Effects of postovipositional hypoxia and hyperoxia on leatherback turtle reproductive success and hatchling performance.

Leatherback egg clutches typically experience lower hatching success (~50%) than those of other sea turtle species (>70%). The majority of embryonic death (>50%) occurs at early stages of development, possibly because embryos fail to break preovipositional embryonic arrest after oviposition. The embryonic arrest is maintained by hypoxia in the oviduct and following oviposition increased availability of oxygen is the trigger that breaks arrest in all turtle species studied to date. We conducted an ex situ incubator experiment and an in situ hatchery experiment to examine the influence of oxygen availability on hatching success and hatchling traits in leatherbacks. After oviposition, eggs (n = 1005) were incubated in either normoxia (21% O2 ), hyperoxia (32%-42% O2 ) for 5 days, or hypoxia (1% O2 ) for 3 or 5 days. As with other turtles, hypoxic incubation maintained embryos in arrest, equivalent to the time spent in hypoxia. However, extending arrest for 5 days resulted in greater early-stage death and a significant decrease in hatching success (4% 5-day hypoxia vs. 72% normoxia). Eggs placed in incubators had greater hatching success than those placed into hatchery nests (67% vs. 47%, respectively). We found no impact of hyperoxia on the stage of embryonic death, hatching success, hatchling phenotype, exercise performance, or early dispersal. Our findings indicate that delayed nesting and the subsequent extension of embryonic arrest may negatively impact embryonic development and therefore the reproductive success of leatherbacks. They also indicate that incubation under hyperoxic conditions is unlikely to be a useful method to improve hatching success in this species.

Mitigating the effects of climate change on the nests of sea turtles with artificial irrigation.

For sea turtles, like many oviparous species, increasing temperatures during development threaten to increase embryonic mortality, alter offspring quality, and potentially create suboptimal primary sex ratios. Various methods are being implemented to mitigate the effects of climate change on reproductive success, but these methods, such as breeding programs, translocations, and shading, are often invasive and expensive. Irrigation is an alternative strategy for cooling nests that, depending on location, can be implemented relatively quickly and cheaply. However, multiple factors, including ambient conditions, nest substrate, and species characteristics, can influence irrigation success. Additionally, irrigation can vary in duration, frequency, and the volume of water applied to nests, which influences the cooling achieved and embryonic survival. Thus, it is critical to understand how to maximize cooling and manage risks before implementing irrigation as a nest-cooling strategy. We reviewed the literature on nest irrigation to examine whether artificial irrigation is feasible as a population management tool. Key factors that affected cooling were the volume of water applied and the frequency of applications. Embryonic responses varied with species, ambient conditions, and the timing of irrigation during development. Nest inundation was the key risk to a successful irrigation regime. Future irrigation regimes must identify clear targets, either primary or adult sex ratios, that maximize population viability. Monitoring population responses and adjusting the irrigation regime in response to population characteristics will be critical. Most studies reported on the manipulation of only one or two variables, further research is required to understand how altering multiple factors in an irrigation regime influences the cooling achieved and embryonic responses.

