Weights of hatchlings across different temperature treatments

Global warming is one of the most significant and widespread effects of climate change. While early life stages are particularly vulnerable to increasing temperatures, little is known about the molecular processes that underpin their capacity to adapt to temperature change during early development. Using a quantitative proteomics approach, we investigated the effects of thermal stress on octopus embryos. We exposed Octopus berrima embryos to different temperature treatments (control 19 °C, current summer temperature 22 °C, or future projected summer temperature 25 °C) until hatching. By comparing their protein expression levels, we found that future projected temperatures significantly reduced levels of key eye proteins such as S-crystallin and retinol dehydrogenase 12, suggesting the embryonic octopuses had impaired vision at elevated temperature. We also found that this was coupled with a cellular stress response that included a significant elevation of proteins involved in molecular chaperoning and redox regulation. Energy resources were also redirected away from non-essential processes such as growth and digestion. These findings, taken together with the high embryonic mortality observed under the highest temperature, identify critical physiological functions of embryonic octopuses that may be impaired under future warming conditions. Our findings demonstrate the severity of the thermal impacts on the early life stages of octopuses as demonstrated by quantitative proteome changes that affect vision, protein chaperoning, redox regulation, and energy metabolism as critical physiological functions that underlie the responses to thermal stress.

Methods
Female Octopus berrima (Stranks & Norman 1992) (n = 9) were obtained in October 2021 (austral spring) from an artisanal octopus fishery at Venus Bay, South Australia using unbaited octopus pots. Unlike other merobenthic octopus species that produce small planktonic hatchlings, Octopus berrima is a holobenthic species that produce large, well-developed hatchlings (Hua, Nande, Doubleday, & Gillanders, 2023). Following capture, octopuses were transported in individual, 12 L aerated buckets of local seawater (15 °C) kept in insulated bags. Dens in the form of sectioned PVC pipes (65 mm diameter, 20 cm length) were provided for each octopus during transport. Octopuses were transported to the South Australian Research and Development Institute (SARDI) in Adelaide, where all experiments were conducted in a controlled environment room. All octopuses remained in their respective dens during transport and were transferred in their dens from the buckets into separate glass tanks (50 cm × 25 cm × 30 cm) with filtered (0.5 μm) flow-through seawater, and a constant photoperiod (12:12 h). Adults were pre-acclimated at 16 ± 1 °C (mean ± SD) and were fed three live shore crabs (Grapsidae) daily supplemented with occasional live mussels (Mytilidae) and oysters (Ostreidae). All tanks were cleaned daily and covered with shade cloth to reduce excessive light and to induce spawning. All females spawned in their dens between two and 66 days after being transported to the facility and stopped feeding following spawning. We expected different individuals to begin spawning at different times due to natural intra-female variation. To ensure that no females (and embryos) were exposed to the treatment temperatures longer than others pre-spawning, we only exposed them to their respective temperatures once eggs were laid. Females were pre-acclimated at 16 ± 1 °C (mean ± SD) until spawning, after which temperatures were raised by 1.4 ± 0.8 °C per day until respective temperature treatments were reached. Temperature treatments were: 19.3 ± 0.6 °C (control; equivalent to the lower end of current summer average temperature in South Australia, hereafter referred to as control), 22 ± 0.1 °C (higher end of current summer average temperature in South Australia, hereafter referred to as current temperature) and 24.6 ± 0.2 °C (higher end of future projected summer average temperature based on projections from IPCC (2022), hereafter referred to as future temperature) (Table 1). Eggs received maternal care for the entire embryonic duration (average 62 ± 7 days; Table 1) until all eggs had hatched. Tanks were checked daily for hatchlings. Embryos were deemed viable or non-viable by checking the state of the embryos by visual inspection (e.g. heart palpitation and continued development). One-day-old hatchlings were euthanised by immersion in 1.5% magnesium chloride (MgCl2) for 10 min and then in 3.5% MgCl2 for 30 min. Hatchlings were then lightly dried before their wet weights were measured using a micro-balance. Each specimen was placed in a cryogenic vial and snap-frozen in liquid nitrogen before storing at -80°C until subsequent analyses.