].

Clutches of eggs laid at high-density sea turtle nesting beaches such as green turtles on Raine Island, Australia, and olive ridley turtles (Lepidochelys olivacea) nesting in arribada aggregations in Costa Rica, typically experience much higher mortality of embryos (30–80% mortality) than clutches of eggs laid at low nest density beaches like Heron Island, Australia (0–10% mortality) [11–14]. Much of this mortality occurs due to clutch destruction by subsequent nesting females digging up previously constructed nests during their nesting process [14]. However, even in nests that remain undisturbed throughout incubation, embryo mortality is typically much higher than in nests constructed at low-density nesting beaches, with the majority of embryos dying at a very early stage of development [14].

The physical attributes of the nest environment have been hypothesised as the cause of the elevated early stage embryo mortality [11–14]. Nests constructed at high-density beaches may experience lower oxygen, higher carbon dioxide and higher temperature conditions compared to low nest density beaches [11–14]. Although late stage sea turtle embryos are tolerant to exposure to decreased oxygen, elevated carbon dioxide and elevated temperatures [5, 15, 16], early stage embryos may not be tolerant, possibly because they have not yet developed the ability to induce a heat-shock protein response which has protective effects against environmental induced stress [17]. Hence, if a new clutch of eggs is laid adjacent to a maturing clutch as may frequently occur at high-density nesting beaches, the newly laid eggs may be exposed to low oxygen, high carbon dioxide and elevated temperature conditions which could increase the frequency of early embryo death. Indeed, high early stage embryo mortality has been reported in green turtle nests that were laid near maturing nests, however, it was not possible to determine if the respiratory gas conditions or elevated temperatures or a combination of these two factors were responsible for the increase in embryo death [14]. In the current study, through a set of controlled incubation experiments, we investigate if incubation conditions that vary significantly from optimal with respect to oxygen and carbon dioxide, elevated temperatures or a combination of these factors increase the frequency of early embryo mortality in sea turtle embryos.

Methods

Ethics statement

This study was approved by the University of Queensland NEWMA animal ethics committee (certificate SBS/396/18), and eggs were collected under a scientific purposes permit issued by the Queensland Government National Parks Service (permit PTU18-001406).

Sources of eggs

Loggerhead turtle (Caretta cartetta) eggs were collected (four clutches consisting of 99, 102, 103 and 105 eggs) during oviposition at Mon Repos beach (24.8059°S, 152.4416°E) between 4/12/2018 and 15/12/2018 and green turtle (Chelonia mydas) eggs were collected (four clutches consisting of 98, 104, 106 and 110 eggs) at Heron Island (23.4423°S, 151.9148°E) between 06/01/2019 and 19/01/2019. We performed all experiments at the research facility located within the Mon Repos Conservation Park within 100 m of Mon Repos beach. We transferred eggs from loggerhead turtle nests on Mon Repos beach to the laboratory by hand-carried bucket immediately after collection to be processed. We transferred green turtle eggs immediately after oviposition into an insulated plastic container (60 cm x 35 cm x 35 cm, LxWxH) with its lid open and held overnight in a cool room at 5°C to 8°C. This temperature slows down embryo development so that the embryos do not undergo movement-induced mortality during transportation and the hatching rates of eggs transported in this way are similar to untreated eggs [18, 19]. One hour prior to boat departure from Heron Island, the container lid was closed and transported by a 2-hour boat trip to Gladstone followed by a 2.5-hour car trip to Mon Repos where we processed eggs before placing them into incubators, 16 hours after we collected the eggs. We collected two clutches of eggs each night for each species, and we repeated this process once for each species. Hence, in total, we used four clutches of loggerhead turtle eggs and four clutches of green turtle eggs in our experiments.

Egg incubation

Once at Mon Repos laboratory, we rinsed each egg briefly in distilled water to remove grains of sand, ensuring not to submerge eggs for more than a minute. We weighed eggs and labelled them with a unique identification including clutch and egg number using a 2B pencil. We then placed eggs into a gas tight plastic container (Sistema Klip, 4L, 20cm x 20cm x 10cm) labelled with its temperature and respiratory gas mixture treatment. Each container contained ~8 eggs from one female and ~8 from the other female sampled that night (total number of eggs in each container depended on total number of eggs in a clutch). Within the containers, we buried eggs in sand sourced from Raine Island that had been heat sterilized. We used this sand because Raine Island is host to a high nest-density population of green turtles and many nests experience high early stage embryo mortality at this location [14]. We added distilled water (60g distilled water to 1kg of sand– 6% w/w) to the sand to ensure eggs remained well hydrated during incubation. We placed containers into their designated incubator with unsealed lids exposing eggs to room air for the initial 36 hours of incubation. This procedure insured embryos broke developmental arrest before we exposed eggs to the respiratory gas treatments. After this initial 36 h period, we examined eggs to check if development had begun as indicated by the appearance of a white-patch on top of the egg. In turtle and crocodile eggs, a white-patch occurs in the eggshell immediately above the embryo12-36 hours after the start of incubation as fluid is extracted from the eggshell, causing it to dry and turn white [20, 21]. As embryonic development continues, it is thought that dehydration of the albumen below the shell caused by transfer of water from the albumen to the sub-embryonic fluid that surrounds the embryo causes expansion of the white-patch until eventually the white-patch completely surrounds the entire egg [21]. Thus, the rate of expansion of the white-patch is assumed to reflect the rate of embryo development. We assumed that any eggs in which a white-patch was not visible after the initial 36 hours of incubation in air were dead and removed them from the container. We dissected these dead eggs, and examined their contents under a dissecting microscope in order to assign the embryo to a development stage based on morphological criteria described by Miller et al. [22]. In developing eggs, we traced the outside edge of the white-patch using a 2B pencil and photographed them from above.

After the 36 h initiation period, we returned the eggs to their incubators and applied the gas treatments. For loggerhead turtle eggs, 12 treatments were used, a Latin square design of three incubation temperatures, 27°C, 30°C and 33°C, and four respiratory gas mixtures, O₂ = 21%, CO₂ = 0% (room air = control); O₂ = 17%, CO₂ = 4%; O₂ = 14%, CO₂ = 7% and O₂ = 10%, CO₂ = 11%. Unfortunately, by the time green turtle egg trials were run, the supply of the O₂ = 17%, CO₂ = 4% gas mixture was exhausted, so this treatment was replaced with an air and 34°C treatment. We choose the 27°C, 30°C and 33°C temperatures and respiratory gas concentrations as they reflect the range of conditions typically experienced in natural sea turtle nests [7–10]. The ratio of oxygen to carbon dioxide was based on the ratios found in natural green turtle nests on Raine Island [14]. The 34°C temperature was used because continuous incubation at this temperature is reported to be fatal to sea turtle embryos [5, 23]. Each incubator held a container ventilated at 50ml/min with each of the experimental gas mixtures supplied from premixed gas cylinders. We measured gas concentrations in each container twice a day by aspirating gas leaving the containers into an O₂/CO₂ analyser (Quantek 970, USA) to check that eggs were exposed to the appropriate gas concentrations. We also checked temperature inside each incubator twice daily using calibrated mercury in glass thermometers. We incubated all eggs for a further 5.5 days after initiation of the gas mixture exposure. Because we were only interested in investigating embryo death during the first week of incubation, we did not continue laboratory incubation of eggs past this point.

