Mussels enhance digestive enzyme activity in preparation for stressful fluctuating environments

TL;DR

Mussels adjust their digestive enzyme activity to cope with environmental stress like heat and food scarcity.

Contribution

The study reveals mussels elevate amylase activity during low tide to mitigate energy losses under stress.

Findings

Mussel amylase activity increased unpredictably during low tide under fasting or moderate heat stress.

Multistressor conditions (heat and fasting) significantly reduced amylase activity.

Acute aerial heat-shock negatively impacted amylase activity across all groups.

Abstract

Elevated heat and low phytoplankton abundance are physiologically and ecologically challenging marine organisms of the northeast Pacific, including the mussel Mytilus californianus. During low tide, mussels are exposed to warm air and undergo anaerobic metabolism. Tidal variation coincides with daily fluctuations in food availability requiring flexible response systems (e.g., changes in digestive enzyme activity) to maintain homeostasis. In this study we allowed mussels to acclimate in tidal mesocosms with or without aerial heat across high and low levels of food abundance, after which we measured amylase (carbohydrase) activity and gene expression. Remarkably, enzyme activity was unpredictably elevated during low tide in fasted or moderate heat-stress (+ 8 °C; 23 °C) conditions, thereby potentially mitigating energy losses posed by environmental stress. Heat stress combined with fasting (multistressor) presented a marked challenge for mussels as evidenced by lower amylase activity compared to other treatments. A subsequent acute aerial heat-shock (+ 17 °C; 32 °C) negatively impacted amylase activity across all acclimation groups. Gene expression of amylase was anticorrelated with activity suggestive that mussels’ sense environmental fluctuations and respond by regulating digestive enzyme activity. Although mussels can employ these strategies to dampen short-term stress, the heatwaves predicted to persist in their ecosystems in the future may curtail their effectiveness.

The online version contains supplementary material available at 10.1007/s00360-025-01637-w.

Figures

Experimental design showing temperature and tides of hot tanks (non-isothermal) and cold (isothermal) tanks. Each temperature regime included two food levels (algae-fed and fasted). The red lines represent the ambient temperatures peaking at 23 °C during aerial exposure in the hot tank while temperatures between low and high-tide in the cold tank were isothermic (15 °C). The blue lines signify tide level (high or low) which were on a 12 h:12 h tidal cycle with low-tide starting at hr. 0/24 and high-tide starting at hr. 12 of a 24 h clock. Yellow and black ribbons represent a 12 h:12 h. day nig

Digestive enzyme activity (A) and relative gene expression (B) in digestive glands of *M. californianus, (N = 4–7)*. Mussels were acclimated for 12 days to four regimes: Algae-fed cold (Ac), Fasted cold (Fc), Algae-fed hot (Ah), Fasted hot (Fh). All mussels were subsequently subjected to a two-day common extreme (E) heat-shock treatment. The tidal sequence was 12 h:12 h; low: high-tide. High- and low-tide temperatures in the cold regime were isothermic (15 °C). Peak aerial temperature in hot regime was 23 °C. The common treatment aerial temperate peaked at 32 °C. Box plots are blue and red sig

Mussels within the intertidal zone display indeterminate growth. Scope for growth (SFG) predicts potential growth based upon the equation Assimilation-(Respiration+Excretion). Albentosa et al. (2012) predicted a positive relationship between SFG and amylase enzyme activity in submerged mussels. Mussels acclimated or acclimatized to chronic but not extreme heat or starving, negatively effects growth potential in mussels. We predict that mussels mitigate the costs of these single stressors by increasing digestive performance during low tide—increasingly displaying energy-balancing power with dec

Full text

Introduction

The mussel, Mytilus californianus, aggregates on rocky shores to form dense beds along the western intertidal zone of North America, spanning Baja Mexico to the Aleutian Islands of Alaska. Mytilus californianus serves as a foundation species; its beds harbor a diverse array of invertebrate and vertebrate animals, macro-algae, and microbial organisms (Suchanek 1992). As a filter feeder, it clears nearshore water of phytoplankton, and its waste material provides nutrients for surrounding organisms. Such effective filtering of organic material solidifies bivalves’ role in biogeochemical cycles and as carbon sinks (Baumas and Bizic 2023; Tamburini et al. 2022). As a sessile organism, M. californianus must cope with prevailing environmental regimes of the intertidal zone, making it a quintessential sentinel of climate change (Helmuth et al. 2006). Therefore, understanding the mechanistic strategies mussels employ to survive and sustain positive growth can support efforts by coastal managers to conserve mussel beds and their associated communities while tracking climate change.

