In this study, we examined how the gene expression patterns of M. franciscanus 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 [46]. Although both temperature and pCO2 can influence rates of sea urchin development [34, 53], 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 they may be driven by the quality of the particular male selected to fertilize the eggs. Upon including data from our previous study that examined gene expression patterns during M. franciscanus early development [48], a PCA showed that, although samples primarily grouped by developmental stage, there is a clear distinction between the embryos of the two studies (Additional file 6). This is likely due to a combination of genetic and environmental differences between the two source populations, as the adult urchins were collected from different sites and during different years. Indeed, in the purple sea urchin S. purpuratus, genetic variation has been shown to influence transcriptomic responses to temperature and pCO2 stress during early development [36, 54]. Given that the data presented here represents a limited selection of the genetic variation that exists in this species, the results should be interpreted with caution. 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.
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 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.
Gene expression 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 matm pCO2) and documented significant transcriptomic differences between gastrula and prism stages [48]. 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, although gene expression analyses performed independently of stage (i.e., without analyzing gastrula and prism stages separately) are reported in Additional file 2. Nevertheless, embryos at each developmental stage exhibited different transcriptomic responses to temperature and pCO2 treatments. For instance, many more genes were differentially expressed due to temperature at the prism stage than at the gastrula stage (Fig. 3). 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). Similarly, the morphometric response to temperature and pCO2 treatments varied by stage, in which pCO2, but not temperature, affected gastrula embryos by reducing body size under elevated pCO2 conditions (i.e., 1050 matm) [46]. 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 [46]. 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 [23]. 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 [55], a fundamental process known as gastrulation that is essential for successful development in metazoans [56]. At the prism stage, the embryo differentiates its digestive tract and develops skeletal rods, which are vital structures required for the embryos to eventually become feeding, planktotrophic larvae [57, 58]. 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 [59, 60]. 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 [61]. 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. Temperature explained 20.3% of the observed variance (p = 0.001) with 2049 genes up-regulated and 1955 genes down-regulated in embryos raised under 17 °C relative to those raised under 13 °C (Figs. 1d and 3a). Furthermore, PC1, which captured 23.8% of the gene expression variance at the gastrula stage, was significantly correlated to the temperature treatment (Fig. 2a). 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 [36], whose biogeographical distribution overlaps with that of M. franciscanus. Here, DE analysis revealed that gastrula raised in the higher temperature treatment (i.e., 17 °C) expressed genes associated with a 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 [36]. We also found that M. franciscanus gastrula embryos raised under the warmer treatment exhibited transcriptomic patterns indicative of increased transmembrane transport, 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 [36]. Lastly, temperature appeared to impact the regulation of gene expression in M. franciscanus gastrula embryos, including genes related to epigenetic 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 [36].
Despite similarities in how temperature influenced gastrula gene expression, unlike in M. franciscanus, there was an effect of temperature on S. purpuratus morphology. Specifically, S. purpuratus gastrula embryos raised at warmer temperatures were significantly smaller in size [36], whereas M. franciscanus gastrula embryos did not significantly differ in size as a result of temperature [46]. 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. Below, 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 (Gastrula temperature)
Response to stress at the cellular level often includes processes to mitigate or remove cell damage [62]. Cell death protein 3, which encodes a protease involved in apoptosis [63], was up-regulated in gastrula embryos raised at 17 °C, indicating that the warmer temperature may have caused stress-induced programmed cell death. DE analysis 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 [61, 64]. Here, GO terms related to DNA metabolic process and DNA recombination were enriched with up-regulated genes under warmer temperature conditions (Fig. 4a). 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 [65, 66]. Taken together, 17 °C temperature conditions appear to induce stress within the gastrula embryos, which undergo response mechanisms to combat cellular damage.
Transmembrane transport (Gastrula temperature)
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. 4a). This is also supported by PC loadings, in which genes related to ion binding and cation channel contributed variance to PC1 (Additional file 4a), which was significantly correlated to the temperature treatment (Fig. 2a). 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 [67].