After 5.5 days of gas exposure we removed containers from incubators and examined the eggs. If the initial white-patch had not expanded in size, we assumed the embryo was dead and removed the egg, which we dissected and staged. If the white-patch had expanded we assumed the embryo was alive and traced the new white-patch boundary with a 2B pencil and photographed the egg from the side. An estimate of white-patch coverage as a percent of the entire egg surface was made using ImageJ (https://imagej.nih.gov/ij/). The increase in the area of the white patch from 36h to 5.5 days was used as an estimate of growth rate of the embryo. To confirm our assumption that embryos within eggs were alive we selected one egg at random from each treatment to be dissected and staged. We used the white-patch size and developmental stage of embryos of these opened eggs to confirm that white-patch size is correlated with embryo development. Because both loggerhead and green turtles are endangered or threatened species, we did not kill all eggs to ascertain that they were living and to assess their embryonic development stage. We relocated the surviving eggs into artificial nests consisting of 100 to 120 eggs pooled from the different treatments on Mon Repos Beach. Because we could not identify from which treatment hatchlings came from in these artificial nests, we could not assess the effect of treatment during the first 7 days of incubation on hatching success, so this data is not reported.

Statistical analysis

We used Pearson correlation to test for a relationship between relative white-patch area and embryo developmental stage. We arcsin transformed relative white-patch area data before ANOVA analysis. We used a mixed model ANOVA (clutch random factor, incubation temperature and respiratory gas treatment fixed factors) for analysis of relative white-patch area for both loggerhead and green turtle eggs. Statistical significance was assumed if p < 0.05. We performed all statistical procedures using Statistica© version 13 software.

Results

Embryo mortality

Only 9 (5 loggerhead turtle eggs, 4 from one clutch, 1 from another clutch; 4 green turtle eggs, 2 from one clutch and 2 from another clutch) out of 827 eggs (409 loggerhead turtle eggs, 418 green turtle eggs) failed to form a white-patch. Dissections indicated that these eggs were fertile but died in very early stages of development soon after oviposition (developmental stages 6–8 of Miller et al. [22]). For loggerhead eggs that formed a white-patch, with the exception of one embryo incubated at 27°C in 14% O₂, 7% CO₂, which died at development stage 9, there was no embryo mortality between the 36 h to 7 day period of gas exposure across all treatments (Table 1). Similarly, mortality of green turtle embryos was very low across all treatments between 36 h and 7 days of incubation (Table 2) and embryos died between developmental stages 10 and 12.

Table 1. Experimental treatments used to incubate loggerhead turtle (Carretta caretta) eggs and the absolute number as well as the proportion of embryos alive at day 7 after 5.5 days exposure to the gas mixture incubation treatment.

Incubation temperature (^oC)	Gas mixture	Number of eggs set	Number of eggs with white-patches at 36 h	Number of embryos alive at 7 days	Proportion survived (%)
27	21% O₂, 0% CO₂	32	31	31	100
27	17% O₂, 4% CO₂	34	33	33	100
27	14% O₂, 7% CO₂	36	36	35	97
27	10% O₂, 11% CO₂	32	32	32	100
30	21% O₂, 0% CO₂	32	32	32	100
30	17% O₂, 4% CO₂	32	32	32	100
30	14% O₂, 7% CO₂	32	31	31	100
30	10% O₂, 11% CO₂	32	32	32	100
33	21% O₂, 0% CO₂	36	35	35	100
33	17% O₂, 4% CO₂	35	35	35	100
33	14% O₂, 7% CO₂	40	39	39	100
33	10% O₂, 11% CO₂	36	36	36	100

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Table 2. Experimental treatments used to incubate green turtle (Chelonia mydas) eggs and the absolute number as well as the proportion of embryos alive at day 7 after 5.5 days exposure to the gas mixture incubation treatment.

Incubation temperature (^oC)	Gas mixture	Number of eggs set	Number of eggs with white-patches at 36 h	Number of embryos alive at 7 days	Proportion survived (%)
27	21% O₂, 0% CO₂	36	36	36	100
27	17% O₂, 4% CO₂	35	35	35	100
27	10% O₂, 11% CO₂	37	37	37	100
30	21% O₂, 0% CO₂	36	36	36	100
30	17% O₂, 4% CO₂	35	35	35	100
30	10% O₂, 11% CO₂	37	37	36	97
33	21% O₂, 0% CO₂	36	35	35	100
33	17% O₂, 4% CO₂	36	36	36	100
33	10% O₂, 11% CO₂	36	35	33	94
34	21% O₂, 0% CO₂	94	92	89	97

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Embryo development

We assessed embryo development after the 5.5 day exposure to the gas treatments by two methods; direct staging of embryonic development by dissection of one egg from each treatment, and by the relative white-patch area. All embryos were alive as indicated by a beating heart when dissected. Relative white-patch area was correlated with developmental stage in both loggerhead and green turtles (Table 3) indicating that relative white-patch area is a good indicator of developmental stage during early incubation. For this reason, and because the sample size for relative white-patch area was much greater than for developmental stage, only detailed analysis of relative white-patch area is presented. In loggerhead turtle embryos, both incubation temperature and respiratory gas treatment influenced growth of the white-patch, and the interaction terms temperature*gas and clutch*temperature*gas were significant (Table 4). In green turtle embryos, all three factors, clutch, temperature and gas influenced growth of the white-patch, and the interaction terms temperature*gas and clutch*temperature*gas were significant (Table 5). The trend in both species was for growth rate to increase from 27°C to 30°C, and then to remain similar between 30°C and 33°C, and for green turtle eggs that were incubated at 34°C in air, growth of the white-patch was slowest at this temperature compared to eggs incubated in air at other temperatures (Fig 1). The rate of growth of the white-patch decreased when the respiratory gases embryos were exposed to became increasingly different from the optimal atmospheric condition of 21% oxygen, 0% carbon dioxide, for both loggerhead and green turtle embryos (Fig 1). While dissecting eggs to record embryo development stage, we found that one green turtle embryo from the 33°C, 21% O₂, 0% CO₂ treatment was malformed but alive.

Table 3. Data relating the embryonic development stage (Miller et al. [22]) to the relative white-patch area for loggerhead and green turtle embryos after 7 days of incubation.

Loggerhead turtle
Incubation temperature (^oC)	Gas mixture	Developmental stage	Percent of shell covered by the white-patch
27	21% O₂, 0% CO₂	13	88
27	17% O₂, 4% CO₂	13	62
27	14% O₂, 7% CO₂	10	49
27	10% O₂, 11% CO₂	10	34
30	21% O₂, 0% CO₂	14	83
30	17% O₂, 4% CO₂	18	71
30	14% O₂, 7% CO₂	15	58
30	10% O₂, 11% CO₂	13	45
33	21% O₂, 0% CO₂	16	84
33	17% O₂, 4% CO₂	17	69
33	14% O₂, 7% CO₂	15	60
33	10% O₂, 11% CO₂	16	66
Pearson correlation	r² = 0.33, t = 2.21
Pearson correlation	p = 0.049, n = 12
Green turtle
Incubation temperature (^oC)	Gas mixture	Developmental stage	Percent of shell covered by the white patch
27	21% O₂, 0% CO₂	16	84
27	17% O₂, 4% CO₂	15	68
27	10% O₂, 11% CO₂	15	37
30	21% O₂, 0% CO₂	18	85
30	17% O₂, 4% CO₂	19	82
30	10% O₂, 11% CO₂	13	43
33	21% O₂, 0% CO₂	16	77
33	17% O₂, 4% CO₂	19	81
33	10% O₂, 11% CO₂	12	48
Pearson correlation	r² = 0.62, t = 3.34 p = 0.012, n = 9

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Table 4. Mixed factor ANOVA table for loggerhead turtle (Carretta carretta) egg arcsin transformed relative white-patch area after 7 days exposure to incubation temperature, and 5.5 days exposure to gas mixtures.

