Projected ocean temperatures impair key proteins used in vision of octopus hatchlings

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.


| INTRODUC TI ON
Increases in global temperatures have led to habitat loss and altered species distribution and abundance, with many species at risk of extinction (Bellard et al., 2012).Marine ecosystems are particularly vulnerable to global warming as oceans serve as the largest heat sink on Earth and have already absorbed more than 80% of the heat added to the global climate system (Poloczanska et al., 2013).By the end of the century under business-as-usual scenarios, it is predicted that temperatures will continue to rise by another 5°C, while sea surface temperatures are predicted to rise by 3°C (IPCC, 2022).
Determining biological responses to global warming is therefore crucial in understanding how ecosystems may be altered under future environmental conditions (Pan et al., 2015) and therefore how we can best adapt to these changes.
To predict how species will respond to changing temperatures, a thorough understanding of the molecular processes underpinning temperature adaptation is required.Proteomics is an advanced and powerful tool that allows for the comprehensive analysis of all proteins expressed by the genome, which improves our understanding of physiological processes.Natural selection directly operates on phenotypic traits, which largely depend on the proteome.The proteome is responsible for most of the structural and functional determinants of cellular and organismal phenotypes.Therefore, it offers a more immediate reflection of the traits that are being evolutionarily favoured than either the genome or the transcriptome and thus reveals insights into organism adaptation under a given stressor (Diz et al., 2012).Despite the clear advantages of proteomics, its application in ecological studies is still scarce (Tomanek, 2014).
Temperature has a significant effect on physiological processes, and how proteome adjustments counteract temperature changes remains a key question (Domnauer et al., 2021).Sensing and responding to environmental stressors such as thermal changes have a strong cellular and molecular basis, which has been demonstrated through proteomic and other studies.A well-known mechanism for responding to stressors involves the activation of the cellular stress response, such as the induction of heat shock proteins and antioxidant enzymes (Kültz, 2005).Understanding changes in protein expression and the types of proteins that contribute to thermal tolerance or sensitivity can therefore help to predict and assess the impact of global warming on species performance.Moreover, such studies can identify the physiological functions that are likely to represent the bottlenecks of successful adaptation.
The early life stages of many species are particularly susceptible to the effects of global warming, which can have detrimental effects on their survival (Bellard et al., 2012;Cline et al., 2019;Klockmann & Fischer, 2017;Przeslawski, 2004;Rosa, Baptista, et al., 2014).However, the small sizes at early life history stages make it challenging to study them in the wild.Experimental studies can also isolate effects of specific environmental stressors such as temperature from confounding effects of other parameters and provide useful insights into their specific contributions under future conditions.Cephalopods (squid, cuttlefish and octopus) are ecologically important as both predators and prey and comprise multiple species of high commercial interest.While some cephalopods appear to be increasing in abundance (Doubleday et al., 2016), a recent meta-analysis found that the impacts of global warming on cephalopods are largely negative, with warmer oceans arising as a threat to these taxa (Borges et al., 2023).For instance, it is well established that ocean warming reduces survival rate and metabolic rate leading to malformations and oxidative stress in early life cephalopods (Pimentel et al., 2012;Repolho et al., 2014;Rosa et al., 2012;Vidal et al., 2002).In the wild, some cephalopod species are already shifting their ranges poleward that is likely due to climate-driven warming (Oesterwind et al., 2022;Ramos et al., 2014).In spite of these negative, climate-driven impacts, whole proteome analyses of thermally stressed cephalopods to uncover the molecular and physiological underpinnings of these detrimental effects have yet to be done.Here, we experimentally exposed octopus embryos to current and projected future ocean temperatures and examined their proteome using state-of-the-art quantitative proteomics.We demonstrate that this approach can identify many differentially abundant proteins under heat stress and provide insight into key physiological processes underlying the vulnerability of early life stages to future climate conditions.