Metabolism (Gastrula temperature)
In S. purpuratus, gastrula embryos raised under warmer temperatures up-regulated metabolic genes [36]. Here, gastrula raised under the lower temperature treatment expressed genes associated with metabolic depression. Specifically, GO terms identified as negative regulation of biological process and negative regulation of metabolic process were significantly enriched by genes down-regulated in gastrula embryos raised under 17 °C relative to those raised under 13 °C (Fig. 4a). 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 [68]. Generally, higher metabolic rates have been recorded at warmer temperatures in marine ectotherms [69-72]. This positive correlation between temperature and metabolism has been observed in M. franciscanus at the adult stage [73].
Regulation of gene expression (Gastrula temperature)
Temperature also had an evident effect on the regulation of gene expression in M. franciscanus gastrula embryos. GO terms enriched by genes 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) were identified as regulation of transcription by RNA polymerase II, translation regulator, and regulation of gene expression (Fig. 4a). Histone binding, histone modification, chromatin remodeling, and chromatin organization genes were also associated with the 13 °C gastrula treatment. Histone and post-translational modifications are examples of epigenetic mechanisms. These, and other epigenetic modifications can act to regulate gene function without altering the DNA sequence, promoting phenotypic plasticity and potentially modulating the response to different environmental conditions [74-76]. 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 [77], and have been shown to mediate responses to changing environmental conditions in marine organisms [78-80]. Additional analyses such as ChIP-seq (i.e., chromatin immunoprecipitation sequencing) to locate regions targeted by histone modifications and DNA-binding proteins [81, 82] or ATAC-seq (i.e., assay for transposase-accessible chromatin with high-throughput sequencing) to assess genome-wide chromatin accessibility [83] are required to profile specific histone variants or modifications and their impact on gene expression.
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 modifications and chromatin remodeling. These mechanisms are much less studied in marine invertebrates than other modifications such as DNA methylation [74, 75], 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, and the up-regulated genes appeared to have a greater log2 FC in expression (Fig. 3a). Nevertheless, future studies pairing comparative epigenetic analyses with transcriptomic approaches are required to elucidate how these various mechanisms influence gene expression in response to different environmental conditions in M. franciscanus.
pCO2 influenced gene expression 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 13.2% of the observed variance (p = 0.021) with 9 genes up-regulated and 166 genes down-regulated in embryos raised under 1050 matm pCO2 relative to those raised under 475 matm pCO2 (Figs. 1d and 3b). Additionally, PC2, which captured 12.0% of the gene expression variance at the gastrula stage, was significantly correlated to the pCO2 treatment (Fig. 2a). In this study, we anticipated that the 475 matm pCO2 treatment was not stressful, as it represented the average ambient pCO2 levels M. franciscanus regularly experience in their natural habitat [44]. Evidence suggests that calcifying marine organisms such as M. franciscanus are sensitive to declines in ocean pH (i.e., increases in pCO2 levels) [84-86], and while M. franciscanus may periodically experience elevated pCO2 conditions in nature during upwelling events [15, 43, 45, 87], the 1050 matm 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 matm) were significantly smaller than those raised under 475 matm [46]. Therefore, pCO2 appeared to have a greater influence at the organismal level but elicited a relatively muted transcriptomic response compared to temperature. Below we discuss the expression patterns of genes affected by the gastrula pCO2 treatment, which included those related to metabolism and ion transport.
Metabolism and ion transport (Gastrula pCO2)
Gastrula raised under the lower pCO2 treatment (i.e., 475 matm) expressed genes that significantly enriched GO terms related to several macromolecule biosynthetic processes (Fig. 4b). In contrast, those raised under the elevated pCO2 treatment (i.e., 1050 matm) expressed genes that enriched GO terms associated with macromolecule catabolic processes (Fig. 4b). This may, in part, explain the difference in body size that was observed as a result of the pCO2 conditions. Gastrula in the 475 matm pCO2 treatment appeared to construct proteins and other macromolecules to maintain their growth and body size, while gastrula in the 1050 matm pCO2 treatment underwent catabolic processes, possibly to obtain the energy required to respond to elevated pCO2 levels. 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. GO terms related to ion binding, active transmembrane transporter, and ATPase coupled to movement of substances were enriched by genes up-regulated in gastrula raised in the 1050 matm treatment (Fig. 4b). 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 matm) [39], although the transcriptomic response of S. purpuratus embryos to pCO2 stress can be influenced by maternal effects [38].