Effect	SS	DF	MS	F	P
Intercept	208.5	1	208.5	394.2	< 0.001
Clutch (random)	1.6	3	0.5	25.0	0.420
Incubation temperature (fixed)	1.2	2	0.6	11.2	0.009
Respiratory gas (fixed)	15.1	3	5.0	95.9	< 0.001
Clutch*Temperature (random)	0.3	6	0.1	0.6	0.698
Clutch*Gas (random)	0.5	9	0.1	0.6	0.777
Temperature*Gas (fixed)	2.2	6	0.37	4.3	0.007
ClutchTemperatureGas (random)	1.6	18	0.1	6.6	< 0.001
Error	4.6	352	0.01

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Table 5. Mixed factor ANOVA table for green turtle (Chelonia mydas) egg arcsin transformed relative white-patch area after 7 days exposure to incubation temperature, and 5.5 days exposure to gas mixtures.

Effect	SS	DF	MS	F	P
Intercept	189.8	1	189.9	186.1	< 0.001
Clutch (random)	3.1	3	1.0	17.8	0.003
Incubation temperature (fixed)	0.6	2	0.3	12.7	0.007
Respiratory gas (fixed)	17.5	2	8.7	169.1	< 0.001
Clutch*Temperature (random)	0.2	6	0.1	1.3	0.335
Clutch*Gas (random)	0.3	6	0.1	2.7	0.071
Temperature*Gas (fixed)	1.1	4	0.27	14.0	< 0.001
ClutchTemperatureGas (random)	0.2	12	0.1	1.8	0.046
Error	3.0	277	0.01

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Fig 1 — Thick solid line and circles = 21%O₂, 0%CO₂, dotted line and squares = 17%O₂, 4%CO₂, dashed line and triangles = 14%O₂, 7%CO₂, thin solid line and diamonds = 10%O₂, 11%CO₂. Error bars = Standard errors. Numbers associated with symbols = number of eggs in sample. Letters adjacent to symbols for the 21%O₂, 0%CO₂ gas treatment for green turtle eggs indicate significant differences according to a Tukey post-hoc test adjusted for unequal sample sizes.

Discussion

Respiratory gases

Contrary to our expectation, exposure of early stage embryos to low oxygen and high carbon dioxide gas mixtures did not influence mortality during the first week of incubation. Hence, we reject the respiratory gas hypothesis as an explanation of early embryo death syndrome so prevalent in high nest-density sea turtle rookeries. However, we found that as the respiratory gases deviated further from atmospheric air, the rate of embryo development decreased, so clearly there was a deleterious effect of exposure to respiratory gases that deviate from atmospheric conditions at this early stage of embryonic development. If new clutches are laid in close proximity to maturing clutches, this can potentially cause the respiratory gases in the newly laid clutch to deviate significantly from atmospheric air. Under these conditions, the clutch may experience a longer incubation period than other clutches that are not influenced by altered respiratory gases. However, in nature, clutches are only exposed to self-generated low oxygen and high carbon dioxide partial pressures during the last 2 weeks of incubation. After this time, hatchlings escape the nest and oxygen and carbon dioxide partial pressures return to atmospheric conditions. So, even if a new clutch was laid next to a late maturing clutch, embryos in the new clutch are most likely only exposed to adverse respiratory gases for a maximum of two weeks unless they also have multiple clutches of eggs with embryos at different developmental stages next to them.

Incubation temperature

Although incubation temperature appeared to have little effect on embryo mortality over the first 7 days of incubation, continuous incubation at 34°C from the beginning of incubation results in death of all sea turtle embryos [5, 23–25]. The observation that an embryo was still alive, but malformed after 7 days of incubation at 33°C suggests it is possible that even though embryos incubated at this temperature are still alive after a week of incubation, some embryos have developed high temperature induced teratogenic malformations that will result in embryo death later in incubation. Hence, high nest temperatures experienced early in incubation is a possible explanation for the large number of nests experiencing a high rate of early embryo death in high-density nesting aggregations. Indeed, a natural green turtle clutch that experienced 80% early embryo death at Raine Island experienced an average temperature of 35.5°C during its first week of incubation [14]. In natural sea turtle nests, a rise in nest temperature due to metabolic heating is always accompanied by a fall in oxygen and increase in carbon dioxide, and it is generally theorised that a decrease in oxygen would exacerbate the detrimental effect of high temperature [2], and this has been indicated in developing lizard embryos [3]. However, we found no evidence of this exacerbated effect in our early stage sea turtle embryos, with high survivability during the first week of incubation when exposed to hypoxia in combination with hypercapnia across all experimental temperatures.

Although high incubation temperatures were not immediately fatal during the first week of incubation, they did retard the growth of early stage embryos. In ectotherms, including sea turtles, embryonic growth and development rate increase with an increase in incubation temperature within the viable temperature range. This explains the increase in embryo development rate we observed between 27°C and 30°C, however development rate did not continue to increase at 33°C, and actually decreased at 34°C in green turtle eggs when eggs were incubated in air. It would appear that incubation at temperatures of 33°C and higher are sub-optimal and that although development can continue, these high temperatures may cause cellular damage that needs to be repaired, and this damage slows the rate of development. Alternatively, slowing of development at high temperatures may be a tactic to prevent high temperature damage, by waiting until cooler temperatures return before recommencing development. Ultimately, for early stage embryos incubated at temperatures of 34°C and higher for long periods, the high temperature results in embryo death [5, 23–25].

We collected the eggs for our experiments from low density nesting populations in which we knew by checking flipper tag numbers that the females laid their clutch on their first laying attempt. Previous reports [26–28] indicate that retention of eggs for several days within the female reproductive tract due to multiple failed nesting attempts that frequently occurs at high nest-density beaches, causes a decrease in egg viability. Hence, it is possible that at high nest-density beaches a combination of prolonged egg retention and exposure of eggs to low oxygen, high carbon dioxide partial pressures and high nest temperatures result in a higher frequency of early stage embryo mortality.

In summary, although we found the development rate of early stage sea turtle embryos is retarded when exposed to partial pressures of oxygen and carbon dioxide typically encountered in maturing sea turtle clutches, such exposure is not fatal. Likewise, when we exposed early stage sea turtle embryos to incubation temperatures of 33°C and above, development was slowed. At nesting beaches that experience high-density nesting, the close proximity of nests means that many newly laid clutches could experience high temperatures due to the metabolic heat production of nearby maturing clutches, and this might result in elevated rates of early embryo death, especially if temperatures exceed 34°C for long periods of time. Hence, as the number of nesting females increases at high-density rookeries, the number of newly laid clutches exposed to high temperatures increases, and therefore the proportion of clutches experiencing early embryo death syndrome increases as reported for green turtle clutches at Raine Island [14]. A management strategy that could mitigate this increase in early embryo death could be to increase the beach area suitable for nesting so that nests could be spaced further apart, or the use of other strategies such as shading or irrigation that reduce sand temperature.

Supporting information

S1 Dataset

(XLSX)

Click here for additional data file.^{(53.4KB, xlsx)}

Acknowledgments

Experimental facilities were provided by the Queensland Government Department of Environment and Science.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This project was funded by the The Raine Island Recovery Project which is a five-year, $7.95 million collaboration between BHP, the Queensland Government, the Great Barrier Reef Marine Park Authority, Wuthathi and Kemerkemer Meriam Nation (Ugar, Mer, Erub) Traditional Owners and the Great Barrier Reef Foundation. Grant supplied to DTB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0233580.r001

Decision Letter 0

Frank Melzner

24 Jul 2020

PONE-D-20-13033

The effect of respiratory gases and incubation temperature on early stage embryonic development in sea turtles

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: General points:

The manuscript presents a sound study that answers an important biological question with real conservation consequences for threatened sea turtle populations. I congratulate the authors on designing and executing a valuable study to elucidate the causes of abnormally high early-stage embryonic death at high-density sea turtle nesting beaches.

