In this study, we examined how the gene expression patterns of gastrula and prism embryos varied by the developmental temperature and pCO2 conditions under which they were raised. We also assessed whether the transcriptomic results aligned with the morphometric and physiological results previously reported in . Although both temperature and pCO2 can influence rates of sea urchin development [38, 39], any potential differences in developmental timing should not have impacted the results of this study because samples were collected based on developmental progression to the desired embryonic stages as detailed in the Methods, rather than by hours post-fertilization. Overall, we found that while transcriptomic patterns varied by developmental stage, temperature had a dominant effect on changes in gene expression while pCO2 elicited a more subtle transcriptomic response that was largely limited to the gastrula stage. Experimental conditions impacted genes related to the cellular stress response, transmembrane transport, metabolic processes, and the regulation of gene expression.
In terms of experimental design, embryos were obtained by evenly pooling eggs from five females and fertilizing them with sperm from a single male to produce all full or half siblings. Admittedly, there are caveats to this approach. The results presented here may only be representative of a small subset of the population, or it may be driven by the quality of the particular male selected to fertilize the eggs. For instance, in the purple sea urchin S. purpuratus, genetic variation has been shown to influence transcriptomic responses to temperature and pCO2 stress during early development [40, 41]. We therefore recommend that additional studies be performed within other M. franciscanus populations and with multiple male-female crosses to determine if our results are unique to this study. Nevertheless, this approach was implemented in an effort to limit genetic variability and male-female interactions that may have otherwise confounded the molecular results of this study.
All samples used for RNA extractions were each composed of a pool of 5000 individuals and should thus represent the same mixture of genotypes. Therefore, we do not expect differences in gene expression patterns to be due to genetic variability between embryo cultures, particularly because a low incidence of mortality was observed during the experiment, although it was not directly measured in this study. In the absence of selection, the observed variability in gene expression, body size, and thermotolerance between embryos raised under different experimental treatments reflect plasticity exhibited by M. franciscanus during its early development. We discuss this plasticity, and how it may relate to embryo performance under different conditions that M. franciscanus are likely to experience in their natural environments currently and in the future under ocean change scenarios.
Transcriptomic patterns varied by developmental stage: General patterns
Developmental stage (gastrula or prism embryos) was the primary factor driving differences in gene expression patterns across samples (Fig. 1a and 1b). In a past study, we raised cultures of M. franciscanus embryos in a single laboratory environment that mimicked average, non-stressful conditions in situ (i.e., 15 °C and 425 µatm pCO2) and documented significant transcriptomic differences between gastrula and prism stages . Therefore, there are many alterations in gene expression between these stages that occur as a result of development and are independent of differences in environmental temperature and/or pCO2 conditions. This is also evident in Fig. 1a in which gastrula samples do not cluster with prism samples that share the same experimental treatment.
Because comparing gastrula versus prism gene expression patterns was not a goal of this study, no direct differential expression analyses were performed between stages. Nevertheless, embryos at each developmental stage exhibited different transcriptomic responses to temperature and pCO2 treatments. For instance, at the gastrula stage, many more genes were up-regulated than down-regulated in 17 °C relative to 13 °C, whereas at the prism stage, a similar number of genes were relatively up- and down-regulated between the two temperature treatments (Fig. 2). Additionally, the pCO2 treatment explained a significant amount of variance in gene expression in gastrula embryos, but not later at the prism stage (Fig. 1d and 1f). The difference in transcriptomic response by developmental stage was also evident in the WGCNA, in which the gastrula and prism temperature and pCO2 treatments were significantly correlated to many different module eigengenes (Fig. 5). Similarly, the morphometric response to temperature and pCO2 treatments varied by stage, in which only pCO2 affected gastrula by reducing body size under elevated pCO2 conditions (i.e., 1050 µatm) . On the other hand, temperature was the dominant factor at the prism stage, with warmer conditions (17 °C) increasing body size, offsetting the stunting effect of high pCO2 . The observed patterns between gene expression and body size will be described in greater detail later in the Discussion.