Temperature was the dominant factor at the prism stage
At the prism stage, temperature accounted for 27.2% of the observed variance (p = 0.001) with 3842 genes up-regulated and 3434 genes down-regulated in embryos raised under 17 °C relative to those raised under 13 °C (Figs. 1f and 3c). Furthermore, PC1 for the prism stage captured 27.6% of variance in gene expression and was significantly correlated to the temperature treatment (Fig. 2b). Unlike at the gastrula stage, responses to temperature that were 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 [46]. Indeed, prism body size and thermotolerance variables were also highly correlated to PC1 (Fig. 2b). Therefore, the transcriptomic response to temperature appears to have influenced both growth and resistance to heat stress in M. franciscanus prism embryos. Prism embryos raised at 17 °C exhibited increased expression of genes related to the cellular stress response, transmembrane transport, and metabolic processes, while genes related to DNA repair and the regulation of gene expression were associated with the colder temperature treatment (i.e., 13 °C).
Cellular stress response (Prism temperature)
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 [88, 89]. Oxidative stress, and the response to resulting cellular damage, due to elevated temperatures have been documented across a wide variety of taxa, including algae [90], plants [91], mollusks [92], and fishes [93, 94]. GO enrichment from the DE analysis identified terms including oxidoreductase, antioxidant, response to oxidative stress, and response to reactive oxygen species that were enriched with genes up-regulated in response to the warmer temperature treatment (Fig. 5a). At the gastrula stage, embryos in the 17 °C treatment also expressed genes associated with DNA repair, but there was no evidence of increased macromolecule repair gene expression at the prism stage. This could indicate that while both stages initiated a cellular stress response due to warmer temperatures, there was less cellular damage of nucleic acids incurred by the prism stage.
In general, global change biology research in marine systems has focused on the negative consequences of increasing temperatures associated with ocean warming [95]. 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. DE analysis indicated that prism embryos in the 13 °C treatment increased expression of genes related to DNA damage and repair. Specifically, identified GO terms included cellular response to DNA damage stimulus, DNA metabolic process, DNA binding, and catalytic, acting on DNA (Fig. 5a). Increased DNA damage as a result of low temperature stress has been recorded in the Pacific white shrimp Litopenaeus vannamei [96]. Although 13 °C is within the range of temperatures that M. franciscanus experience in the Santa Barbara Channel (SBC) where urchins were collected for this study, it is lower than the annual average of ~15 °C [44] and may have generated stress and cellular damage in the prism embryos, leading to the activation of repair mechanisms.
Transmembrane transport and metabolism (Prism temperature)
Similar to the gastrula stage, warmer temperature conditions caused an increased expression of genes related to transmembrane transport in prism embryos. Specifically, GO terms of ion binding, ion transmembrane transporter, cation channel, and sodium and potassium ion transmembrane transporters were enriched with up-regulated genes (Fig. 5a), 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 metabolic 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. 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 [68-72].
Regulation of gene expression (Prism temperature)
At the prism stage, the colder temperature treatment exhibited an enrichment of genes related to translation regulator, transcription coregulator, regulation of gene expression, and regulation of transcription by RNA polymerase II (Fig. 5a). Additionally, GO terms identified as histone binding, chromatin organization, RNA modification, and methylation-dependent protein binding were enriched in genes down-regulated at 17 °C relative to 13 °C (Fig. 5a). Thus, gene expression in prism embryos raised at 13 °C appeared to be epigenetically regulated by histone and RNA modifications. This differential expression of genes related to gene expression regulation (i.e., down-regulated at the colder temperature relative to the warmer temperature) is similar to the pattern observed at the gastrula stage.
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 [38, 39, 97-100]. Here, the pCO2 treatment had a relatively minimal effect on prism gene expression patterns. The pCO2 treatment explained only 9.3% of the observed variance at this stage and was not significant (p = 0.091) (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 [46]. 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. Although GO terms identified as ion binding and ATPase coupled to transmembrane movement of ions were enriched with genes up-regulated at the elevated pCO2 level (Fig. 5b), we also expected to observe increased stress associated with prism embryos undergoing calcification processes under lowered pH conditions, but we detected no evidence of this.