My main criticism with this manuscript is that, for the two clutches where data was presented for stage of embryonic mortality, the temperature treatments were applied over the entire incubation period and therefore does not reflect a "short-term" early incubation effect of a nearby mature clutch incubating. In the clutches where high temperatures were only applied for the initial period of incubation the authors have not presented the data for stage of embryonic development at which the embryos died. I suggest that the authors either revise the manuscript to include the stage of death data for the unhatched eggs from clutches that were reburied in artificial nests. If these data do not exist, the authors need to address this limitation and I suggest they include a caveat in the discussion to highlight the limitation that embryonic death cannot be conclusively attributed to "short-term early-stage" high incubation temperature. This is the only major change I recommend making to the manuscript, the rest of the points I raise should be easy to address.

What about the impact of extended pre-ovipositional embryonic arrest on embryos ability to survive high nest temperatures and low oxygen partial pressure? At high-density nesting beaches, such as Raine Island and Ostional, it could be likely that there are higher rates of failed nesting attempts due to disturbance from other females. This would mean a higher proportion of females return to nest over subsequent days to attempt oviposition when compared with those nesting at low-density nesting beaches. Inherently, this would mean that embryos are maintained in embryonic arrest for longer periods of time. Previous experiments have found that sea turtle embryos have limited tolerance to remain in embryonic arrest (Rings et al. 2015 Phys. Bio. Zool. and Williamson et al. 2019 Sci. Rep.) and in one population of leatherback turtles longer interesting intervals result in higher proportions of early-stage embryonic mortality (Rafferty et al. 2011 PLoS One). If embryos are potentially compromised by extended arrest they may be more susceptible to higher incubation temperatures and lower oxygen availability when they do recommence development after the arrest. It would be good for the authors to include some acknowledgement of this possibility in the discussion.

Specific points:

Page 1, Line 14: Change to "high death rate" or "high mortality rate".

Page 1, Line 22: Change "sea turtle nest" to "sea turtle nests".

Pages 1 - 2, Lines 23 - 24: Change "did not influence embryo mortality of early stage embryos" to "did not influence the mortality rate of early stage embryos".

Page 2, Line 25: I am unsure what the current word count for the abstract is, but I suggest including a final sentence summarizing the importance and implications of the findings.

Page 2 Line 34: Delete "of" from "increases of lizard"

Page 2, Lines 36-37: Provide a reference for this sentence. Perhaps one of the Ackerman papers on the topic. I suggest also breaking this paragraph into two separate paragraphs. One on the information about developmental arrest and the other on the information about varying gas concentrations and temperature throughout incubation in the nest.

Page 2, Line 39: Change "stalls" to "arrests".

Page 2, Lines 39 - 40: Embryos do not break developmental arrest immediately after oviposition. The study that the authors cite for this sentence suggests that embryos break the arrest after at least 12 hours of exposure to higher oxygen partial pressures. Please change this sentence to reflect this difference.

Page 2, Line 42: Change "development arrest" to "developmental arrest".

Page 3, Line 53: Change "it released carbon dioxide" to "it releases carbon dioxide".

Page 3, Line 57: Change “aggregations such as” to “aggregation beaches, such as”.

Page 3, Line 58: Arribadas is plural in Spanish. Change "nesting in arribadas aggregations" to "nesting in arribadas" or "nesting in arribada aggregations"

Page 3, Line 68: Again, this paragraph is very long. Suggestion to split into two paragraphs after this sentence.

Page 3, Lines 70-72: Provide appropriate references for this section of the sentence. Possibly Nicki Mitchell et al papers on heat-shock proteins in sea turtle embryos e.g. Bentley et al. 2015 Mol. Ecol.; or Tedeschi et al 2015 or 2016.

Page 5, Line 111: Please provide more information on the type of containers used for the application of the gas treatments.

Page 5, Lines 113 - 114: Unless I have missed it, there is no explanation provided for the use of Raine Island sand during incubation. Please explain the reasoning for this.

Page 6, Line 131: Provide reference to the staging criteria that you staged embryos with. I assume Miller's 1985 staging guide?

Page 7, Line 159: Please provide clarification whether these eggs were maintained at their assigned temperature treatment or if all eggs were then incubated at a similar temperature.

Page 7, Line 160: Please explain a little more about what happened to these eggs. Were they opened and staged to assess viability? Were the eggs left to incubate until hatching in the incubators?

Page 8, Lines 188-191: I assume you excavated and staged the unhatched eggs from the clutches that were reburied in artificial nests as well. You should provide a summary of these data also. Or explain why this was not done. If the primary concern of this study is early stage death I think it is quite important to present the data on which stage of development these embryos died at.

Page 9, Line 198: This is an important finding and indicates that growth of the white spot is actively regulated by the embryo rather than passively by desiccation or other environmental influences.

Page 10, Line 228: Change "logger turtle" to "loggerhead turtle"

Page 10, Lines 235-237: It is important the authors have acknowledged this potential limitation in assessing the difference in growth rates between the two species given that they applied different methodologies to the eggs dependent on species.

Page 12, line 274: Change "early stage sea turtle embryos development rate" to "the development rate of early

stage sea turtle embryos".

Page 19, Lines 426 – 427 and Page 20, Lines 430 - 431: Table 1 & 2 Captions are nonsensical to me. The experimental treatments cannot indicate survival in themselves. I suggest changing "turtle eggs indicating survival" to "turtle eggs and embryo survival" or something similar.

Reviewer #2: Generally, I found the study by Booth et al. interesting and relevant from a conservation perspective and the collected data is suitable to answer the main study question. The manuscript itself is concise, well-written with appropriate referencing. However, my major concern is that the experimental design is unclear and does not always seems to address the main study question, which could be amended by a careful revision of the M&M section. In addition the authors draw conclusions that are not supported by their data or their experimental approach: i) by comparing the two study species, which is not supported by statistics or a comparable handling of the embryos, ii) by concluding that development is affected by hypoxia, which was not tested independently from hypercapnia and iii) by confounding the results from a short-term exposure in a common garden experiment with the results from a long term survival trial (where embryos were pre-exposed to different gas tensions, but then kept in ambient air). I think all of these concerns can be addressed, but will require re-analyzing some of the data, changing the presentation of the results and re-writing large parts of the discussion to adhere more closely to the study question and the data at hand. Therefore, I cannot recommend the manuscript for publication in PlosOne in its current form and in the following I list 5 major concerns that I would like the authors to address. In addition, I have listed a number of minor comments that will hopefully help with the revision of this manuscript.

1. The discussion is well-written and meaningful as far as it pertains to the main research question. But to a large extent the authors take the discussion beyond what can be concluded from their data to an extent that is unacceptable for publication. I recommend the authors adhere closely to the main research question and avoid over-interpreting their data. For instance , the authors conclude that one species is more susceptible to hypoxia than the other, based on a metric of developmental rate (L227-245), but this isn’t supported by any statistical analysis, the embryos were not treated in the same way prior to experiments and generally potential interspecific differences in developmental rate are not considered. Furthermore, the authors discuss potential effects of hypoxia on development, but hypoxia was always tested in combination with hypercarbia and therefore such mechanistic distinctions cannot be made (L238-245). I urge the authors to fundamentally revise the interpretation of their data and pay close attention to the limitations in their experimental design.

2. I think the experimental design is appropriate to answer the main study question on whether early embryonic development/survival is affected by the conditions (gas and temperature) created by nearby clutches on crowded beaches. This was assessed in a first experiment over 7 days where embryos were exposed to different combinations of gas tensions and temperature and development was monitored. This experiment answers the main study questions and there were no effects on early embryo survival, but development was delayed. After this, things get more confusing and the further experiments are not in line with this initial hypothesis. The second experiment looked at long-term survival of the embryos at different temperatures, but at ambient gas tension (and using the embryos that had been previously exposed to different gas tensions). Here the authors report significant differences in survival, but it’s unclear whether this longer time-frame addresses the initial question about “early embryo mortality” (a term that should be clearly defined early in the paper) and fails to address the actual conditions on crowded beaches that always include altered gas tensions. The presentation and discussion of the results intermixes the findings from these two experiments and it is often unclear which condition is being discussed. I suggest the authors outline more clearly the justification for this second experiment and how it fits within the main study objective. One option would be to re-analyze the data and test whether an early exposure of the embryos to altered gas tensions had an effect on long-term survival even at atmospheric gas tensions (currently the results from all gas-tension pre-exposures are pooled across the temperature treatments). The findings of this analysis would be interesting regardless of the outcome and could tie the two experiments together to test a common hypothesis.