| Sample collection and experimental design
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.
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 preacclimated 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 2 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 (MgCl 2 ) for 10 min and then in 3.5% MgCl 2 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.
All experiments were approved by The University of Adelaide Animal Ethics Committee (approval no.S-2020-063) and were carried out in strict accordance with the Australian code for the care and use of animals for scientific purposes.

| Sample preparation for mass spectrometry
All proteomic analyses were carried out in the Kültz lab at the University of California, Davis using previously published workflows (Root et al., 2021) with the following changes: Frozen O. berrima embryos were crushed using a porcelain mortar and pestle after chilling with liquid nitrogen.Protein extraction, reduction, alkylation and in-solution trypsin/LysC digestion followed by C18 peptide clean-up were performed using an iST kit following manufacturer instructions (Preomics cat.#P.O.00027; Planegg-Martinsried, Germany).Peptides were lyophilised using a Savant SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in 50 μL liquid chromatography-mass spectrometry Optima grade water containing 0.1% formic acid (Thermo Fisher Scientific).Peptide resuspension was facilitated by bath sonication for 5 min.Peptide concentration was determined using a quantitative peptide assay (Pierce cat.#23290; Thermo Fisher Scientific), peptides were diluted to a concentration of 100 ng/ μL, and samples were transferred to total recovery glass vials (Waters Corporation, Milford, MA, USA) for sample injection.Two microliters (200 ng total peptides) were injected for each sample using a nanoAcquity sample manager (Waters Corporation) and trapped for 1 min at 15 μL/min (Symmetry, Waters Corporation).
Peptides were then separated (1.7 μm BEH C18, 250 mm; Waters Corporation) by reversed phase liquid chromatography at 300 nL/ min over 60 min at a three to 35% acetonitrile gradient (nanoAcquity binary solvent manager; Waters Corporation) before online introduction into the Impact II UHR-qTOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) via a 15 μM orifice picoemitter tip (New Objective, Littleton, MA, USA).All samples were processed as a single batch with Hystar 4.1 (Bruker Daltonics).
Bovine serum albumin standards (68 fmol) were used before and after the sample batch to verify stable instrument performance.

| Data-dependent acquisition for raw spectral library construction
Data-dependent acquisition (DDA) spectra were acquired with OTOF control 3 (Bruker Daltonics), and peak lists generated with DataAnalysis 4.4 (Bruker Daltonics) as previously reported (Root et al., 2021).Unambiguous peptide and protein annotations to spectrum matches were generated with PEAKS 10+ (Bioinformatics Solutions, Waterloo, Canada) using the O.
TA B L E 1 Sample information and experimental results of adult octopuses and their hatchlings.

| DIA and protein quantitation
A second acquisition was performed for each sample in DIA mode.
The same parameters as those used for DDA were applied in addi-

| Data analyses
Hatching rates per brooding octopus were calculated as a measure of embryonic mortality.Hatchling weights were also compared across different temperature treatments and visualised with a box plot in R version 4.2 (R Core Team, 2020) using the ggplot2 package (Wickham, 2016).The Kruskal-Wallis test was then conducted using the kruskal.testand pairwise.wilcox.testfunctions from base R to determine statistical significance for each pairwise comparison of treatments.
To determine the effects of thermal stress on protein expression levels and identify statistically significant proteins, MSstats integrated into Skyline 22.2 was used (Choi et al., 2014;Pino et al., 2020).Significance thresholds were set at adjusted p < .05 after correcting for multiple testing (Benjamini & Hochberg, 1995) at a protein abundance change of less than 0.5-fold or more than 2.0-fold.For statistical analysis using MSstats and Skyline, the confidence level was set to 95%, MS level to 2, scope to protein, normalisation method to equalise medians and summary method to sum of transitions.Volcano plots were generated in Skyline 22.2 for all three pairwise comparisons.To visualise the fold change of proteins of interest across different thermal conditions, a heat map was generated in R using the ggplot2 package (Wickham, 2016).In the heat map for downregulated proteins, fold change values are shown as absolute values for ease of comparison.For instance, a fold change of 0.5-fold is shown as 2.0fold reduction.

