Effects of thermal history on the heat shock response of bull kelp (Nereocystis luetkeana)

1 Introduction

Climate change is affecting marine environments across the globe (Fujita 2013; Galland et al. 2012; Hoegh-Guldberg and Bruno 2010; Kortsch et al. 2015), specifically, global air temperatures have risen by 0.85 °C since 1800 and are projected to increase at least by another 1.5 °C by the end of the century (Boer and Yu 2003; Flato and Boer 2001; Helmuth et al. 2002; Makwana et al. 2024). This increase in air temperature has affected ocean warming indices and circulation patterns (Pacific Decadal Oscillation, El Niño-Southern Oscillation, and North Pacific Gyre Oscillation), which has contributed to an increase in the occurrence of marine heatwaves (Oliver et al. 2019, 2021). The combination of the local weather, oceanographic patterns, and longer-term climate shifts (Oliver et al. 2021) have increased the frequency of marine heatwaves per year by 50 % in the last two centuries (Smale et al. 2019). Recorded thus far, marine heatwaves can vary in area impacted from 200 to 5,000 km², last from 5 to 500 days, and increase the local water temperature by 1–4.5 °C above average (Oliver et al. 2021). For marine organisms with narrow thermal limits and sensitive thermoregulatory systems, marine heatwaves can present a distinct threat to their survival (Reed et al. 2022; Smith et al. 2023).

Between 2013 and 2016, Sonoma and Mendocino Counties along the California coast experienced a sequential series of dramatic environmental changes. The most relevant change was the formation of an unprecedented marine heatwave known as “The Blob” (Di Lorenzo and Mantua 2016; Oliver et al. 2019; Reed et al. 2016; Rogers-Bennett and Catton 2019; Smale et al. 2019). The Blob formed in 2014 from a combination of a weak North Pacific High – a semi-permanent cyclone that sits above the Pacific Ocean north of Hawaii and west of California (Kenyon 1999) – and a weakened coastal upwelling season in the Northern California Current. Whereupon, it proceeded to raise the sea surface temperature in the area by 7 °C within an hour of its arrival (Leising et al. 2015; Peterson et al. 2017). Winds that would normally displace surface water in the upwelling process were too weak to move the buoyant warm water, so the water simply stayed in place and grew in mass over time (Bond et al. 2015), making it one of the longest-lasting and the largest marine heatwaves on record (Oliver et al. 2021). Coinciding with the formation of the Blob was an outbreak of sea star wasting disease and a boom in the purple sea urchin (Strongylocentrotus purpuratus) population (Di Lorenzo and Mantua 2016; Oliver et al. 2019; Reed et al. 2016; Rogers-Bennett and Catton 2019; Smale et al. 2019).

This series of events resulted in a massive loss of over 90 % of bull kelp (Nereocystis luetkeana Postels et Ruprecht 1840) coverage across its historic range in Sonoma and Mendocino Counties (Catton et al. 2016), more than 350 km of coastline (Rogers-Bennett and Catton 2019). Much of the area formerly inhabited by bull kelp transformed into sea urchin barrens; large areas of rock populated primarily by sea urchins and crustose coralline algae (Beisner et al. 2003; Ellison 2019). Without the kelp, there was a distinct lack of availability of food and shelter for less voracious organisms, such as fish, mollusks, and crustaceans (Catton et al. 2016), leading to a loss of biodiversity. This “perfect storm” of events (Catton et al. 2016) demonstrated the true importance of bull kelp as a foundation species to California’s coastal environments.

Northern bull kelp can be found within a ∼4,800 km range on North America’s west coast, stretching from Point Conception, California to Unimak Island, Alaska (Contolini et al. 2021) and tends to grow at depths of 3–20 m, inhabiting bedrock, rocky reefs, or boulder fields, which they use to recruit and develop holdfasts (Springer et al. 2007). Bull kelp is considered a foundation species by which a food web and/or dynamic ecosystem may be structured or built (Ellison 2019). Bull kelp consists of a microscopic haploid stage – known as the gametophyte – and a macroscopic diploid stage – known as the sporophyte (Dobkowski and Crofts 2021). It has been suggested that the upper thermal limit for bull kelp from central California is above 15 °C, as it will inhibit sporophyte growth (Wheeler et al. 1984), while more recent studies have found the optimal growth temperature can range between 2 and 17 °C (Contolini et al. 2021; Supratya et al. 2020). Like most kelp species, bull kelp has been affected by the increasing frequency of marine heatwaves, but the effects themselves have been different depending on location. For example, bull kelp populations in Alaska (USA) showed variable to stable responses during the Blob (Starko et al. 2025), but populations in the lower northern California range lost significant abundance (Catton et al. 2016; Smale 2020). So far, the only metrics used to measure the thermal response of bull kelp have been physical or ecological, such as reproductive success (Korabik et al. 2023; Muth et al. 2019), blade growth and structure (Supratya et al. 2020). If we can use molecular tools to measure the physiological response of bull kelp to environmental changes, we may be able detect how perturbations, like temperature, affect an organism’s ability to minimize mortality rather than only relying on physical assessment (Ryan and Hightower 1999).