3. In general, the way the authors have described the experimental design of their study is convoluted and even after reading the manuscript again it is not entirely clear to me why certain conditions were tested. I recommend that the authors include a dedicated section in the M&M on experimental design, where they lay out clearly what the experimental treatments were, when each treatment was sampled and how the treatments were replicated (the authors use eggs as N whereas really each clutch should be an experimental unit, where N=4). I also recommend dividing the experiments into clearly defined stages (phases or series). The first from 0-36 h after which measurements were taken, the second 36 h- 7d which tested the effects of gas tensions and temperature on development and finally a long-term exposure 7-46 d in which only survival was assessed.

4. I recommend the authors revisit the presentation of their data. Figures 2 and 3 show both species in one graph, whereas gas tensions were pooled for each temperature. I don’t think this is a valid presentation of the data as there were significant differences between the gas tensions and therefore, they shouldn’t be pooled in the first place. In addition, there was a significant interaction effect between gas tensions and temperature and therefore all combinations of temp*gas need to be shown separately. I recommend showing the p-values of main effects and interactions in the figure as well, as this reflects the outcome of the statistical analysis and Tables 3 and 4 could be omitted. And finally, I suggest presenting one figure per species, as no statistical comparisons are made between the two species.

5. In one of the treatments the pre-mixed gases ran out and the authors chose to convert this treatment into a high-temperature exposure (as far as I understood this section) where embryos were exposed to 34°C instead of 33°C. It is not clear why this additional increase in 1°C was chosen and, without any context, it is surprising that it had large effects on animal survival. I recommend the authors justify their approach more clearly. Since the other gas tensions were pooled by temperature this 34°C exposure is the only treatment that received ambient air throughout development (correct?) and therefore is not necessarily comparable to the other treatments. One way to avoid this confounding effect of pre-exposure to different gas tensions would be to compare the 34°C exposure only to the other treatments that were exposed to ambient air throughout (controls). I suspect the results would still hold up and this would make for a more meaningful comparison (if the results don’t hold up this is reason for concern in itself).

Specific comments:

Abstract

L13-14 Here and in the introduction, please define the term “early stage embryo death”. This is especially important since the study assessed embryo development over two distinct time-frames and throughout the manuscript it is unclear whether both parts of the study address the issue of the early mortality of embryos.

I find the last sentence of the abstract a bit cryptic and, based on this, I was unsure whether there was a clear effect of temperature on embryo mortality. Consider rephrasing this sentence for clarity. Also, I’d like to see a concluding statement in the abstract that is based on the current findings. What does it all mean for wild turtles that nest in crowded beaches?

Introduction

L34 delete “of”. Also, it’s worthwhile mentioning that these were embryos of live-bearing lizards and I would note the level of hypoxia and the temperatures, as well.

L45 replace that” with “those”

L49 I would not use the term peak metabolism, because it implies that metabolism would decrease again throughout embryonic development. Presumably this refers to the last stage in development before hatch.

L53 “releases”

L51-54 I agree that hypoxia and hypercarbia typically go hand-in-hand in natural settings, but the effects on the organisms differ greatly between these two conditions. Overall, I found the paper focused only on O2 whereas the experimental treatments manipulated both gases. I suggest the authors introduce briefly the effects of hypercapnia on embryonic development as well; many good physiological studies have recently dealt with this.

L54-54 What are typical temperatures in the sand? Provide some context so the reader knows if we’re talking about a 30% (sand temp ~10°C) increase in temperature or a 10% increase (sand temp ~30°C).

L56 final stages of incubation

L57-60 I found this sentence rather vague, but it is a critical justification for conducting this study. Please state specifically whether within one species crowded nesting beached have higher embryo mortality rates compered to empty beaches. Be specific and mention how large of an effect this is for each species. As written, it is unclear whether comparisons are made across the two species or these very different habitats.

L72 Are there any studies that indicate an inability of young embryos to mount a heat-shock protein response? Please cite.

L72-79 This is a repetition of earlier statements and I suggest integrating it with previous mentions.

L79 I liked that the authors put forward a testable hypothesis in the abstract and I suggest doing the same here, in the introduction. I also recommend that the authors specifically introduce the conditions that are considered “optimal” for embryo development and how they are going to be manipulated in this study (i.e. replace “suboptimal”).

Materials and Methods

L90-106 In this paragraph, mention the numbers of eggs that were collected. I was surprised to read in the results that there were several hundred eggs per clutch. If anything, this strengthens the paper.

L97 I would avoid mentioning brand names. An insulated cooler or container.

L95-106 I understand that there were some logistical challenges to getting the green turtle eggs into the lab. Presumably the careful transport of these embryos did not affect their development, but it would be good if the authors could justify this in a sentence or two. Were mortality rates generally comparable to clutches that were left undisturbed? Similar to those of the loggerhead turtles that did not experience the transport procedure? Or were they typical for what has been reported for this species in the past?

L136-139 I think the authors should provide a justification for why exactly these gas tensions were used. I understand that they span the conditions that turtles may experience in the wild, but why were exactly these combinations of O2/CO2 chosen? I.e. O2 is reduced in steps of 80%, 66% and 47% relative to normoxia, whereas CO2 is increased in steps of 4%, 7% and 11%. What’s the basis for choosing these ratios? Also, is the 21%/0% treatment really the best control, as wild turtles would probably never experience this in the sand. I’m not saying that there is something fundamentally wrong with these experimental conditions, but I think the authors need to justify specifically why they were chosen.

L149-160 This section is a bit confusing as written and the authors should state more clearly what measurements were performed at which times? How do they know if the embryos grew (and were alive) before tracing the patch and comparing it to the previous measurement?

It seems that two different methods were used to assess whether an embryo was alive, tracing a white spot on the egg and staging embryos by dissection. Why this redundancy is there a reason to think that one method would yield different or unreliable results? And if so, why not use the more reliable of the two methods. And what exactly was assessed during staging of the embryos?

It’s also not clear to me why the embryos were only exposed to the gas mixtures for 7 days. And it sounds like some embryos were returned to their natural nests thereafter, but others were not. It’s not entirely clear to me how this helped addressing the research question. Please clarify.

L157-160 The authors mentioned earlier that one of the gas mixtures ran out and presumably this is why the last clutch of green turtle eggs was kept at ambient gas concentrations throughout, but instead temperature was manipulated, right? There is no mention of that here, which I found confusing.

L159-160 Include, how many eggs were sampled at each timepoint.

Results

L178 The authors have not introduced the metric of developmental stages or how they were assessed. This should be described in the M&M section.

L187-188 It’s still not clear to me exactly how these long-term experiments were performed. It sounds like all loggerhead eggs were returned to the wild after the 7 day exposure, but the green turtle eggs went back into the incubators. At this point gas tensions were ambient for all treatments (L159), but in figure 1 it says that data was pooled across gas treatments. And that we have one additional treatment that was only exposed to air throughout the experiment (because the gas ran out) and this treatment was kept at 34°C instead of 33°C (L138-140). I had to go back and forth several times to piece this information together and I’m still not sure whether this is all correct. I’m also unsure whether the comparisons that are being made are actually meaningful (see major comment about including a dedicated M&M section on experimental design).