Different life stages are predicted to have different sensitivities to stress . The variability between gastrula and prism stress responses may be explained by a difference in stage-specific vulnerability. During the gastrula stage, the archenteron is formed from invagination of the embryo’s vegetal plate , a fundamental process known as gastrulation that is essential for successful development in metazoans . At the prism stage, the embryo develops its digestive tract and skeletal rods, which are vital structures required for the embryos to eventually become feeding, planktotrophic larvae [44, 45]. Accordingly, differences in responses to environmental conditions between these two stages are likely reflective of the distinct processes undergone by these embryos to ensure their continued developmental progression.
The variability between stages could also be due to the timing and duration of exposure to stress. The effects of a stressor can become increasingly deleterious as the length of exposure continues, and organisms not permitted adequate time to recover may exhibit increasingly poor performance. Furthermore, during development there may be negative carry-over effects that persist into later life stages [46, 47]. Alternatively, organisms may acclimate to stressful conditions over time, and are therefore less adversely affected by a stressor following the initial exposure. For example, in the coral Acropora hyacinthus, the immediate transcriptomic response to heat stress was much higher than the transcriptomic response following 20 hours of exposure to warmed conditions . Thus, it remains important to acknowledge that organisms may responds differently to various environmental stressors depending on their life history as well as the timing and duration of the exposure.
Temperature influenced gastrula embryos on a molecular level
Temperature was the dominant factor influencing changes in gene expression at the gastrula stage, explaining 19.2% of the observed variance (p < 0.001) with 4626 genes up-regulated and 1735 genes down-regulated in embryos raised under 17 °C relative to those raised under 13 °C (Figs. 1 and 2). In general, the observed temperature effects on gene expression at the gastrula stage were approximately akin to those reported for the purple sea urchin S. purpuratus , whose biogeographical distribution overlaps with that of M. franciscanus. Here, DE analysis and WGCNA revealed that gastrula raised in the higher temperature treatment (i.e., 17 °C) expressed genes associated the cellular stress response. Gastrula embryos of S. purpuratus that were raised under an 18 °C temperature treatment exhibited a comparable cellular stress response by up-regulating genes associated with cellular responses to reactive oxygen species and unfolded proteins . We also found that M. franciscanus gastrula embryos raised under the warmer treatment exhibited transcriptomic patterns indicative of increased transmembrane transport and metabolism, while embryos from the colder treatment appeared to decrease metabolic processes. Similarly, S. purpuratus increased expression of ion channel, cell-cell signaling, and metabolism genes at warmer temperatures . Lastly, temperature appeared to impact the regulation of gene expression in M. franciscnus gastrula embryos, including genes related to epigenetic and epitranscriptomic mechanisms. Temperature also appeared to influence how gene expression was regulated in S. purpuratus gastrula embryos with higher temperatures leading to a down-regulation of genes related to transcription and RNA processing .
Despite similarities in how temperature influenced gastrula gene expression, unlike in M. francicanus, there was an effect of temperature on S. purpuratus morphology. Specifically, S. purpuratus gastrula embryos raised at warmer temperatures were significantly smaller in size , whereas M. franciscanus gastrula embryos did not significantly differ in size as a result of temperature . This could reflect differences in experimental design between the studies (e.g., treatment temperatures, breeding designs, and urchin collection sites), or it could reflect differences that exist at the species level. Unlike S. purpuratus, the temperature response of M. franciscanus gastrula embryos that occurred at the molecular level was not reflected at the organismal level. We postulate that the transcriptomic differences between gastrula raised at 17 °C and 13 °C served to compensate for direct temperature effects and allowed the embryos to maintain the same size despite the temperature treatments. For the remainder of this section, we explore with greater detail how temperature affected the expression patterns of genes associated with the cellular stress response, transmembrane transport, metabolism, and gene expression regulation in M. franciscanus gastrula embryos.
Cellular stress response
Environmental stress can lead to the production of reactive oxygen species (ROS), which can cause oxidative stress if ROS production exceeds the organism’s antioxidant or damage repair capacity [49, 50]. Oxidative stress, and the response to resulting cellular damage, due to elevated temperatures have been documented across a wide variety of taxa, including algae , plants , mollusks , and fishes [54, 55]. Gastrula embryos raised at 17 °C were positively correlated to the blue WGCNA module that included genes associated with oxidoreductase activity (Figs. 5 and 6). Oxidoreductase enzymes catalyze the transfer of electrons from a reductant to an oxidant  and can contribute to the production of ROS [57–59]. The blue module also included genes related to response to oxidative stress and response to ROS (Fig. 6). Thus, at 17 °C, gastrula embryos appear to be responding to oxidative stress at a molecular level.