| Mortality under future warming
An average (±SD) of 173 ± 36 embryos per octopus brood were laid, with three broods per treatment (Table 1).More than 95% of all octopus embryos raised under control and current temperature treatments hatched.For two out of three broods raised under future temperatures, none of the embryos hatched; the third brood had a hatch rate of 43%.The average (±SD) hatching rates under control, current, and future temperatures are 98.1 ± 2.3%, 100 ± 0% and 14.2 ± 24.6%, respectively.The two brooding females at 25°C died naturally while their eggs were in the early stages of development, while the remaining female showed visible signs of stress (e.g., remained out of den, abnormal body posture and morphology) and was euthanised shortly after her first eggs hatched.In contrast, all six brooding females acclimated to 19 and 22°C did not exhibit any signs of stress and were euthanised after their last eggs hatched.

| Changes in protein abundance under different thermal conditions
The spectral library for the DIA mass spectrometry quantitative analysis of O. berrima samples included 1372 proteins comprising 5237 peptides and 31,712 transitions.Volcano plots showed that, compared with the control, there were few proteins (i.e.four proteins reduced, 28 proteins elevated) changing in abundance under current temperatures (Figure 1a).An intermediate number of proteins (i.e., 59 proteins reduced, 52 proteins elevated) changed under future temperatures when compared to current temperatures (Figure 1b).However, when compared to the control, 22 and five times more proteins significantly reduced and elevated respectively (i.e.88 proteins reduced, 138 proteins elevated) under future temperatures (Figure 1c).A complete list of all proteins is available in the Supporting Information for all three pairwise comparisons (Tables S1-S6).

| Future temperatures may impair octopus vision
Proteins with important roles in vision, including collagen, retinol dehydrogenase, S-crystallin and glutathione-S-transferase, were consistently downregulated under future temperatures compared to either the control or current temperatures (Figure 2).Fold

| Heat stress activates the cellular stress response
We found significantly elevated proteins that were involved in the cellular stress response including molecular chaperone activity, cell death, energy metabolism, redox regulation and protein syn-

| Reduction in non-essential processes under future temperatures
Significant downregulation was found in proteins involved in di-

| Size differences of hatchlings across treatments
The median weight of hatchlings significantly decreased as temperatures increased (Kruskal-Wallis chi-squared = 102.51,df = 2, p < .0001)(Figure 4).Hatchlings exposed to control (Kruskal-Wallis, p < .0001)and current (Kruskal-Wallis, p < .0001)temperatures were approximately twice the weight of hatchlings exposed to future temperatures.Hatchlings exposed to control conditions were slightly heavier (1.1× heavier) than those exposed to current temperatures (Kruskal-Wallis, p < .0001).