L199-200 It’s ok to present only these data, but mention in a sentence whether the two metrics of embryo development agree with one-another or not. Perhaps the staging data could be made accessible in a supplement, to avoid cherry-picking one dataset over the other?

L205-207 This sentence is confusing as written, please re-phrase.

Tables

In general, I would avoid the vertical lines in tables for publication.

In the table captions I would mention the full species names, instead of just “loggerhead or green eggs”.

Figure 1 I would avoid using the thick line in the 34°C treatment as is covers the actual datapoints. Chose a different linetype that is less occlusive. In my version, the R2 appeared as a box. If the data were pooled across gas treatments, I’d like to see some measure of variability included around the means (SD or SEM).

Were there any significant differences in survival between the embryos that had previously been exposed to the different gas tensions? If not mention this here as it justifies the pooling of these data. Otherwise report the result as it would be interesting to know whether hypoxia or hypocapnia during early development has an impact on long-term survival rates.

Figure 2 Since there were significant effects of gas tensions on embryo development these treatment should not be pooled. The presentation of these data will need to be changed. The figure will need to show the development for each gas tension individually and to highlight that there was a significant effect of temperature the authors could include the p-values for the main effects and the interaction term in the figure as well, which would replace Table 3.

The same considerations apply to Figure 3. I recommend the authors revisit the presentation of their data. I suggest presenting one figure per species, as no statistical comparisons are made between the two species. And each of these figures will need to show all combinations of experimental treatments as they were significantly different and there was a significant interaction between gas treatments and temperature, in each case. A more common way to express changes in development with temperature would be to calculate the Q10 values for each step. This is somewhat of a personal preference, but the authors should consider this type of analysis.

Discussion

L217 the gases were not “sub-atmospheric” for CO2

L222-226 I disagree with this statement as it neglects the possibility that there could be a number of adjacent clutches that are staggered in their developmental stage and therefore a single clutch of eggs may experience hypoxia and hypercapnia throughout development or for any fraction of this time.

L227-230 I’m not sure this comparison is backed by the data and no statistical evidence is provided to support this. In addition, differences in the development of a “white patch”, a rather subjective marker, could simply be due to differences between egg size, egg-shell composition, etc. between the species. Based on the provided data it cannot be concluded that one species is more susceptible to the altered gas tensions than the other. This applies to the entire following paragraph from L227-245.

L235-238 I think it’s great that the authors discuss critically the possibility that cooling the embryos of one species before the trials may have confounded the results. But I still think it would be good to dissipate these concerns earlier in the M&M section by providing a sound justification of this approach. I understand that field conditions are usually a compromise and if it was simply not possible to treat the eggs from both species in the same way that can be sated as well. Based on this discrepancy I would further discourage the authors from drawing any conclusions by comparing the development of the two species. The experimental design simply does not support these comparisons.

L238-245 The authors focus on an O2 limitation as the probable cause for an impaired development. Also this cannot be concluded from the data, as O2 and CO2 were always altered in concert. I urge the authors to fundamentally revise the interpretation of their data and pay close attention to the limitations in their experimental design. I think the experimental design is appropriate to answer the main study question on whether embryonic development is affected by the conditions (gas and temperature) of nearby clutches on crowded beaches. I recommend the authors adhere closely to this question and avoid overinterpreting their data. The effects of O2 and CO2 on development of embryos can be discussed and some speculation is ok to suggest future experiments, but the effect of these gases cannot be disentangled unambiguously.

L261-263 Again, based on the presented data I’d like to know whether early hypoxia or hypercapnia had an effect on late embryo mortality in ambient gas tensions. Did the authors test for such an effect? Whatever the finding, this would be interesting to report in the results.

L269-272 I’m not convince that the slower development at higher temperatures (by itself) is an indication of cellular damage or morbidity. In fact, it could be an adaptive response of the embryos to prevent a further deterioration of the conditions surrounding the clutch and perhaps to allow for neighboring clutched to hatch first by delaying their own development. The only data that really suggests that the embryos may be pushed beyond their physiological tolerance is the survival data. I suggest the authors make this distinction more clearly.

L276 “clutches”

L278 at 34°C long-term survival was affected. Again, here the results of the short-term and long-term experiments are intermixed. I recommend differentiating more clearly between effects on development (within the first 8.5 days) and long-term survival, which was affected by temperature.

L281-288 I like this last paragraph that interprets the major findings in an applied context and provides clear management strategies for turtle conservation. I suggest ending the abstract in a similar way.

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Reviewer #1: Yes: Sean Williamson

Reviewer #2: No

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PLoS One. 2020 Dec 2;15(12):e0233580. doi: 10.1371/journal.pone.0233580.r002

Author response to Decision Letter 0

3 Sep 2020

All issues raised by the reviewers have been addressed in the response to reviewer's document.

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PLoS One. doi: 10.1371/journal.pone.0233580.r003

Decision Letter 1

Frank Melzner

29 Sep 2020

PONE-D-20-13033R1

The effect of respiratory gases and incubation temperature on early stage embryonic development in sea turtles

PLOS ONE

Dear Dr. Booth,

While both reviewers acknowledge substantial improvements in the revised version of this ms, they also feel that mortality data should be supplied, along with a critical discusssion of mortality observed in this vs. older studies. I agree with their assessment and suggest you provide the required data and engage in a more critical discussion of mortality in relation to temperature.

Please submit your revised manuscript by Nov 13 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Frank Melzner

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: I am concerned that my main criticism of this manuscript has still not been fully addressed. The study aim is to assess the impact of changes in suboptimal gas mixtures and temperatures during early-embryonic development to ascertain whether this could explain high rates of early embryonic death at high-density nesting beaches. Data on stage of embryonic death has not been presented for sufficient comparison of differences between the treatments. Furthermore, in this revised version, there is no longer any substantial mortality data included. This makes it difficult to definitively conclude that any of the treatments applied caused higher rates of early-embryonic death and poorer hatching success in general. The authors did apparently open 2 eggs (or potentially 4?) from each treatment to assess stage of embryonic development at the end of the treatment but this data has not been reported in the manuscript.

Given the above, if data on stage of embryonic death cannot be provided, the authors need to temper interpretation of results that temps above 34℃ are more harmful than non-optimal gas concentrations. There was minimal difference in the proportion of embryos that you assumed were still alive at 7 days across all treatments, including temperature treatments. Furthermore, the data you report on relative growth of the white-patch indicate that non-ideal gas mixtures (10%O2, 11%CO2) had a more pronounced negative impact on white-patch growth than temperatures of 34 degrees. There were also no major differences in early-embryo survival across your temperature treatments.

I am concerned by the response to my initial suggestion in the first round of review:

“Page 8, Lines 188-191: I assume you excavated and staged the unhatched eggs from the

clutches that were reburied in artificial nests as well. You should provide a summary of these

data also. Or explain why this was not done. If the primary concern of this study is early stage

death I think it is quite important to present the data on which stage of development these

embryos died at." To which the authors state: "This part of the study has now been deleted from the manuscript.”

This part of the study has not been deleted from the manuscript. The authors still state that eggs were reburied in artificial nests on Mon Repos Beach. Is there excavation data with data on stage of death for the eggs? Given the central concern of the study is early stage death, it is important that data on the stage that embryos died at is reported in as much detail as possible. Or provide explanation why this was not done.

Reviewer 2 brings up a similar point which could be answered if the hatching success and stage of embryonic death data is supplied for the eggs that were reburied in artificial nests at Mon Repos:

“L261-263 Again, based on the presented data I’d like to know whether early hypoxia or

hypercapnia had an effect on late embryo mortality in ambient gas tensions. Did the authors

test for such an effect? Whatever the finding, this would be interesting to report in the results.

The data and discussion on late embryo mortality has been deleted from the revised

manuscript.”