ROS can cause cellular damage to lipids, proteins, and nucleic acids [59, 60]. Genes related to unfolded protein binding and glycosylation (Fig. 3a) were up-regulated in gastrula embryos raised at 17 °C, indicating a response to protein alteration and damage. The unfolded protein response acts to restore protein folding in the endoplasmic reticulum and reestablish protein homeostasis [61, 62], while glycosylation is important for regulating the structure, function and stability of proteins [63, 64]. DE analysis and WGCNA also provided evidence of DNA damage and repair. This is similar to observations in Acropora corals in which heat stress caused an up-regulation of DNA replication and repair genes [48, 65]. Here, the turquoise WGCNA module, which was positively correlated to the gastrula 17 °C treatment (Fig. 5), included GO terms related to cellular response to DNA damage stimulus as well as DNA replication and repair, such as DNA polymerase, DNA metabolic process, DNA binding, and helicase (Fig. 6). Genes related to DNA metabolic process and DNA recombination were also up-regulated under warmer temperature conditions (Fig. 3a). DNA metabolic processes can include both DNA synthesis and degradation for the purposes of replication and repair. Furthermore, DNA recombination in somatic cells has been identified as a critical mechanism for DNA damage repair [66, 67]. Taken together, 17 °C temperature conditions appear to induce oxidative stress within the gastrula embryos, which undergo stress response mechanisms to combat cellular damage.
Transmembrane transport and metabolism
Gastrula embryos raised under warmer temperatures also increased expression of genes related to transmembrane transport, potentially both within and between cells (i.e., cell-cell communication). For instance, genes related to G protein-coupled receptor, cation channel, cell surface receptor signaling pathway, and vesicle-mediated transport were up-regulated in gastrula embryos raised under 17 °C relative to those raised under 13 °C (Fig. 3a). Additionally, GO terms within the blue WGCNA module, which was positive correlated to the gastrula 17 °C treatment (Fig. 5), included sodium and potassium ion transmembrane transporters, ATP hydrolysis coupled cation transmembrane transport, proton transmembrane transport, and neurotransmitter transport (Fig. 6). Increased transport of materials, particularly ions, across cell membranes may indicate osmoregulation and maintenance of homeostasis. This aligns with reports in juvenile sea urchins of the species Loxechinus albus, in which gene expression alterations under elevated temperatures provided evidence of increased active transmembrane transport of sodium and potassium ions .
Active transport of ions across cell membranes is an energetically expensive process that can incur substantial metabolic costs during sea urchin early development . The blue module also contains genes related to lipid metabolic process, peptide metabolic process, carbohydrate metabolic process, and ATP metabolic process (Fig. 6). This up-regulation of metabolic genes is similar to observations in S. purpuratus gastrula embryos raised under warmer temperatures . In contrast, gastrula raised under the lower temperature treatment expressed genes associated with metabolic depression. Specifically, genes up-regulated in gastrula embryos raised under 13 °C relative to those raised under 17 °C included those related to negative regulation of biological process and negative regulation of metabolic process. In this study, metabolic rates of embryos raised under different treatments were not measured at the gastrula stage, but we may expect that, given the effect of temperature on biochemical reaction kinetics, metabolic rate should increase predictably with temperature . Generally, higher metabolic rates have been recorded at warmer temperatures in marine ectotherms [71–74].
Regulation of gene expression
Temperature also had an evident effect on the regulation of gene expression in M. franciscanus gastrula embryos. GO terms identified as regulation of transcription by RNA polymerase II, translation regulator, and regulation of gene expression are relatively down-regulated in gastrula embryos raised under 17 °C relative to those raised under 13 °C (i.e., genes are comparatively up-regulated in the colder temperature treatment) (Fig. 3a). The turquoise WGCNA module, which was positively correlated to the 17 °C gastrula treatment and negatively correlated to the 13 °C gastrula treatment (Fig. 5), contained genes associated with transcription and several epigenetic processes. GO terms identified within the turquoise module included DNA templated transcription process, transcription coactivator, RNA polymerase binding, histone binding, chromatin binding, and RNA methylation (Fig. 6).