The intertidal zone is the area of coastline between high and low tidal height. Mussels affixed to rocky structures within this horizontal band of shoreline reside in a fluctuating environment driven by twice-daily tidal oscillations (i.e., shifts in temperature, oxygen, and food availability), monthly changes in plankton abundance (i.e., food), and seasonal temperatures (Helmuth 1998; Helmuth and Hofmann 2001; Mass et al. 2022; Yu et al. 2019). During high tide, mussels are submerged in seawater concentrated with phyto- and zooplankton upon which the mussels filter feed. At low tide, mussels are exposed to air and cannot consume food. However, digestion of ingested organic material can proceed during aerial exposure (Langton 1977). Thus, a mussel’s feeding period is primarily driven by the ebb and flow of the daily tidal cycle. During aerial exposure, mussels close their shell-valves to avoid desiccation as they endure prevailing solar radiation and convective heat exposure (Helmuth 1998). Shell-valve closure, however, separates tissues from oxygen, thereby activating low ATP-yielding anaerobic metabolic pathways (Connor and Gracey 2012; De Zwaan 1977). Hence, emersion/immersion cycles are the principal modulators of intertidal mussels’ core metabolic architecture, from transcribed genes to enzyme activity. However, most studies investigating feeding and digestion in intertidal mussels have been conducted in submerged conditions only, thereby limiting our understanding of these vital functions within a mussel’s natural environment.

As ectotherms, the body temperatures of mussels closely match that of the prevailing environment. Hence, sufficiently elevated air temperatures during low tide can denature cellular proteins, impacting their function and thereby signaling the activation of cellular mitigation responses (i.e., the heat-shock response [HSR]). Protein restabilization and the destruction of irreversibly denatured peptides are two aspects of HSR. These processes require relatively high levels of ATP (Gracey et al. 2008; Halpin et al. 2004; Lindquist and Craig 1988). Hence, heat stress can have detrimental effects on an animal’s energy balance (i.e., the quantitative difference between energy reserves and maintenance costs). The degree of aerial heat stress a mussel encounters changes both over seasonal timescales due to variation in average monthly air temperature and day-to-day transitions between diurnal and mixed-semidiurnal tides. Although water temperatures in the northeastern Pacific Ocean are consistently within the optimal range for M. californianus, thermoclines can form during summer heatwaves, curtailing the upwelling of deep, nutrient-dense cold water that supports primary production (Arteaga and Rousseaux 2023). Thus, the environmental variables of the intertidal zone play a strong role in modulating the energy balance of mussels over temporal scales. To this point, mussels show indeterminate growth−primarily dependent upon environmental factors (Koehn and Gaffney 1984). The energy acquired from nutrient assimilation is allocated toward positive growth (somatic and reproductive) after maintenance and repair costs are accounted for, resulting in a temporally coordinated energetic trade-off between growth and heat stress (Fitzgerald-Dehoog et al. 2012). Therefore, comprehensive experimentation in field and controlled settings can be harnessed to understand the biological strategies mussels exploit to acquire resources in the face of aerial heat stress and variable nutrient access (Talevi et al. 2023).

As a result of daily changes in maximal tidal height, mussels residing on elevated portions of vertically sloping rocky shores experience longer periods in air (i.e., without access to food) and higher levels of heat exposure than low-shore mussel populations (Helmuth and Hofmann 2001). Therefore, mussels at relatively high shore elevations are challenged by prolonged heat stress, feed less, and acquire less energy than low-shore mussels. This results in slower indeterminate growth rates for high-shore mussels compared to their low-shore conspecifics (Connor and Robles 2015; Petes et al. 2008). Ultimately, a mussel’s energy balance is negatively affected by both heat-shock recovery costs and lower nutrient availability, reflecting bioenergetic restriction and reduced growth rates that are scaled by its tide-height position on the shore (Fitzgerald-Dehoog et al. 2012; Menge et al. 1997).