These are the only major concerns I still have with the manuscript, the rest below should be straightforward to fix.

Specific points:

Page 2, Line 35: Spacing either side of "/" needs to be fixed.

Page 4, Line 73: "low aggregation nesting aggregation beaches" needs to be changed. Suggest "low density nesting beaches" or "low density nesting beach aggregations".

Page 4, Line 78: Change "with the majority of embryos die at an early stage" to "with the majority of embryos dying at an early stage of development".

Page 5, Lines 106-110: Maybe I've got this wrong. But it seems confusing to me that you ran out of one of the gas mixtures for the treatments for the green turtle experiment when the dates you've reported here suggest this experiment was undertaken first. I suggest double checking your dates for egg collections you have listed in the methods. Or revisit the wording you have used to explain the reason why this treatment was not included in the green turtle experiment.

Page 8, Lines 182-184: Here in the methods, the authors state that two eggs were randomly selected from each treatment (one from each of the "two clutches") to be opened and staged, but further down in the results they state that four eggs from each treatment (one per clutch) were opened and staged. Similarly, at the start of the methods you report four clutches per species being collected at the start of the methods. Furthermore, in the results you report sample numbers of eggs that were opened, 9 and 12 for green turtles and loggerheads respectively. These numbers don’t add up to me, please provide clarification.

Page 9, Line 213: You should present a summary of the data on developmental stage of these opened eggs. The data is not supplied in the raw data file either.

Page 10, Line 221: "all three factors, clutch, temperature and clutch influenced growth" needs to be changed to "all three factors, clutch, temperature and gas influenced growth".

Page 10, Line 226-227: "became increase different" needs to change to "became increasingly different"

Page 15, Line 339: The journal title is abbreviated here whereas other journal titles in the list are not abbreviated. Change for consistency / journal style.

Page 19, Line 435: Change "treatment in for green" to "treatment for green".

Reviewer #2: The authors have done a good job in responding to my previous comments and I see most issues addressed in their revised version. Overall, the manuscript reads much better and is structured in a more intuitive way. Also, the M&M section describes the experiment much more clearly now (largely because it is a simpler experiment). I think, deleting the long-term survival data, which was outside of the proposed study question makes for a simpler more concise paper and avoids much of the previous confusion surrounding the definition of “early stage embryo mortality”. The authors have re-analyzed their data, as suggested, and the new figures convey the major results accurately. To a large extend the discussion adheres more closely to the actual data and much of the previous over-interpretation of the results has been amended. However, in their discussion of the effects of incubation temperature, the authors conclude that elevated temperatures are the likely cause for early embryo mortality, which is not supported by the data presented here. In my mind, this needs to be corrected.

I think the manuscript is much improved from its previous version and I still think the study and it’s findings are interesting and worthwhile publishing. However, I urge the authors to amend the one main comment I have outlined below, before I can recommend the manuscript for publication with PLOS ONE. In addition, I’ve listed a few minor, largely editorial comments below, for the authors to consider (all line numbers refer to those in the revised version with tracked changes)

Main comment

In line L295 and following, it reads that there was no effect of high temperatures on early embryo mortality, but that continued incubation at 34°C caused the death of all sea turtles. As far as I can tell, this is based entirely on the results from previous work. In the present study, embryos incubated at the highest temperature (34°C) had a 97% survival rate. The only adverse temperature effects reported here are based on the observation of one malformed embryo incubated at 33°C. Therefore, it should be stated clearly that the study found no effect of high temperature on early embryo survival (just like there was no effect of adverse gas tensions on early embryo survival). If anything, these results stand in contrast to those of the previous studies cited here and I’d like to see a critical discussion of this discrepancy and its implications.

Again, later in the discussion (L325-328), the authors repeat the conclusion of these high-temperature effects, saying that early stage embryos incubated for a long period at 34°C would experience higher mortality. I find this misleading, because at longer incubations we’re not talking about early embryo mortality anymore, which is the main study question. And again, this is not supported by the present data.

I think the authors should conclude that neither adverse gas tensions or high temperatures had an effect on early embryo survival (at least with the experimental protocol used here) and should rather focus their discussion of the effects of temperature and gases on embryo growth rates, which were significant and are supported by the data. I think these are interesting findings that will inform on the physiological status of the developing embryos, and that are barely discussed in the current version.

Minor comments

Abstract

L19…increase in mortality rate.

L31…but there is evidence suggesting high…I think this is too vague for an abstract. Why not mention what that evidence is?

Introduction

L78 aggregation is repeated

L83 dying

L84 I would clearly define the term “early stage embryo mortality here”. This was done in the abstract, but not the introduction.

M&M

L140 delete “it”

Discussion

L340 Change “In contrast” to “In addition”. The results from the adverse gas exposure and the high temperature were similar in that they both slowed development, but didn’t increase mortality.

L342-348 Again, I urge the authors to discuss the adverse effects of high temperature more critically. The present study found no increased early embryo mortality to speak of and the conclusions here are derived from previous work. I think the authors need to discuss the discrepancy between these findings in more detail. These results are stated correctly in the abstract, but are convoluted in the discussion.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Sean Alexander Williamson

Reviewer #2: No

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PLoS One. 2020 Dec 2;15(12):e0233580. doi: 10.1371/journal.pone.0233580.r004

Author response to Decision Letter 1

22 Oct 2020

Response to reviewers’ comments.

Please thank the reviewers for their feedback and comments and suggestions. Below we detail our response to the reviewer’s comments. Reviewers’ comments are in italics and our responses are in regular font.

Reviewer 1:

General points:

I am concerned that my main criticism of this manuscript has still not been fully addressed. The study aim is to assess the impact of changes in suboptimal gas mixtures and temperatures during early-embryonic development to ascertain whether this could explain high rates of early embryonic death at high-density nesting beaches. Data on stage of embryonic death has not been presented for sufficient comparison of differences between the treatments. Furthermore, in this revised version, there is no longer any substantial mortality data included. This makes it difficult to definitively conclude that any of the treatments applied caused higher rates of early-embryonic death and poorer hatching success in general. The authors did apparently open 2 eggs (or potentially 4?) from each treatment to assess stage of embryonic development at the end of the treatment but this data has not been reported in the manuscript.

Response: The focus of the manuscript is early stage embryonic development, development that occurs within the first 7 days of the eggs being laid. The reason for this highly focused short period of incubation is that at high density nesting beaches, a high proportion of embryo mortality occurs during this period of development. There was virtually no early embryo death detected in any of our treatments during the first 7 days of incubation. However, for the few embryos that did die, we now report the stages that these embryos died at in the revised manuscript. A small sample of eggs were opened at the end of the 7 day treatment period, and all of these embryos were found to be alive. This is now reported in the revised manuscript: “All embryos were alive as indicated by a beating heart when dissected”.

Response: We agree with the reviewer’s comments, but our comments about temperatures above 34oC are based on previously published studies. We have adopted the reviewer’s suggestion and “tempered” our conclusion about high temperature death of early stage embryos, by replacing the would “likely” with “possible” in the revised manuscript: “Hence, high nest temperatures experienced early in incubation is a possible explanation for the large number of nests experiencing a high rate of early embryo death in high-density nesting aggregations.” And we have also tempered our conclusion later in the discussion: “At nesting beaches that experience high-density nesting, the close proximity of nests means that many newly laid clutches could experience high temperatures due to the metabolic heat production of nearby maturing clutches, and this might result in elevated rates of early embryo death, especially if temperatures exceed 34oC for long periods of time.”

I am concerned by the response to my initial suggestion in the first round of review:

“Page 8, Lines 188-191: I assume you excavated and staged the unhatched eggs from the

clutches that were reburied in artificial nests as well. You should provide a summary of these

data also. Or explain why this was not done. If the primary concern of this study is early stage

death I think it is quite important to present the data on which stage of development these

embryos died at. This part of the study has now been deleted from the manuscript.”