Epigenetic modifications, primarily consisting of DNA methylation, chromatin organization (e.g., histone and posttranslational modifications), and noncoding RNAs, are mechanisms of nongenetic variation by which the phenotypes of organisms can be altered faster than, and without the need of, changes in genotype [75, 76]. The dynamic changes in different epigenetic marks can act to regulate gene function without altering the DNA sequence, promoting phenotypic plasticity and potentially modulating the response to different environmental conditions [77–79]. Histone variants and modifications may activate or repress transcription processes by altering chromatin structures, impacting the regions of the genome that are available for transcription , and have been shown to mediate responses to changing environmental conditions in marine organisms [81–83]. Here, histone binding and chromatin remodeling genes are positively correlated with the 17 °C gastrula treatment. However, additional analyses such as ChIP-seq (i.e., chromatin immunoprecipitation sequencing) to locate regions targeted by histone modifications and DNA-binding proteins [84, 85] or ATAC-seq (i.e., assay for transposase-accessible chromatin with high-throughput sequencing) to assess genome-wide chromatin accessibility  are required to profile specific histone variants or modifications and their impact on gene expression.
Another layer to gene expression regulation includes the posttranscriptional modification of RNA molecules, an emerging concept known as RNA epigenetics or epitranscriptomics [87, 88]. The turquoise WGCNA module that was positively correlated to the 17 °C gastrula treatment included genes related to RNA modification, RNA methyltransferase, RNA methylation, and pseudouridine synthesis (Fig. 6). RNA modifications can occur in ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA) and small nuclear RNA (snRNA) [89, 90]. In addition to regulating gene expression, RNA modifications can modulate RNA transport and degradation, alternative splicing, protein binding, and development, among other essential biological processes [90–93]. RNA modifications such as the methylation of cytosine or adenosine are dynamic and reversible, allowing for alteration in protein-RNA interactions and rapid responses to changes in environmental conditions. For example, N6-methyladenosine (m6A), a very common RNA modification in eukaryotes, appears to play a role in the mammalian temperature stress response by promoting translation initiation of heat shock response genes . Another common modification is pseudouridine (ψ), the first modified nucleoside discovered in RNA . An up-regulation of genes related to pseudouridine synthesis could indicate enhanced transcript stability under elevated temperatures, as was observed in yeast undergoing heat shock . Despite over 100 types of RNA modifications having been recorded , epitranscriptomic studies in invertebrates and marine species remain limited. Nonetheless, recent technological advances such as m6A-seq [98, 99] and ψ-seq  have provided exciting future avenues to explore epitranscriptomics in non-model systems.
Although evidence of transcription regulation was observed, it is difficult to conclude if this led to an increase or decrease of gene expression, particularly with respect to the functional significance of histone and RNA modifications. These mechanisms are much less studied in marine invertebrates than other modifications such as DNA methylation [77, 78], and although our data support that these modifications occurred in response to the environment, additional approaches are required to determine the precise modifications, their locations, and their impact on gene expression. In this study, there were more up-regulated than down-regulated genes in the 17 °C versus the 13 °C gastrula treatment. Nevertheless, future studies pairing comparative epigenetic and epitranscriptomic analyses with transcriptomic approaches are required to elucidate how these various mechanisms influence gene expression in response to different environmental conditions in M. franciscanus.
p CO 2 influenced gene expression and body size of gastrula embryos
The pCO2 treatment influenced gene expression patterns at the gastrula stage, although to a lesser degree than temperature, and the interaction between the two factors was not significant (Fig. 1d). Gastrula pCO2 conditions explained 12.5% of the observed variance (p = 0.034) with 49 genes up-regulated and 202 genes down-regulated in embryos raised under 1050 µatm pCO2 relative to those raised under 475 µatm pCO2 (Figs. 1 and 2). In this study, we anticipated that the 475 µatm pCO2 treatment was not stressful, as it represented the average ambient pCO2 levels M. franciscanus regularly experience in their natural habitat . Evidence suggests that calcifying marine organisms such as M. franciscanus are sensitive to declines in ocean pH (i.e., increases in pCO2 levels) [100–102], and while M. franciscanus may periodically experience elevated pCO2 conditions in nature during upwelling events [15, 28, 30, 103], the 1050 µatm pCO2 treatment was expected to induce a stress response.