| DISCUSS ION
Here, we present the first proteomic analysis of octopus embryos exposed to current and future projected temperatures.The results shed light on the molecular determinants of the potential vulnerability of octopus embryos to future climate conditions, mainly in the form of impaired vision and activation of the energy-expensive cellular stress response.
Expression levels of proteins with major roles in vision were significantly reduced under future temperatures.Crystallins are important structural proteins found in high abundance in animal eye lenses that preserve lens transparency and optical clarity, but their aggregation can cause the formation of cataracts (de Jong et al., 1989;Tan et al., 2016).S-crystallins are found in cephalopods such as octopuses and have evolved from the cellular detoxification enzyme, glutathione-S-transferase, which transfers glutathione to xenobiotic electrophilic substrates like reactive oxygen species, thereby protecting cells from macromolecular damage (Tan et al., 2016).As a result of this evolutionarily conserved sequence, S-crystallins have a strong binding affinity to glutathione which is also typically found in high concentrations in the lens.This binding contributes to the stability of S-crystallins and prevents their aggregation and resulting cataract formation in cephalopod lens (Tan et al., 2016).Since  Jong et al., 1989), the significantly reduced expression of all three S-crystallin proteins in this study suggests that future temperatures may result in impaired vision in the octopus embryos.
In addition, retinol dehydrogenase 12, which was significantly downregulated, is an important retinal reductase found in high abundance in the inner segments of photoreceptors and is required for the regeneration of visual pigments as part of the visual cycle (Belyaeva et al., 2005;Sarkar & Moosajee, 2019).It is also a major antioxidant protein that has a protective effect against toxic lipid peroxidation products which can damage photoreceptors.In addition to its role in lens transparency, S-crystallin also functions as a heat shock protein crucial for the proper folding of proteins (de Jong et al., 1989).Even though these two proteins serve additional functions in antioxidant defences and protein refolding throughout the body, they are found in the highest levels in the eyes, and their levels in this study are thus representative of their expression in the eyes.
Therefore, reduced levels of these multi-functional proteins (retinol dehydrogenase 12 and S-crystallins) not only contribute to a reduced capacity for protein refolding and oxidative stress responses especially in the eyes but also result in impaired vision under future temperatures.
Glutathione-S-transferase, which plays a crucial role in lens detoxification and protection against peroxidation damage (de Jong et al., 1989), was likewise downregulated significantly.Regulating the redox potential of the lens and keeping lens proteins in the reduced state requires protection by antioxidants and oxidation defence systems (Lou, 2003).Cephalopods are visual predators virtually immediately upon hatching and unsurprisingly, embryos already possess large eyes relative to their body size (Wild et al., 2015).Hence reduced levels of glutathione-S-transferase in the eyes could also contribute to impaired vision.
Furthermore, collagen is an abundant protein found in connective tissues, providing structural support in tissues such as the skin, tendons and cartilage.The proper functioning of the eye is dependent on the structural roles and properties of collagen that contribute to the transparency and refraction of corneas and lens (Ihanamäki et al., 2004).Marneros et al. (2004) has also shown that collagen is important for vision and the function of the retinal pigment epithelium.Although information on cephalopod-specific collagen is still scarce (Imperadore et al., 2018), we speculate that based on the current knowledge of collagen functions, significantly reduced levels of collagen in octopus embryos under future temperatures can severely affect their vision.
F I G U R E 3 Heat map of significantly elevated protein levels across different thermal conditions.Similar proteins were significantly upregulated under future temperatures when compared to the control (left) and current (right) temperatures, as well as under current temperatures when compared to the control (middle) (adjusted p < .05 in MSstats).Proteins are grouped according to their key functions which are labelled on the right of the heat map.Fold change values are labelled for each protein and each treatment for ease of comparison.Grey bars refer to non-significant changes for the protein in that treatment.
We note that the reduced expression of proteins involved in vision only occurred under future temperatures, suggesting the severity of future thermal stress on octopus vision.To the best of our knowledge, the impact of thermal stress on vision has not yet been reported in octopuses.Thus, our study breaks new ground for investigating temperature effects on this specific physiological function in cephalopods.From an ecological perspective, visual impairment can have profound impacts on octopus survival as they rely on vision for camouflage, detecting prey and predators, and communication (Hanlon, 2007;Mäthger et al., 2009;Villanueva et al., 2017).
Octopuses are highly visual predators with a diverse diet and compromised fitness in this taxon could trigger cascading effects in the food web.
Exposure to higher temperatures imposes strain on cells that often result in protein denaturation, damage to DNA and membranes as well as increased oxidative stress (Kültz, 2003).Increased heat also enhances metabolic rates and stimulates physiological processes, thus organisms require more energy to counteract damage at the expense of growth, reproduction and immunity (Alfonso et al., 2021).Damage to macromolecules (proteins, DNA, membranes) activates the cellular stress response which involves defence mechanisms in cells to repair the damage (Kültz, 2005).
Defence mechanisms utilise an evolutionarily conserved set of genes and pathways to maintain cellular and organismal integrity.