Stress-inducible heat shock proteins are a specific group of molecular chaperones used for examining temperature, and are highly conserved across species (Camarena-Novelo et al. 2019). When most eukaryotic organisms are subjected to environmental stress, cells will undergo a process called the heat shock response (HSR; Krivoruchko and Storey 2010). This response serves the dual purpose of repairing cellular damage brought on by physiological stress and preparing for the next instance of stress, rather than only serving as a preventative measure (Verghese et al. 2012). It acts as a series of changes in gene expression to suppress normal protein biosynthesis, and induces cytoprotective genes, which code for heat shock proteins (Vierling 1991). Heat shock proteins (Hsps) are responsible for preventing other proteins from denaturing under stress (Adham et al. 1991). The most extensively studied family of heat shock proteins is Hsp70 (Vayda and Yuan 1994), which has been observed in almost all organisms and has both constitutive and inducible isoforms (Brown et al. 1993; Hu et al. 2022). The constitutive proteins are expressed and transcribed at the same rate as any protein, and are responsible for folding new cytoplasmic proteins and secretory proteins (Verghese et al. 2012), while the inducible forms aid in many physiological stress responses, such as protecting thermally damaged proteins from aggregation, unfolding aggregated proteins, either refolding damaged proteins or selecting them for degradation, and moving proteins to their needed locations in the event of physiological stress (Place et al. 2004). All Hsp70s require the hydrolysis of ATP, making the process energetically costly (Vierling 1991).

The mechanistic machinery of the HSR is widely studied in the field of ecophysiology, as it can influence – indirectly – physiological patterns from the cellular level up to the whole organism (Lindquist 1986). These patterns, in turn, affect how organisms are able to cope with living in a dynamic and ever-changing environment, such as the ocean (Hofmann 2005). Special attention has been paid to the gene expression and protein abundance components of the HSR in intertidal invertebrates (Brokordt et al. 2015; Giudice et al. 1999; Sorte and Hofmann 2004; Tomanek and Somero 1999, 2002–to name a few), and fish from different thermal niches (Lewis et al. 2016; Place et al. 2004). Pushing the stress response to its limit will define where an organism’s thermal homeostatic range lies, and how that may vary throughout an organism’s lifetime (Giudice et al. 1999). Sessile organisms, in particular, can be more vulnerable to thermal stress but have been shown to adjust their HSRs seasonally (or on a long-term scale), as they are unable to escape temperature fluctuation, and therefore must acclimatize to their surroundings on a regular basis (Hofmann 1999).

In marine algae, the gene expression of hsp70 has been studied more extensively (Hara et al. 2022; Hereward et al. 2020; Henkel and Hofmann 2008a, 2009; Jueterbock et al. 2014; King et al. 2019; Liu et al. 2019) than protein abundance. However, the few studies with Hsp70 protein were conducted in a variety of green (Ulva prolifera; Fan et al. 2020; Zhang et al. 2012), red (Gracilaria vermiculophylla; Hammann et al. 2016) and brown algae (Fucus serratus; Ireland et al. 2004), where it has been noted that Hsp70 protein concentration can rapidly increase within 1–2 h of exposure to thermal stress, and as late as 24 h. Given the likelihood of continued increase in the occurrence of marine heatwaves (Oliver et al. 2019), this suggests measuring Hsp70 protein abundance may be a useful indicator detecting cellular temperature stress in bull kelp. To the best of our knowledge, there has been little to no research on the thermal tolerance of bull kelp at the molecular level. In this current study, we examine the physiological response of N. luetkeana to thermal stress using individuals from two bull kelp populations on the northern California coast. By measuring Hsp70 protein abundance from blades acclimated to ambient seawater temperature (13 °C) compared to those acclimated under warmer seawater temperature (17 °C), we can assess whether bull kelp populations mount a HSR during short-term heating events, similar to that of marine heatwaves, and if thermal history and population play a role in determining thermal tolerance.