Indeterminate growth rate and terminal size are important life-history traits of mussels because larger mussels are superior in competition for space, fecundity, and predation-escape in comparison to smaller individuals. Flexibility in gut-related physiological processes, such as digestive enzyme function, may be a strategy to mitigate environmental bioenergetic restrictions and improve growth potential. In this context, the nutrient balancing hypothesis of digestive physiology states that organisms needing critical but scarcely available nutrients may scavenge for these resources by over-producing corresponding enzymes (Clissold et al. 2010; Raubenheimer and Simpson 1997). Although this strategy has not explicitly been observed in mussels, we postulate that mussels may engage in a form of ‘energy-balancing’ which suggests using real-time energy balance as a stimulus, rather than nutrients, to regulate the level of digestive enzyme activity in the gut—in line with hunger and satiety mechanisms observed in vertebrates (Friedman-Einat and Seroussi 2019; Morley 1995). Energy levels negatively impacted by way of environmental perturbations, such as high heat exposure or lack of food in the prevailing sea, would promote increased digestive enzyme activity to replenish losses. Theoretically, the investment in ramping-up digestion pays off as improved energy balance, provided that all other biological factors are functioning normally. When engaged in energy-balancing, compared to nutrient-balancing, the priority is to support homeostasis by maintaining a net positive energy level, rather than optimizing intake of a specific nutrient.

A study of digestive physiology (Connor et al. 2016) in M. californianus observed high-shore mussels experiencing greater than average environmental temperatures (high heat stress) and longer periods of aerial exposure (low feeding) than low-shore mussels. Cellulase enzyme activity was unexpectedly robust in high-shore mussels compared to low-shore conspecifics—consistent with the energy-balancing hypothesis. Cellulase digests cellulose, a ‘scarce’ energy resource, suspended in very low concentrations in coastal marine environments. Comparatively, amylase is a digestive enzyme that disassembles starch polymers, which are ‘abundant’ in the phytoplankton dispersed across northeastern Pacific coastal habitats. Unlike their cellulase activity, however, the mussels in Connor et al. (2016) did not show variability in average amylase activity across microhabitats (both thermal and emersion gradients). Still, the high-shore mussel groups showed the least variance in amylase activity, insinuating a much more limited range of gut functionality and more stable digestive activity compared to low-shore mussels. Possibly as a result of limited food availability and elevated average temperatures in the high intertidal zone. Thus, the authors postulated that mussels sense environmental factors that negatively impact their energy budgets, such as low food or high heat, or can evaluate their internal level of energy. Mussels may respond to these environmental or endogenous cues by boosting the activities of specific enzymes (e.g., cellulase) in anticipation of opportunities to feed on scarce nutrients. Simultaneously, they may limit the synthesis and secretion of other digestive enzymes targeted for polymers that are more readily available in the environment, as a means to conserve energy (e.g., decrease the synthesis of amylase).

Precise measurements of heat and food levels can be onerous in field conditions, thereby challenging the interpretation of results. A handful of laboratory-based studies revealed lower amylase activity in bivalves submerged in warm water (Khan et al. 2020; Shang et al. 2022). However, an investigation of amylase activity by Pham et al. (2023) found evidence that mussels acclimated to moderate convective heat in air, and moderate food levels did not exhibit energy-balancing processes (i.e., overproduction of enzyme activity), perhaps due to the lack of nutritive disruption or adequate level of heat stress needed to signal for the activation of this strategy. Cellulase activity remained constant across temperature treatments, whereas amylase activity was slightly depressed in warm-acclimated mussels compared to those kept in cool conditions-indicating energy conservation during moderate heat stress in the face of food availability. The proximate mechanisms driving lower amylase activity in warm-acclimated mussels are intriguing, especially considering the absence of its observation in the environmentally challenged, high-shore mussels in the field. This knowledge gap offers the opportunity to study thermal digestive physiology in mussels across a variety of temperatures and food levels with precision in laboratory conditions.