These mechanisms include induction of heat shock proteins such as molecular chaperones, which had significantly elevated levels in the octopus embryos only under future temperatures.Chaperones are essential proteins that recognise unfolded proteins and either designate them for removal or assist in their refolding (Gething & Sambrook, 1992).Considering that the cleavage and polyadenylation specificity factor subunit 1 and serine/threonine-protein kinase PRP4 homolog in our study are among the most highly expressed proteins at 32 and 27 times, respectively, compared to the control, it can be inferred from their roles in pre-mRNA splicing that levels of protein synthesis are significantly enhanced (Gross et al., 1997;Murthy & Manley, 1995).Since proteins serve a variety of functions including growth, immunity, regulation of gene expression and structural support, the indirect role of chaperones in maintaining these functions is particularly important in ensuring integrity at the cellular and organismal level and ultimately survival of the organism especially under stress.
DNA damage can occur in the form of double-or single-strand breaks, which can be sensed by DNA topoisomerases that are responsible for altering DNA topology (Kültz, 2005).For instance, DNA topoisomerase II, which was upregulated under future temperatures, introduces double-strand breaks to eliminate overwinding in DNA (Nitiss, 2009).This upregulation suggests marked DNA damage in this treatment.Macromolecular damage also occurs within cell membranes, manifesting as changes in permeability, lipid arrangement and peroxidation.Phospholipid-transporting ATPase VD is the catalytic component of the P4-ATPase flippase, which is responsible for the transport of phospholipids to maintain lipid composition of cell membranes (Timcenko et al., 2019).Upregulation Oxidoreductases assist in oxidative damage repair by producing reducing equivalents for antioxidant enzymes (Kültz, 2005), thioredoxins are also part of the oxidoreductase family that serve as electron donors in antioxidant defences (Udayantha et al., 2021).
Changes in the cellular redox state serves as a trigger of the cellular stress response, thus the activation of these free radical scavenging systems is expected under exposure to thermal stress.The increase in energy required to maintain molecular chaperoning and damage repair activities is also reflected by the elevation of two metabolic enzymes, pyruvate dehydrogenase E1 component subunit beta and acetyl-CoA acetyltransferase A in the mitochondria.Pyruvate dehydrogenase complexes are a key link between glycolysis and the Krebs cycle in the cellular production of energy by converting pyruvate to acetyl-CoA (Zhou et al., 2001).Besides glycolysis, acetyl-CoA can also be derived from ketogenesis via fatty acid beta-oxidation.
Acetyl-CoA acetyltransferase A can then catalyse the interconversion of acetoacetyl-CoA and acetyl-CoA, which is then available for energy production (Fukao et al., 1997;Goudarzi, 2019).Taken together, these findings suggest that in octopus embryos exposed to temperatures reflective of 2100, these adaptive mechanisms need to be activated to increase energy production to meet the elevated energetic demands required for cellular stress responses including the repair of macromolecular damage.
The reduction in levels of proteins involved in digestion and nucleic acid metabolism also reflects the diversion of precious energy resources from less essential processes to more critical physiological processes.For instance, under future temperatures, both chymotrypsinogen A, the inactive precursor of chymotrypsin 1, and its active counterpart were significantly downregulated.These enzymes are primarily responsible for catalysing the breakdown of proteins into smaller peptides within the intestines (Appel, 1986).
Furthermore, the marked reduction in the levels of cytosolic 10-formyltetrahydrofolate dehydrogenase suggests a decrease in nucleotide biosynthesis (Krupenko et al., 2019).The significant increase in levels of serine/threonine-protein kinase 11-interacting protein-like isoform X1 also indicates a redirection of resources away from growth promotion as serine/threonine-protein kinase 11 negatively controls cell growth and acts as a tumour suppressor (Shackelford & Shaw, 2009).
Although stress responses can help organisms adapt to environmental stressors, these physiological adjustments may not be enough under exceedingly challenging conditions that the organism cannot habituate to (Alfonso et al., 2021), which seems to be the case in this study.This was also evident from the high mortality rate observed in both maternal adults and octopus embryos exposed to future temperatures, suggesting that they were near thermal tolerance limits in this condition.Given that maternal care of embryos occurs in octopuses, thermally stressed females under future warming conditions may have potentially affected embryo survival in addition to direct thermal effects on the embryos.Having shown visible signs of stress, females under future temperatures were likely under thermal stress, which has an important indirect effect on hatching success and the molecular phenotype, that is proteome of the offspring.In the case of cephalopods and other organisms with parental care, adults succumbing to high temperatures suggests that global warming has a simultaneous impact on multiple generations.While proteins under future warming conditions could only be analysed using a single brood due to the death of other broods, the few surviving hatchlings still showed an immense amount of thermal stress and are unlikely to survive into adulthood.
Moreover, our results coincide with other research that also examined the thermal impacts on early life stages, reiterating the vulnerability of this developmental period to the effects of climate change (Klockmann & Fischer, 2017;Przeslawski, 2004;Rosa, Baptista, et al., 2014;Rosa, Trübenbach, et al., 2014).While we note experimental studies are a simplification of reality and impacts observed in the lab may not directly translate to the wild, oxygen isotope data, analysed in the statoliths of wild-caught O. berrima, suggests that the species may already be seeking thermal refugia at current summer temperatures (Martino et al., 2022), further supporting our study that heat stress negatively impacts octopuses.
Given the findings of this study, we predict that global warming has detrimental effects on the embryonic stages of octopuses.
These cephalopods are of both public interest as a charismatic animal, and of commercial interest as an alternative protein source.