It is possible that while there was a clear phenotypic difference in prism embryos raised under high versus low pCO2 conditions, the transcriptomic changes underlying this difference were much more subtle. Alternatively, the prism stage may simply lack a robust transcriptional response to the 1050 matm pCO2 treatment. For instance, the Mediterranean sea urchin Paracentrotus lividus exhibits different transcriptomic responses depending on the magnitude of the pH stressor [101]. Decreased pH conditions caused P. lividus embryos to increase their expression of calcification genes, but not once the pH dropped below a certain threshold [101]. A similar result was observed in S. purpuratus in which embryos raised under a high pCO2 treatment designed to reflect near-future levels exhibited a muted transcriptomic response relative to those raised under a more moderate pCO2 treatment designed to reflect present-day low pH conditions [39]. 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 [39]. While a failure of embryos to respond at the transcriptomic level may permit continued successful development under high pCO2 conditions, there may be important physiological consequences such as the observed reduction in body size [46]. Thus, the lack of a transcriptomic response to high pCO2 may have important fitness consequences for M. franciscanus.
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 [54]. Thus, minimal transcriptomic responses to pCO2 may be due to the genetic structure of the sea urchins used in this experiment. 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 [38]. 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 act on populations that are regularly exposed to high pCO2 conditions, such as those that often experience upwelling conditions within the CCS [15], and that these populations may harbor genotypes that are resistant to low pH conditions [40, 98, 102-105]. 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 [40]. 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 [40]. While the site where the adult sea urchins were collected does experience periods of low pH due to upwelling [43, 45], low pH events occur less frequently than at more northern sites within the CCS [15, 106, 107]. Therefore, the urchins used in this study may be comparatively less adapted towards mounting a transcriptomic response to high pCO2.
HSP gene expression
Heat shock proteins (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 [108, 109]. Higher levels of HSPs have been shown to confer increased thermotolerance across a variety of marine taxa [110-113]. Therefore, we may have expected an up-regulation of HSP genes linked with the slight increase in thermotolerance measured at the prism stage [46]. However, there were mixed results regarding differential expression patterns of HSP genes. At both stages, the heat shock 70 kDa protein 12-A like gene was up-regulated in the 17 °C treatment relative to the 13 °C treatment, whereas heat shock 70 kDa protein cognate 5 and heat shock 70 kDa protein 14 were down-regulated. No Hsp70 genes were differentially expressed due to pCO2, and no Hsp90 genes were differentially expressed due to temperature or pCO2.
Our results contrast with a study in S. purpuratus that found expression of Hsp70 and Hsp90 increased at higher temperatures [36]. However, 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 [32]. A study in S. purpuratus found induction of Hsp70 only occurred at temperatures above 21 °C [37]. Therefore, the 17 °C treatment may not have been extreme enough to induce a clear differential expression of HSP genes. Furthermore, in the green sea urchin Psammechinus miliaris, expression of HSP genes was low during early development relative to expression in adults [114]. The authors suggested that HSP expression was limited during this time [114] because over-expression of HSPs could have negative consequences for successful early development [115]. Therefore, large increases in HSP expression may be restricted during M. franciscanus early development.
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 [46]. The warmer temperature treatment could even mitigate the stunting effect of elevated pCO2 on prism body size [46]. 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 [116]. Prolonged heat exposure may eventually become detrimental, and negative carry-over effects can arise at later life stages [59, 60]. 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 [31]. Nevertheless, our study revealed that M. franciscanus exhibited a sizeable transcriptomic response to 17 °C at both developmental stages. At the urchin collection site, temperatures of 17 °C are currently recorded during the summer months [44], 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 [117, 118].
During early development, M. franciscanus appear to be more susceptible to rising pCO2 levels than to rising temperatures. The lack of a large transcriptional response, particularly at the prism stage, 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 [46, 119-121]. However, during seasonal upwelling events, M. franciscanus are exposed to corrosive pH conditions that lack the mitigating effects of warmer temperatures [15, 16]. Spawning that occurs during or immediately prior to an upwelling event will subject developing embryos to high pCO2 conditions paired with colder temperatures. 1050 matm pCO2 levels have been measured at the urchin collection site during upwelling events [44], so populations of M. franciscanus are likely already impacted by elevated pCO2. Given ocean acidification and the increase in upwelling frequency and intensity that is predicted with continued climate change [122-124], the likelihood of M. franciscanus developing under stressful pCO2 conditions should rise in the future.