Como sucede con muchas especies ovíparas, el incremento en las temperaturas durante el desarrollo embrionario puede aumentar la mortalidad embrionaria, alterar la calidad de la descendencia y tiene el potencial de crear proporciones sexuales primarias poco óptimas en las tortugas marinas. Se están implementando varios métodos para mitigar los efectos del cambio climático sobre el éxito reproductivo, aunque dichos métodos (p. ej.: los programas de reproducción, reubicaciones y sombreado) suelen ser invasivos y costosos. La irrigación es una estrategia alternativa para el enfriamiento de los nidos, la cual, según la ubicación, puede implementarse de manera rápida y económica. Sin embargo, factores como las condiciones ambientales, el sustrato de anidación y las características de la especie pueden influir sobre el éxito de la irrigación. Además, la duración, frecuencia y volumen del agua aplicada a los nidos durante la irrigación puede variar, lo que influye sobre el enfriamiento y la supervivencia embrionaria. Por todo esto, es importante entender cómo maximizar el enfriamiento y gestionar los riesgos antes de implementar la irrigación como estrategia de enfriamiento de nidos. Revisamos la literatura sobre la irrigación de nidos para analizar si la irrigación artificial es una herramienta viable de manejo poblacional. Los factores clave que afectaron el enfriamiento fueron el volumen aplicado de agua y la frecuencia de las aplicaciones. Las respuestas embrionarias variaron según la especie, condiciones ambientales y el momento de irrigación durante el desarrollo. El principal riesgo para un régimen exitoso de irrigación fue la inundación del nido. Los próximos regímenes de irrigación deben identificar objetivos claros, ya sean las proporciones sexuales adultas o primarias, que maximicen la viabilidad poblacional. Para esto, serán muy importantes el monitoreo de las respuestas poblacionales y el ajuste del régimen de irrigación en respuesta a las características de la población. La mayoría de los estudios reportaron la manipulación de una o dos variables, por lo que se requiere de mayores estudios para entender cómo la alteración de varios factores en el régimen de irrigación influye sobre el enfriamiento obtenido y las respuestas embrionarias.

Increasing hypoxia progressively slows early embryonic development in an oviparous reptile, the green turtle, Chelonia mydas.

Green turtle (Chelonia mydas) embryos are in an arrested state of development when the eggs are laid, but in the presence of oxygen, arrest is broken and development resumes within 12-16 h. However, the precise oxygen level at which embryos break arrest and continue development is not known. To better understand the impact of oxygen concentration on breaking of arrest and early embryonic development, we incubated freshly laid eggs of the green sea turtle for three days at each of six different oxygen concentrations (less than or equal to 1%, 3%, 5%, 7%, 9% and 21%) and monitored the appearance and growth of white spots on the shell, indicative of embryonic development. As reported previously, white spots did not develop on eggs incubated in anoxia (less than or equal to 1% oxygen). For all other treatments, mean time to white spot detection and white spot growth rate varied inversely with oxygen concentration. In nearly all cases the difference between eggs at different oxygen levels was statistically significant (p ≤ 0.05). This suggests that sea turtle embryonic development may respond to oxygen in a dose-dependent manner. Our results indicate that the development of green turtle embryos may be slowed if they are exposed to the most hypoxic conditions reported in mature natural nests.

Transcriptomic analysis of preovipositional embryonic arrest in a nonsquamate reptile (Chelonia mydas).

After gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with development recommencing in an apparently normal fashion after subsequent exposure to normoxia. To examine the transcriptomic events associated with embryonic arrest in green sea turtles (Chelonia mydas), RNA-sequencing analysis was performed on embryos from freshly laid eggs and eggs incubated in either normoxia (oxygen tension ~159 mmHg) or hypoxia (<8 mmHg) for 36 h after oviposition (n = 5 per group). The patterns of gene expression differed markedly among the three experimental groups. Normal embryonic development in normoxia was associated with upregulation of genes involved in DNA replication, the cell cycle, and mitosis, but these genes were commonly downregulated after incubation in hypoxia. Many target genes of hypoxia inducible factors, including the gene encoding insulin-like growth factor binding protein 1 (igfbp1), were downregulated by normoxic incubation but upregulated by incubation in hypoxia. Notably, some of the transcriptomic effects of hypoxia in green turtle embryos resembled those reported to be associated with hypoxia-induced embryonic arrest in diverse taxa, including the nematode Caenorhabditis elegans and zebrafish (Danio rerio). Hypoxia-induced preovipositional embryonic arrest appears to be a unique adaptation of turtles. However, our findings accord with the proposition that the mechanisms underlying hypoxia-induced embryonic arrest per se are highly conserved across diverse taxa.

Nearshore neonate dispersal of Atlantic leatherback turtles (Dermochelys coriacea) from a non-recovering subpopulation.