While the effect of pCO2 on gastrula gene expression patterns was less than the effect of temperature, there was a pronounced impact of pCO2 conditions on gastrula body size. Gastrula raised under elevated pCO2 conditions (i.e., 1050 µatm) were significantly smaller than those raised under 475 µatm . Interestingly, WGCNA module-trait relationships showed that the trait for gastrula body size shared highly similar module correlations to that of the lower pCO2 treatment (475 µatm) (Fig. 5). Therefore, pCO2 elicited a relatively muted transcriptomic response compared to temperature, but appeared to have a much greater influence at the organismal level. Below we discuss the expression patterns of genes affected by the gastrula pCO2 treatment, which included those related to metabolism, ion transport, and the cellular stress response.
Metabolism and ion transport
Gastrula raised under the lower pCO2 treatment (i.e., 475 µatm) expressed genes associated with macromolecule biosynthetic processes. In contrast, those raised under the elevated pCO2 treatment (i.e., 1050 µatm) expressed genes associated with macromolecule catabolic processes (Fig. 3b). This may, in part, explain the difference in body size that was observed as a result of the pCO2 conditions. Gastrula in the 475 µatm pCO2 treatment appeared to construct proteins and other macromolecules to maintain their growth and body size, while gastrula in the 1050 µatm pCO2 treatment underwent catabolic processes, possibly to obtain the energy required to respond to pCO2 stress. The pCO2 stress response may include an increase of ion transport as a means of maintaining acid-base equilibrium given elevated H+ concentrations under high pCO2 conditions. DE analyses and WGCNA revealed that gastrula in the 1050 µatm treatment up-regulated genes related to ion binding, active transmembrane transporter, and ATPase coupled to movement of substances (Figs. 3b and 6). Similarly, increased expression of ion transport genes has been observed in gastrula embryos of S. purpuratus exposed to moderately elevated pCO2 levels (e.g., ~ 800 µatm) , although the transcriptomic response of S. purpuratus embryos to pCO2 stress can be influenced by maternal effects .
Cellular stress response
Elevated pCO2 exposure also often impacts expression of cellular stress response genes in marine metazoans . The cellular stress response is associated with the increased synthesis of molecular chaperones , including heat shock proteins (HSPs). Here, gastrula embryos raised under 1050 µatm pCO2 differentially up-regulated genes related to heat shock protein binding (Fig. 3b). This result conflicts with another study in M. franciscanus that found that expression of the molecular chaperone Hsp70 decreased under elevated pCO2 levels , although this was measured at the larval stage. However, increased expression of HSP genes under elevated pCO2 conditions has been observed in the Antarctic pteropod Limacina helicina antarctica , in juveniles of the stony coral Acropora millepora , and in the cold-water coral Desmophyllum dianthus . HSPs act as molecular chaperones in the cellular stress response by assisting in protein transport, protein folding and unfolding, stabilization of denatured proteins, and degradation of misfolded proteins [111, 112]. Thus, gastrula in the 1050 µatm pCO2 treatment appeared to respond to stressful conditions via increased expression of HSP genes to establish or maintain physiological equilibrium.
Temperature was the dominant factor at the prism stage
At the prism stage, temperature accounted for 22.8% of the observed variance (p < 0.001) with 4132 genes up-regulated and 4286 genes down-regulated in embryos raised under 17 °C relative to those raised under 13 °C (Figs. 1 and 2). Unlike at the gastrula stage, responses to temperature measured at the molecular level were also observable at the organismal level. Development at the warmer 17 °C treatment led to an increase in prism body size as well as a modest increase in prism thermotolerance . Indeed, prism body size and thermotolerance traits exhibited highly similar WGCNA module correlations as the prism 17 °C treatment (Fig. 5). Therefore, the transcriptomic response to temperature appears to have influenced both growth and resistance to heat stress in M. franciscanus prism embryos.