2 Materials and methods

2.1 Bull kelp sampling and blade preparation

We collected vegetative sporophyte blades from Nereocystis luetkeana from two sites: Big River, Mendocino County, CA, USA (39° 18′ 5.292ʺ N123° 45′ 26.784ʺ W) and Russian Gulch, Sonoma County, CA, USA (39° 19′ 53.472ʺ N 123° 47′ 21.696ʺ W; Figure 1) in October 2022, with Big River designated as the northern site and Russian Gulch designated the southern site. The two sites are approximately 129 km from each other toward the southern range of distribution for bull kelp. The Big River site is located at the shallower northwest end of Mendocino Bay near the mouth of the Big River behind the Mendocino Headlands, while the Russian Gulch site is located at Russian Gulch State beach, a relatively isolated cove with a steep, sloping shore. No environmental conditions or metrics were measured; site descriptions were based on visual observation and field notes (personal observation, Brent Hughes).

Figure 1:

Bull kelp field collection sites. Vegetative sporophyte blades (n = 5) were collected from two field sites: northern site = Big River Beach, Mendocino County, CA (USA) and southern site = Russian Gulch State Beach, Sonoma County, CA (USA), denoted by a black dot. The black line represents bull kelp distribution along the western Pacific coast extending up into the Aleutian Islands.

We collected vegetative blades from five randomly selected individual sporophytes per site, placed the blades in coolers between paper towels soaked with seawater, and transported them to the University of California, Davis Bodega Marine Laboratory (USA) for preparation. We cut the collected vegetative sporophyte into 25.4-cm strips from the middle of the blades to ensure blade development was consistent. If tissues were taken closer to the pneumatocyst, this would indicate the younger development and if tissues were collected near the end of the blade, then the tissues would be the oldest part and could contain reproductive tissues (spore-packets/sori). We briefly soaked the cut blades in an iodine-filtered seawater solution (1:9 iodine at 1-µm filtered and UV sterilized seawater) before the blades in 38.48 × 20.32-cm glass Pyrex dishes filled with filtered seawater (1-µm filtered and UV sterilized seawater).

3 Results

There was a significant difference in total Hsp70 protein abundance between sporophytes acclimated to the ambient seawater temperature compared to those acclimated to the warmer seawater temperature (F_1,72 = 7.32, p = 0.009; Figure 2A). Specifically, sporophytes acclimated to 17 °C exhibited 20.2 % greater protein abundance than those acclimated to 13 °C, with 14 % of the variability due to the random effect of individual bull kelp sporophytes (Wald p-value = 0.19). These results suggest that the bull kelp acclimated to the warmer temperature mounted a stronger response to the short-term temperature change, while sporophytes acclimated to the ambient temperature had a lower response.

Figure 2:

Bull kelp sporophyte Hsp70 total protein abundance. Hsp70 protein abundance is represented as relative light units (RLU) on the y-axis with (A) acclimation temperature (°C) and (B) collection site on the x-axis. The asterisk (*) denotes a significant difference (p = 0.009) while the dagger (†) denotes a marginal significance (p = 0.058). Box plot values represent the means ± SEM of Hsp70 protein abundance from the sporophytes (n = 10) for each collection site and acclimation temperature, with the middle line representing the median protein abundance and the dots indicating outliers in the dataset.

Collection site had a marginal difference in total Hsp70 protein abundance between sporophytes collected from Big River and those collected from Russian Gulch (Figure 2B). Specifically, sporophytes from Russian Gulch exhibited approximately 22 % more Hsp70 protein abundance than those from Big River (F_1,8 = 4.85, p = 0.058), with 6.6 % of the variability due to the random effect of individual bull kelp sporophytes (Wald p-value = 0.41). These results would suggest that the sporophytes from the southern site of Russian Gulch mounted a greater heat shock response than those sporophytes from the northern site of Big River.

Total Hsp70 protein abundance did not differ between any of the heat shock temperatures (F_4,72 = 0.74, p = 0.565; Figure 3), and none of the interactions between collection site, acclimation treatment, and heat shock temperature were significant (Table 1).