The cryptic 'lost years' of sea turtles challenge conservation efforts due to unknown movements and habitat utilisation of young life stages. Behavioural information strengthens dispersal and habitat utilisation models estimating unidentified movements. In this study, leatherback hatchlings were actively tracked with miniature acoustic tags off the east coast of Costa Rica for 83.15 min (± 9.12 SD) to determine their movements and swimming behaviour. Drifters were deployed throughout the tracking process to obtain surface current data. Hatchling (n = 42) over-ground and in-water swimming speed and bearing were calculated. Mean over-ground distance travelled was 2.03 km (± 0.71 km SD) with an over-ground average swim speed of 0.41 m/s (± 0.15 m/s SD). Mean bearing was 108.08° (± 20.19° SD) compared to the 137.56° (± 44.00° SD) bearing of nearshore ocean currents during tracking. Hatchling mean in-water swimming speed was 0.25 m/s (± 0.09 m/s SD). The lower in-water speed suggests hatchlings were advected by the currents, with overall movement strongly influenced by the current direction. This information can be assimilated into broader spatiotemporal distribution models to interpret the influence of directional swimming on ecosystem utilisation and help to achieve informed management decisions across all life stages of the population.

Synchronised nesting aggregations are associated with enhanced capacity for extended embryonic arrest in olive ridley sea turtles.

Sea turtle species in the genus Lepidochelys exhibit an unusual behavioural polymorphism, nesting in both aggregations and solitarily. Aggregated nesting events, termed 'arribadas', involve hundreds of thousands of females congregating at a single nesting beach over a few days to oviposit their eggs. Aggregate and solitary nesting behaviours are associated with distinct inter-nesting intervals, three and four weeks for non-arribada and arribada nesters respectively. Consequently, embryos are maintained in pre-ovipositional embryonic arrest in the hypoxic oviduct for different lengths of time depending on the mother's reproductive behaviour. However, sea turtle embryos are limited in their capacity to remain in arrest and will subsequently die if held in hypoxia too long. Here, we tested whether embryos oviposited during arribada or non-arribada nesting differ in their capacity to be maintained in pre-ovipositional arrest. Olive ridley turtle (Lepidochelys olivacea) eggs from eight clutches (four from each nesting tactic) were divided among seven treatments after oviposition; normoxia (control; 21% O2), or hypoxia (1% O2) for 3, 3.5, 4, 8, 15 or 30 days, before being returned to normoxia. Arribada eggs were capable of extending pre-ovipositional arrest for longer, with some eggs from the 8- and 15-day hypoxia treatment still hatching while no non-arribada eggs hatched after more than four days in hypoxia. This difference in embryonic capacity to survive extended periods of arrest may be an important mechanism facilitating arribada behaviour by allowing longer inter-nesting intervals. Our finding provides an intriguing insight into the physiological mechanisms that are integral to this unique mass-nesting behaviour.

When Is Embryonic Arrest Broken in Turtle Eggs?

Turtle embryos enter a state of arrested development in the oviduct, allowing the mother greater flexibility in her reproductive schedule. Development recommences once eggs transition from the hypoxic oviduct to the normoxic nest. Significant mortality can occur if turtle eggs are moved between 12 h and 20 d after oviposition, and this is linked to the recommencement of embryonic development. To better understand the timing of developmental arrest and to determine how movement-induced mortality might be avoided, we determined the latency (i.e., time elapsed since oviposition) to recommencement of development following oviposition by exposing the eggs of green turtles (Chelonia mydas) to hypoxia (oxygen tension <8 mmHg) for 3 d, commencing 30 min to 48 h after oviposition. Embryonic development-including development of the characteristic opaque white spot on the eggshell-was halted by hypoxic incubation. When the delay before hypoxic incubation was 12 h or less, hatching success did not differ from a control group. If the hypoxic treatment began after 16 h or more in normoxia, then all embryos died. Thus, by returning eggs to a hypoxic environment before they have broken from arrest (i.e., within 12 h of oviposition), it is possible to extend embryonic arrest for at least 3 d, with no apparent detriment to hatching success. Therefore, hypoxic incubation may provide a new approach for avoidance of movement-induced mortality when conservation or research efforts require the relocation of eggs. Our findings also suggest that movement-induced mortality may have constrained the evolution of viviparity in turtles.