As voracious predators that occupy a broad trophic niche, the impaired survival of such an ecologically significant taxon can alter ecosystem functioning by influencing food-web dynamics.
Not only are octopuses facing fishing pressure (Hua, Thomson, et al., 2023), but the predicted negative impacts of global warming on octopus survival from our study also suggest that they are to thermal stress (Hua, Young, et al., 2023).A mechanistic understanding of how species would perform under future warming will be critical in developing effective mitigation strategies to help protect vulnerable species and ecosystems.
tion to the following DIA-specific parameters: mass range of 390-1015 m/z; scan rate at 25 Hz; isolation width of 10 m/z with 0.5 m/z overlap; scan interval of 2.5 s; mass error threshold of 20 ppm; resolving power was 30,000; quantitation scope = protein; normalisation = equalise median; summary method = sum of transitions.At least four (generally six) transitions were scored for each peptide.All proteomics raw data (DDA, DIA), Skyline target lists, spectral libraries and quantitative results are available under restricted access at PanoramaPublic (accession #QQ0001kl) and ProteomeXchange (accession #PXD040795).
change of collagen ranged from −3 to −18 when compared to the control, and from −3 to −14 when compared to current temperatures.Fold change of retinol dehydrogenase 12 was twice as low under future temperatures at −13 than under current temperatures at −6. Collagen alpha-1 (XXVI) and retinol dehydrogenase 12 were the proteins with the highest fold change (−18 and −13 respectively) among the significantly downregulated proteins (p < .0001).S-crystallin proteins had a fold change of approximately −2 underfuture temperatures, similar to that under current temperatures.Glutathione-S-transferase enzymes had a fold change range of −2 to −6 under future warming, also similar to that of −2 to −4 under current temperatures.None of the above proteins showed significant change under current temperatures when compared to the control.
gestion and DNA/RNA metabolism (Figure 2).Chymotrypsin 1 had similar levels of reduction under future temperatures when compared to the control (fold change = −3, p < .0001)and current (fold change = −2, p = .004)temperatures.Some proteins showed significant reduction under future temperatures only F I G U R E 1 Volcano plots showing relative changes in protein levels between temperature treatments.(a) Current (22°C) versus control (19°C) temperatures, (b) future (25°C) versus current (22°C) temperatures and (c) future (25°C) versus control (19°C) temperatures.Each plot represents data from one temperature treatment, which is bolded, compared with other temperatures.The y-axis represents statistical significance, and the x-axis represents magnitude of change, both of which are −log 10 and log 2 transformed, respectively, for easier visualisation.Each triangle or dot represents a protein.Orange triangles represent proteins that are significantly elevated under the higher temperature (adjusted p < .05 and fold change >2).Blue triangles represent proteins that are significantly reduced under the higher temperature (adjusted p < .05 and fold change <0.5).Grey circles represent proteins with non-significant changes.when compared to the control and not when compared to current temperatures.These included chymotrypsinogen A which is involved in digestion (fold change = −4, p < .0001),ATP-dependent RNA helicase DDX51 which is involved in RNA metabolism (fold change = −7, p < .0001),and cytosolic 10-formyltetrahydrofolate dehydrogenase which is involved in nucleotide synthesis, (fold change = −3, p < .0001).Lastly, the tumour suppressor protein involved in negative growth control, serine/threonine-protein kinase 11-interacting protein-like isoform X1, was significantly upregulated under future temperatures when compared to current temperatures (fold change = 2, p = .005)(Figure 3).
lens transparency and proper light refraction require a very high F I G U R E 2 Heat map of significantly reduced protein levels across different thermal conditions.Similar proteins were significantly downregulated under future temperatures (in bold) when compared to control (left) and current (right) temperatures (adjusted p < .05 in MSstats).Proteins are grouped according to their key functions which are labelled on the right of the heat map.Fold change values are shown as absolute values and labelled for each protein and treatment for ease of comparison.Grey bars refer to non-significant changes for the protein in that treatment.concentration of proteins like crystallins in the lens fibre cells (de of this catalytic component by 31 times under future temperatures indicates significant damage to cell membranes.Membrane damage in the form of lipid peroxidation and other oxidative damage in general could also be supported by the elevation of proteins involved in redox regulation, namely putative oxidoreductase GLYR1 isoform X1 and thioredoxin domain-containing protein 17.
under additional environmental pressure as they face an uncertain future in a changing climate.In addition, we have shown the usefulness of quantitative proteomics in identifying and quantifying changes in protein expression levels and thereby assessing the adaptability or vulnerability of organisms to environmental stressors.As the climate continues to warm, it is important to measure the biological responses of different species, particularly during their vulnerable early life stages, to global warming through the use of advanced tools that capture sublethal effects on fitness such as proteomics.Future studies should apply such powerful and predictive techniques in the field which are important and useful in measuring the proteomic responses of wild octopuses Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; software; validation; visualization; writing -original draft; writing -review and editing.Dietmar Kültz: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; resources; software; validation; writing -original draft; writing -review and editing.Kathryn Wiltshire: Investigation; resources; writing -review and editing.Zoe A. Doubleday: Conceptualization; funding acquisition; supervision; writing -review and editing.Bronwyn M. Gillanders: Conceptualization; funding