Similarities to the transcriptomic response at the gastrula stage
Like the gastrula stage, prism embryos raised at 17 °C exhibited increased expression of genes related to oxidative stress, transmembrane transport, and metabolic processes. As previously discussed, oxidative stress caused by the production of ROS can lead to cellular damage of lipids, proteins, and nucleic acids [49, 50, 60]. GO enrichment from the DE analysis and WGCNA identified terms including oxidoreductase, antioxidant, response to oxidative stress, and response to reactive oxygen species from genes up-regulated in response to the warmer temperature treatment (Figs. 4 and 6). At the gastrula stage, embryos in the 17 °C treatment also expressed genes associated with protein and DNA repair, but there was no evidence of increased macromolecule repair gene expression at the prism stage. This could indicate that while both stages responded to oxidative stress due to warmer temperatures, there was less cellular damage of proteins and nucleic acids incurred by the prism stage.
Also similar to the gastrula stage, warmer temperature conditions caused an increased expression of genes related to transmembrane transport in prism embryos. Specifically, up-regulated genes were related to ion binding, active transmembrane transporter, ATP hydrolysis coupled cation transmembrane transport, and sodium and potassium ion transmembrane transporters (Figs. 4 and 6), which may indicate osmoregulation and the maintenance of homeostasis. Prism embryos in the 17 °C treatment also increased expression of genes related to energetic processes (e.g., ATP metabolic process, organic acid metabolic process, lipid catabolic process, cyclic nucleotide metabolic process, and carbohydrate metabolic process) possibly to generate the energy required to support active transmembrane transport of ions and other materials. The up-regulation of genes related to energy production may have also supported the increased growth of prism embryos under warmer temperatures. Genes within the blue WGCNA module, which was positively correlated to the 17 °C prism treatment, were related to amide and nucleoside phosphate biosynthetic processes. This may indicate anabolic metabolism in which additional synthesis of proteins and nucleic acids supported larger body sizes. In contrast, DE analysis revealed an increase in expression of genes related to the negative regulation of biological and metabolic processes in prism embryos raised at 13 °C. This supports the predicted expectation that organisms exhibit decreased metabolism under colder temperatures [70–74].
DNA damage and gene expression regulation at lower temperatures
In general, global change biology research in marine systems has focused on the negative consequences of increasing temperatures associated with ocean warming . However, given the expected rise in variable and extreme weather events, the impact of decreased temperatures is also an important consideration, especially in regions dominated by upwelling. Both DE analysis and the WGCNA indicated that prism embryos in the 13 °C treatment increased expression of genes related to DNA damage and repair (Figs. 4 and 6). Specifically, identified GO terms included cellular response to DNA damage stimulus, DNA helicase, DNA metabolic process, DNA binding, and DNA polymerase. Increased DNA damage as a result of low temperature stress has been recorded in the Pacific white shrimp Litopenaeus vannamei . Although 13 °C is within the range of temperatures that M. franciscanus experience in the Santa Barbara Channel where urchins were collected for this study, it is lower than the annual average of ~ 15 °C  and may have generated stress and cellular damage in the prism embryos, leading to the activation of repair mechanisms.
There also appeared to be a relative up-regulation of genes associated with gene expression regulation in prism embryos raised under the lower temperature treatment, including genes related to translation regulator, transcription coregulator, regulation of gene expression, and regulation of transcription by RNA polymerase II (Fig. 4). Additionally, the turquoise WGCNA module, which contained genes related to epigenetic and epitranscriptomic modifications, was positively correlated to the 13 °C prism treatment (Fig. 5). Gene expression in prism embryos raised at 13 °C appeared to be regulated by histone and chromatin modifications, which can influence genome accessibility, and by RNA modifications such as RNA base methylation via methyltransferase activity (Figs. 4 and 6). Interestingly, the turquoise WGCNA module was positively correlated to the warmer temperature treatment at the gastrula stage, but was positively correlated to the colder temperature treatment at the prism stage. Thus, how the M. franciscanus epigenome and epitrancriptome respond to temperature conditions can vary during development. This difference in gene expression regulation may have contributed to temperature affecting body size at one developmental stage and not at the other.