Response: If we had data on hatching success of eggs from each of our treatments, we certainly would have reported it, but we do not have this data. The reviewer has asked why we did not obtain this data. The reason is because of the logistics of obtaining such data. We had limited incubator space and time – we could not continue to incubate our eggs in incubators until they hatched – there was not enough incubator space. Our typical experiment group size for each clutch/treatment combination was 8-10 eggs. As the reviewer knows, such a small number of eggs incubated naturally at nest depth on a beach would not produce enough hatchlings to be able to dig themselves to the surface after they hatch, a group size of 25-30 hatchlings is needed. So, in our study we combined eggs from several clutch/treatments to form new clutches of 100/120 eggs that were incubated on the beach. Because within each of these combined clutches, we could not determine which hatchling came from which treatment, there was no point in evaluating the hatching success of these combined nests. The only reason we stated that eggs were buried to complete incubation on the beach was to assure readers that these eggs were not destroyed after our experimental manipulations. We have now added some further details to the methods section: “We relocated the surviving eggs into artificial nests consisting of 100 to 120 eggs pooled from the different treatments on Mon Repos Beach. Because we could not identify from which treatment hatchlings came from in these artificial nests, we could not assess the effect of treatment on hatching success, so this data is not reported.”

Reviewer 2 brings up a similar point which could be answered if the hatching success and stage of embryonic death data is supplied for the eggs that were reburied in artificial nests at Mon Repos:

“L261-263 Again, based on the presented data I’d like to know whether early hypoxia or

hypercapnia had an effect on late embryo mortality in ambient gas tensions. Did the authors

test for such an effect? Whatever the finding, this would be interesting to report in the results.

The data and discussion on late embryo mortality has been deleted from the revised

manuscript.”

Response: Unfortunately, we cannot make comment on this aspect because we do not know that the hatching success of eggs were. The only data we have on later embryo stage death was from the last two clutches of green turtle eggs incubated. We do not have this data for any of the loggerhead turtle experiments, or the first two clutches of green turtle eggs. So, as suggested by the reviewers in the first round of reviews, presenting this data added confusion to the main aim of assessing temperature and gas mixture effects on early stage death,. It is for this reason that we now do not report the data on later stage death of the last two clutches of green turtle eggs in the revised manuscript.

This is the only major concern I still have with the manuscript; the rest below should be straightforward to fix.

Specific points:

Page 2, Line 35: Spacing either side of "/" needs to be fixed. Response: done.

Page 4, Line 73: "low aggregation nesting aggregation beaches" needs to be changed. Suggest "low density nesting beaches" or "low density nesting beach aggregations". Response: This change has been made. Changed to “low density nesting beaches”.

Page 4, Line 78: Change "with the majority of embryos die at an early stage" to "with the majority of embryos dying at an early stage of development". Response: This change has been made as requested.

Page 9, Line 213: You should present a summary of the data on developmental stage of these opened eggs. The data is not supplied in the raw data file either. Response: This data is now reported in a new Table 3 in the revised manuscript.

Page 10, Line 221: "all three factors, clutch, temperature and clutch influenced growth" needs to be changed to "all three factors, clutch, temperature and gas influenced growth". Response: This change has been made.

Page 10, Line 226-227: "became increase different" needs to change to "became increasingly different" Response: This change has been made.

Page 15, Line 339: The journal title is abbreviated here whereas other journal titles in the list are not abbreviated. Change for consistency / journal style. Response: This change has been made.

Page 19, Line 435: Change "treatment in for green" to "treatment for green". Response: This change has been made.

Attachment

Submitted filename: Response to reviewers October 2020.docx

Click here for additional data file.^{(34KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0233580.r005

Decision Letter 2

Frank Melzner

17 Nov 2020

The effect of respiratory gases and incubation temperature on early stage embryonic development in sea turtles

PONE-D-20-13033R2

Dear Dr. Booth,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Frank Melzner

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: I am satisfied that the authors have made sufficient changes to the manuscript to address my previous concerns. I congratulate the authors again on completing an important and needed study. I believe that this article should now be accepted for publication in PLOS One.

I don’t have any further suggestions for changes or edits to the manuscript. My only comment is, for future studies, the authors should consider marking individual eggs with pencil in multiple locations prior to reburial when logistics, such as limited incubator space and time, require that eggs be reburied in nests with eggs from different treatments or females mixed together. It is not guaranteed to work, but provided the eggshells haven’t degraded too much when you excavate the nest you can identify which individual eggs hatched and which did not.

Reviewer #2: The authors have done a great job in addressing my previous comments and I see all my major points addressed in this revised version of their manuscript. The discussion and main conclusions follow nicely from the available data and there is a clear “red line” throughout the paper. I only have two minor suggestions for the authors to consider as they see fit, but have no further comments.

L204 and 209 The authors refer to Miller’s developmental stages throughout this section of the results, a metric which has not been introduced previously in the manuscript. For the naïve reader, like myself, it may be worthwhile describing the method in a sentence and mention what it’s based on and the range it uses (e.g. 0 is oviposition and 15 or so is hatch?).

One of the major conclusions of the paper is that warmer nesting temperatures are more likely to decreasing hatching success compared to adverse gas conditions. The authors talk about the effect of metabolically produced heat in high nesting areas that could cause such increased mortality rates, but not whether rising temperatures in the environment are accelerating these trends. Do the authors have any evidence that average nesting temperatures have increased due to climate change? If such data exist it may be worthwhile mentioning this in a sentence or two, perhaps in the last section on management strategies and perspectives. In my mind, this would be a nice way to round up the study and raise awareness about the need for conservation strategies, which would further strengthen the paper.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Sean Alexander Williamson

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0233580.r006

Acceptance letter

Frank Melzner

20 Nov 2020

PONE-D-20-13033R2

The effect of respiratory gases and incubation temperature on early stage embryonic development in sea turtles.

Dear Dr. Booth:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Frank Melzner

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Dataset

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

The effect of respiratory gases and incubation temperature on early stage embryonic development in sea turtles

David Terrington Booth

Alexander Archibald-Binge

Colin James Limpus

Roles

Abstract

Introduction

Methods

Ethics statement

Sources of eggs

Egg incubation

Statistical analysis

Results

Embryo mortality

Table 1. Experimental treatments used to incubate loggerhead turtle (Carretta caretta) eggs and the absolute number as well as the proportion of embryos alive at day 7 after 5.5 days exposure to the gas mixture incubation treatment.

Table 2. Experimental treatments used to incubate green turtle (Chelonia mydas) eggs and the absolute number as well as the proportion of embryos alive at day 7 after 5.5 days exposure to the gas mixture incubation treatment.

Embryo development

Table 3. Data relating the embryonic development stage (Miller et al. [22]) to the relative white-patch area for loggerhead and green turtle embryos after 7 days of incubation.

Table 4. Mixed factor ANOVA table for loggerhead turtle (Carretta carretta) egg arcsin transformed relative white-patch area after 7 days exposure to incubation temperature, and 5.5 days exposure to gas mixtures.

Table 5. Mixed factor ANOVA table for green turtle (Chelonia mydas) egg arcsin transformed relative white-patch area after 7 days exposure to incubation temperature, and 5.5 days exposure to gas mixtures.

Fig 1. Plot of relative white-patch size on day 7 after 5.5 days of exposure to different gas mixes at different incubation temperatures for loggerhead (Carretta carretta) and green turtle (Chelonia mydas) eggs.

Discussion

Respiratory gases

Incubation temperature

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Frank Melzner

Roles

Author response to Decision Letter 0

Decision Letter 1

Frank Melzner

Roles

Author response to Decision Letter 1

Decision Letter 2

Frank Melzner

Roles

Acceptance letter

Frank Melzner

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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