Figure 3:

Bull kelp Hsp70 total protein abundance compared across different heat shock temperatures, sites, and acclimation treatments: sporophytes from two collection sites (Big River = BR, Russian Gulch = RG) were acclimated to 13 °C (AC13) and 17 °C (AC17) for 7 days. Hsp70 protein abundance is represented as relative light units (RLU) on the y-axis with heat shock temperature (°C) on the x-axis. Box plot values represent the means ± SEM of Hsp70 protein abundance for the sporophytes (n = 5) at each heat shock temperature, with the middle line representing the median protein abundance. No significant interactions were detected in any of the interactions.

4 Discussion and conclusion

This study demonstrates that temperature is one factor that can drive the heat shock response in N. luetkeana, but we recognize that organisms such as bull kelp living in a dynamic and changing environments are likely impacted by other stressors that could play a role in an organism’s physiological response to thermal stress that were not tested in our study. For example, the microbiome community associated with specific kelp species ((golden kelp- Ecklonia radiata; Vadillo Gonzalez et al. 2024; Veenhof et al. 2025) and (giant kelp- Macrocystis pyrifera; Minich et al. 2018)) have shown to impact thermal tolerance at specific stages. However, our study suggests acclimation temperature and/or thermal history (Collins et al. 2020; Tomanek 2010) are important factors that can influence the heat shock response of bull kelp.

The warmer-acclimated bull kelp had 20 % more total Hsp70 protein abundance compared to the ambient-acclimated kelp (Figure 2A) irrespective of collection site. It has been well documented that a strong HSR can occur when marine organisms are acclimated to warmer water (Tomanek and Somero 1999, 2002 – marine snails; Lewis et al. 2016 – salmonids; Hereward et al. 2020; King et al. 2019 – oarweed), which could be because growth temperature, or the temperature of the environment the organism germinated or developed in, has been known to affect the plasticity of the HSR in most organisms, especially sessile ones (Barua et al. 2003). An organism that inhabits warmer temperatures or faces frequent heatwaves is likely to experience repeated triggering of the HSR over the course of its life, resulting in an increase in the base level of the inducible isoform within the cytosol (Maloyan et al. 1999). The buildup of heat shock proteins in the cytosol provides greater support for protein folding, leading to a delay in the HSR being triggered (King et al. 2019). As a result, the higher the acclimation temperature, the greater the available pool of heat shock proteins (Craig and Gross 1991; Henkel and Hofmann 2009) and thus the need to trigger the HSR to deal with any misfolded proteins is lessened. The warmer acclimation temperature (of 17 °C) was meant to mimic the marine heatwave conditions of the Blob (Di Lorenzo and Mantua 2016), thus, to capture the physiological response of bull kelp during an artificial warming event. Warmer temperatures are known to increase the half-life of inducible Hsp70 proteins from 2 to 7 h, therefore increasing the likelihood of the inducible form being present in the cytosol at a given time, especially during the heat shock session (Duncan and Hershey 1989). Though we were not able to distinguish between the two isoforms of Hsp70, it is conceivable that the warm-acclimated kelp might have a greater abundance of the inducible isoform because the sporophytes were being subjected to conditions similar to a week-long heatwave, and at a temperature that has shown to be the thermal limit for bull kelp (Contolini et al. 2021; Coppin et al. 2020; Supratya et al. 2020; Wheeler et al. 1984), therefore adding to the increase in total Hsp70 protein abundance (Tomanek and Somero 2002). The difference we see in protein abundance between the two acclimation temperatures suggests that the acclimation period of seven days appears to have been enough time to build a sufficient “pool” of heat shock proteins in the cytosol for sporophytes acclimated at 17 °C, and that the warmer acclimated kelp had built up a stock of Hsp70 protein to deal with these heatwave-like conditions (Henkel and Hofmann 2008b). This could represent a snapshot of the state of the cells at the time of the event (Mahmood and Yang 2012). The differences we saw between collection sites is further evidence for the effect of thermal history on the HSR for Nereocystis (Buckley et al. 2001; Smith et al. 2024), however we are aware other factors could be impacting bull kelp’s physiological response not tested in this study.