Temperature did not influence HSP gene expression
Higher levels of HSPs, which act as molecular chaperones that protect cells from stress-induced damage, have been shown to confer increased thermotolerance across a variety of marine taxa [115–118]. Therefore, we may have expected an up-regulation of HSP genes linked with the slight increase in thermotolerance measured at the prism stage . However, genes related to HSPs were not differentially expressed as a result of temperature at either the gastrula or prism stages. Our results contrast with a study in S. purpuratus that found expression of Hsp70 and Hsp90 increased at higher temperatures . It is important to note that our study implemented a comparative approach that did not detect a significant difference in the relative expression of HSP genes. This approach may not be capable of detecting more subtle changes in HSP expression, which can vary between genes. For example, in the Antarctic urchin Sterechinus neumayeri, thermal stress led to an increase in Hsp90 expression, while Hsp70 expression remained unchanged . A quantitative approach (e.g., using qPCR) may provide more insight into the absolute expression of genes like Hsp70 and Hsp90 during M. franciscanus development.
In other investigations of sea urchin early development, increased Hsp70 expression was generally not observed under moderate warming scenarios. One study found that Hsp70 was not transcriptionally up-regulated in M. franciscanus until larvae were exposed to temperatures at or above 20 °C . A study in S. purpuratus only found induction of Hsp70 occurred at temperatures above 21 °C . Therefore, the 17 °C treatment may not have been extreme enough to induce differential expression of HSP genes. Furthermore, in the green sea urchin Psammechinus miliaris, expression of HSP genes were low during early development relative to expression in adults . The authors suggested that HSP expression was limited during this time  because over-expression of HSPs could have negative consequences for successful early development . Therefore, large increases in HSP expression may be restricted during M. franciscanus early development.
The prism stage exhibited a limited transcriptomic response to pCO2
In other studies, echinoderms raised under elevated pCO2 conditions have exhibited altered expression of genes related to skeletogenic pathways, spicule matrix proteins, cellular stress response, ion regulation and transport, apoptosis, metabolism and ATP production [104, 105, 123–126]. Here, the pCO2 treatment had a relatively minimal effect on gene expression patterns, particularly at the prism stage. The pCO2 treatment explained only 9.6% of the observed variance at this stage and was not significant (p = 0.084) (Fig. 1f). This contrasts with observations made at the organism level in which elevated pCO2 resulted in smaller prism embryos, although this could be offset by the positive effect of temperature, which acted as the dominant factor influencing body size . It is interesting that the transcriptomic response to elevated pCO2 was more evident in gastrula than in prism embryos particularly because at the prism stage, skeletal rod formation occurs. We may expect increased stress associated with prism embryos undergoing calcification processes under lowered pH conditions, however, evidence of this was not detected by changes in gene expression.
It is possible that while there is a clear phenotypic difference in prism embryos raised under high versus low pCO2 conditions, the transcriptomic changes underlying this difference are too subtle to be identified statistically. Alternatively, the prism stage may simply lack a robust transcriptional response to the 1050 µatm pCO2 treatment. For instance, the Mediterranean sea urchin Paracentrotus lividus exhibits different transcriptomic responses depending on the magnitude of the pH stressor . Decreased pH conditions caused P. lividus embryos to increase their expression of calcification genes, but not once the pH dropped below a certain threshold . A similar result was observed in S. purpuratus in which embryos raised under a high pCO2 treatment designed to reflect near-future levels was relatively muted relative to those raised under a more moderate pCO2 treatment designed to reflect present-day low pH conditions . The authors speculated that the transcriptional response required for acclimating to a more extreme pCO2 level was too metabolically expensive, and the embryos instead opted to conserve energy to ensure short-term survival, perhaps until environmental conditions became more favorable . While a failure of embryos to respond at the transcriptomic level may allow for continued successful development under high pCO2 conditions, there may be important physiological consequences such as the observed reduction in body size . Thus, the lack of a transcriptomic response to high pCO2 may have important fitness consequences for M. franciscanus.