Ecological and evolutionary significance of a lack of capacity for extended developmental arrest in crocodilian eggs.

Hypoxia within the oviducts maintains embryonic arrest in turtles at the pre-ovipositional stage, which expands the timeframe over which nesting can occur without compromising embryo survival. The arrest can be extended post-oviposition through incubation of eggs in hypoxia. We determined whether crocodilian embryos have this same capacity. We also tested whether increased oxygen availability during incubation alters hatching success. We incubated freshly laid saltwater crocodile (Crocodylus porosus) eggs (N = 83) at 32°C in one of five treatments; control (normoxia; 21% O2), 3-day and 6-day hypoxia (1% O2), or 3-day and 6-day hyperoxia (42% O2). Incubation (approx. 82 days) was then completed in normoxia. There was a significant effect of treatment on survival of embryos through to hatching (p < 0.001). The hypoxic treatments resulted in almost no hatching (6.7% and 0% survival for the 3- and 6-day treatments, respectively), while the hyperoxic and control treatments resulted in normal to high hatching success (86.6%, 100% and 64.2% for the control, 3- and 6-day hyperoxic treatments, respectively). Unlike turtles, hypoxic incubation of crocodile eggs failed to delay development. Our results provide the first experimental evidence that, unlike turtles, crocodiles do not exhibit embryonic arrest when incubated under hypoxic conditions immediately following oviposition. An absence of embryonic arrest is of ecological and evolutionary significance, as it implies that crocodilians lack an ability to avoid adverse environmental conditions through delayed nesting and that, unlike turtles, embryonic arrest may not be a potential explanation for the lack of viviparity in the order Crocodylia.

Hematology and Serum Biochemistry for Free-ranging Freshwater Crocodiles ( Crocodylus johnstoni ) in Western Australia.

We determined reference ranges for hematology and serum biochemistry in 39 free-ranging freshwater crocodiles ( Crocodylus johnstoni ). Animals were captured from Windjana Gorge in Western Australia and physically restrained for blood sampling from the supravertebral vein. No significant sex differences were found. Four animals were infected with a haemogregarine-like intraerythrocytic parasite.

Is gastrointestinal plasticity in king quail (Coturnix chinensis) elicited by diet-fibre or diet-energy dilution?

Phenotypic plasticity of organ size allows some animals to manage fluctuations of resource quality or availability. Here, we examined the phenotypic plasticity of the gastrointestinal tract of king quail (Coturnix chinensis) in a diet-fibre manipulation study. Quail were offered either a control low-fibre (high-quality) food (8.5% neutral-detergent fibre; NDF), or one of two experimental diets of higher fibre contents of 16% NDF (i.e. low-quality food). To examine whether phenotypic plasticity of organ size was associated with the fibre content per se, or as a consequence of diluting the diet energy contents by adding fibre, one of the high-fibre feeds was 'balanced' with additional energy to match that of the low-fibre control diet. Total empty dry mass of the gastrointestinal tract was significantly heavier among birds offered the unbalanced high-fibre diet as compared with those offered the control diet, with birds offered the fibrous but energy-balanced diet having guts of intermediate size. The heavier entire-gut mass (dry) of quail offered the unbalanced high-fibre diet was associated mainly with these birds having significantly heavier gizzards. Notably, the larger gizzard in the birds offered the unbalanced high-fibre diet was associated with marked increases in their metabolisability (digestion) of diet fibre. Our findings suggest that the available energy in the diet may be more important for eliciting phenotypic changes in the gut of these herbivorous birds rather than simple physical effects of diet fibre on feed intakes or on muscular compensation to fibrous ingesta.