We were surprised to find that there was no significant difference in Hsp70 protein abundance when bull kelp was subjected to the 1-h heat shock (Figure 3), considering studies have defined a ‘typical’ HSR as a bell-curve (Kellermann et al. 2019; Schulte et al. 2011) when exposed to acute thermal stress. It is common for Hsp70 protein abundance to start low when temperatures are close to ambient, and not stressful for the organism, then rise as temperature increases to a critical threshold, after which protein abundance decreases as temperature continues to warm (Tomanek and Somero 1999). Previous studies on other species of marine algae displayed a HSR during acute heat shock sessions after anywhere from 4 h to 2 weeks of acclimation (Henkel and Hofmann 2008a; Ireland et al. 2004; King et al. 2019). A possibility for the lack of a ‘typical’ HSR in our study could be due to the specificity of the primary antibody used in the study, such that it could not distinguish the difference between the constitutive and inducible isoforms in bull kelp. To the best of our knowledge, and with multiple troubleshooting, we were not able to find an inducible (only) antibody that would work on bull kelp tissues. Therefore, our results are more conservative by taking the sum of constitutive and inducible isoforms together (calling it total Hsp70 protein) since we cannot definitively determine whether the antibody is binding to the inducible form only. Fortunately, data supports that the combination of both constitutive and inducible Hsp70 isoforms is still an effective way to measure stress for an organism (Giudice et al. 1999; Ireland et al. 2004; Lindquist 1986; Mayer and Bukau 2005), but we understand that our results may be underestimating the true response of bull kelp in our study.

There is also the question of whether the acclimation period itself influenced the lack of difference we saw in HSR during the heat shock session. We saw a difference after a 7-day acclimation, wherein the kelp acclimated at 17 °C had greater protein abundance than kelp acclimated at 13 °C. But we did not see a difference between either the acclimation groups or the different heat shock temperatures (within or between acclimation groups) during an hour in the face of an acute heat shock event. A possible explanation for this could lie in the nature of the HSR itself as it is an energetically costly process that operates on a negative feedback loop (Angilletta 2009; Brown et al. 1993; Feder and Hofmann 1999; Krebs and Loeschcke 1994); therefore, it has a threshold of activation, maintenance, and deactivation (Currie et al. 2014; Krakowiak et al. 2018). It could be that the sporophytes acclimated to 17 °C were stressed enough over the course of the week to mount a HSR, while those acclimated to 13 °C were not; hence the general difference we saw between the two groups. The warm-acclimated sporophytes could have been too stressed at the end of the week to mount any greater of a HSR when subjected to an additional heat shock session. However, we were surprised to find that there was a lack of significant difference in Hsp70 protein abundance between heat shock temperatures. Previous studies have shown the HSR can be both immediate and delayed depending on factors such as length of exposure, depth, or location (Henkel and Hofmann 2008a; Ireland et al. 2004; King et al. 2019). Sometimes different subspecies within a shared genus (Clusella-Trullas et al. 2014; Serafini et al. 2011; Tomanek and Somero 1999) can produce a different heat shock response. Unfortunately, the lack of a heat shock response cannot be explained as this time. But it is possible producing more Hsp70 proteins could have been too energetically expensive by the end of a week at 17 °C or conversely, the ambient-acclimated sporophytes might also not have been stressed enough by the acute heat shock to produce more Hsp70 proteins.

Since this is the first time such an experiment has been attempted with this species, further investigation would benefit to help understand the lack of conclusive results with heat shock. One suggestion might be to collect bull kelp from more southern and northern sites along its range as this could provide more insight into the HSR of Hsp70 due to specific geographical variations (Helmuth et al. 2002), while also identifying the effects of increased warming events (Smale 2020). This could illustrate a more complete picture in the HSR by suggesting if some southern populations of bull kelp are more thermally tolerant than others by acclimatizing better (Harley et al. 2006) or if further northern populations of bull kelp (e.g., Washington or Alaska) are more thermally sensitive and delay the activation of their HSR until temperatures are warmer (King et al. 2019).

Overall, these data suggest that an organism’s thermal history can affect the HSR to short-term temperature changes, which means it is the specific temperatures the organism and those within its community and population experience during their lives that may determine how they respond to a short heating event. For example, a recent study found that different bull kelp populations within California showed a differential effect on abundance, length, and development when reared at warmer temperatures (Fattori 2024). This emphasizes the importance of an organisms’ ability to locally acclimatize, especially as marine heatwave occurrences become more frequent, and how these perturbations could affect not only the same species across a wide range of habitats but also their impacts on local community-level biodiversity (Oliver et al. 2019, 2021). Conducting future acclimation studies, especially those at the molecular level, has the potential to build a comprehensive prediction on which organisms in what populations could tolerate and survive future heatwave events, and which may not (Somero 2010).