The genetic structure of the sea urchins used in this experiment may have contributed to the lack of an expected response to the pCO2 treatment. In the green sea urchin Strongylocentrotus droebachiensis, a quantitative genetic breeding design implemented by Runcie and colleagues demonstrated that changes in gene expression as a result of differences in pH exposure were minor relative to gene expression differences as a result of parentage . Thus, minimal transcriptomic responses to pCO2 may be due to genetic variation in M. franciscanus. Furthermore, the environmental exposure history of the adult urchins may have generated non-genetic parental effects (i.e., transgenerational plasticity), which can also generate a limited transcriptomic response to high pCO2 . While all embryo cultures for this experiment were composed of the same mixture of progeny from a cross between one male and five females, it is possible that the sea urchins collected for this experiment may be from a population with a relatively muted transcriptomic response to high pCO2 conditions.
It has been proposed that selection and local adaptation acts on populations that are regularly exposed to high pCO2 conditions, such as those that often experience upwelling conditions within the California Current System (CCS) , and that these populations may harbor genotypes that are resistant to low pH conditions [124, 128–132]. In S. purpuratus, transcriptomic responses to high pCO2 levels can vary by the frequency in which the sea urchin populations are exposed to upwelling conditions . In particular, urchins from populations frequently exposed to low pH have greater transcriptomic responses to high pCO2 than those that experience low pH less often . The adult sea urchins used in this study were collected from a site in the Santa Barbara Channel. While this area does experience periods of low pH due to upwelling [28, 30], low pH events occur less frequently than at more northern sites within the CCS [15, 133, 134]. Therefore, the urchins used in this study may be comparatively less adapted towards mounting a transcriptomic response to high pCO2.
Performance under current and future ocean conditions
Moderate ocean warming may be favorable for M. franciscanus early development by providing larger body sizes and increased thermotolerance at the prism stage . The warmer temperature treatment could even mitigate the stunting effect of elevated pCO2 on prism body size . This effect of warmer temperatures, however, may only be beneficial on a short-term basis. Gene expression analyses indicated that embryos raised under 17 °C responded to cellular stress, and while there were no indications of negative impacts at the phenotypic level, there may be trade-offs and consequences to developing under warmer temperatures such as increased incidences of disease . Prolonged heat exposure may eventually become detrimental, and negative carry-over effects can arise at later life stages [46, 47]. Additionally, the observed plasticity at 17 °C may not extend to more severe warming scenarios. For example, a study in adult M. franciscanus found that although mortality did not vary between urchins acclimated to 15 °C or 18 °C, mortality was significantly higher at a more extreme temperature of 21 °C . Nevertheless, our study revealed that M. franciscanus may be quite resilient to warming, and may even benefit from relatively brief and modest increases in temperature during early development. At the urchin collection site, temperatures of 17 °C are currently recorded during the summer months , and in the future, this temperature is likely to be reached more often given unmitigated climate change. More research is required to determine how M. franciscanus will be impacted as ocean warming continues, particularly as marine heatwaves increase in frequency [136, 137].
During early development, M. franciscanus appear to be more susceptible to rising pCO2 levels than to rising temperatures. The lack of a robust transcriptional response paired with a decrease in body size indicates that exposure to elevated pCO2 is detrimental to developing embryos. Continued ocean acidification is therefore likely to have adverse impacts on future M. franciscanus populations, although this could be offset somewhat by the positive effects of simultaneous ocean warming [31, 138–140]. However, during seasonal upwelling events, M. franciscanus are exposed to corrosive pH conditions that lack the mitigating effects of warmer temperatures [15, 141]. The upwelling season in this region typically extends from early spring until late summer or fall, and is characterized by variable fluctuations between periods of upwelling and the relaxation of upwelling [141, 142]. This overlaps with the natural spawning period of M. franciscanus that occurs annually during spring and early summer months [143–145]. As such, spawning that occurs during or immediately prior to an upwelling event will subject developing embryos to high pCO2 conditions paired with colder temperatures. Given ocean acidification and the increase in upwelling frequency and intensity that is predicted with continued climate change [146–148], the likelihood of M. franciscanus developing under stressful pCO2 conditions should rise in the future.