Mother ID Mean seawater temperature ± SD (°C) Mother wet weight (g) Embryonic duration (days between first spawning and first hatching) Hatching duration (days between first and last hatching) No. of eggs laid Hatching rate (%)
bimaculoides NCBI RefSeq proteome downloaded on 15 Jan 2023.This database consisted of 29,162 proteins.It was supplemented with an equal number of randomly scrambled decoys and 282 common contaminants.PEAKS 10+ database search parameters were applied as previously specified except for mass tolerance limits (10 ppm for precursors and 0.03 Da for fragment ions).DDA data (mzxml and pepxml) were exported from PEAKS 10+ and Skyline 22.2 was used to generate a non-redundant and unambiguous raw MS2 spectral library (blib).The initial target list of proteins, peptides and transitions included in this raw MS2 spectral library was automatically filtered to remove interferences and low-quality entries.Filter criteria were as follows: ion three to last ion; fragment ion charge one to two; precursor charge range one to five; ion types y and b; minimum four product ions, retention time within 5 min of MSMS IDs; MS2 mass accuracy threshold was within 20 ppm of the expected mass; maximum missed cleavages of one; excluding uncommon post-translational modifications; and minimum dotp value of 0.8.The final data-independent acquisition (DIA) assay library represents a target list of 31,712 transitions, 5715 precursors, 5237 peptides and 1372 proteins.This assay represents a tier two assay (Abbatiello et al., 2017).
thioredoxin domain-containing proteins which are related to