The observation of amylase activity depression in previous studies is likely the result of a complex integration of food availability, temperature, and individual energy balance that modulates digestive gland function. A broader examination of this dynamic at the biochemical and molecular level will bridge the paucity of data relating to energy allocation during stress, emphasizing the importance of the current study. Here, we sought to understand the combined effects of abundant food availability versus its absence with moderate aerial heat on the enzyme activity and gene expression of alpha-amylase in M. californianus. Comparing the enzyme activity and gene expression of amylase under variable conditions of nutritive and heat stress enables the evaluation of an energy-balancing versus energy-conservation strategy. We acclimated mussels in the laboratory to simulated tidal regimes designed to impose either isothermic (constant temperature) or moderate aerial heat exposure (fluctuating temperature) combined with variable food abundance (algae-fed vs. fasted). Digestive enzyme performance and gene expression were subsequently measured. We predicted that mussels experiencing optimal temperatures across low and high tide (15 °C) and abundant food would require steady levels of amylase activity across tidal cycles, in line with robust filtration, metabolism, and persistent growth (Bayne et al. 1993). Conversely, we expected well-fed, moderately heat-stressed mussels (23 °C) to down-regulate amylase gene expression during low tide, thereby depressing enzyme activity as a means of decreasing protein synthesis. In this context, decreasing enzyme activity would be consistent with the reallocation of energy from ATP-consuming processes related to digestion toward protein synthesis of HSR enzymes such as heat-shock proteins—exhibiting energy-conservation. Alternatively, we anticipated that starvation would trigger compensatory physiological responses to acquire energy during metabolic stress. Such a state would provoke an overproduction or over secretion of amylase, especially in combination with ATP consuming heat stress−exhibiting energy-balancing.

Following tidal acclimations, we expanded the test of the functional performance of amylase by subjecting all acclimated mussels to a common treatment of extreme aerial heat-shock. We predicted that experiencing a more intense heat stress would curtail energy-balancing strategies. These environmental scenarios align with the stressors mussels face in field conditions (Hofmann and Somero 1995). Therefore, these results refine models of how mussels might respond to their changing environments in the context of thermal and nutritive stress.

Materials and methods

Collection

In June 2022, we collected 134 M. californianus during low-tide from a south-facing, wave-exposed, mid-shore site at Ventura Beach, CA (34.266 N, −119.278 W). We transported the mussels to the University of California, Irvine, and housed them in a 15 ± 1 °C temperature-controlled room. We submerged them in one of two tanks filled with 40 L of seawater (40:60 filtered: artificial seawater) over four days. The tanks were affixed with non-stop underwater filters, air stones, and circulation pumps to depurate until there was no further expulsion of visible material. Each day, from the start of depuration to the end of the experiment, we removed detritus and changed 25% of the water. On day three of depuration, at least one mussel released milt, requiring a complete water change.

Before the start of the acclimation, we weighed and measured the length, width, and height of each mussel, then randomly assigned it to one of four regimes of feeding and low-tide temperature treatments to which it would be acclimated: algae-fed cold (Ac, N = 34), fasted cold (Fc, N = 35), algae-fed hot (Ah, N = 33), and fasted hot (Fh, N = 35). We situated the mussels of each group in a single layer across the bottom of a mesh basket. We then placed the two algae-fed treatment baskets in a corresponding ’algae-fed’ tank and the two fasted treatment baskets in a corresponding ‘fasted’ tank.

Acclimation detail

We simulated a 12 h: 12 h low: high-tide cycle in a 15 °C cold room. The lights were on during low-tide (hr. 24 to hr. 12) and off during high tide (hr. 12 to hr. 24) to match the potential circadian effects of previous laboratory experiments (Connor and Gracey 2012; Pham et al. 2023). To simulate cool low tide, we removed the treatment baskets from the water at hr. 24 to sit on the counter in the cold room until hr. 12 and returned the baskets to the water from hr. 12 to hr. 24. To produce a hot low tide, we placed mussels into an enclosure with a temperature controller (Omega, Norwalk, CT) set to increase 1.5 °C/hr. from room temperature (15 °C), until the air temperature reached 23 °C (Fig. 1). The heat-stressed mussels endured 6 h of heat at 23 °C until the start of high tide at hr. 12. We kept water filters on while the tanks were empty of mussels and used a protein skimmer to remove waste. At the beginning of each high tide (hr. 12), we provided the algae-fed tank with live algae. The tanks of seawater were supplied with air stones and circulation pumps to increase dissolved oxygen and ensure water flow.

We provided the algae-fed mussels with a mixture of live Nannochloropsis, Tetraselmis, Porphyridium, and Isochrysis algae (Filter Feeder Formula, Algae Research and Supply, Carlsbad, CA). Before each feeding, we microscopically examined a sample of the algal culture using a hemocytometer to estimate cells. The hemocytometer ensured we could feed the mussels at a concentration of 5300 cells/mL, compared to 2631 cells in Pham et al. (2023). We chose this concentration to maximize mussel filtration, as evidenced by estimates of 800 cells/mL for M. galloprovincialis (Maire et al. 2007) and 5000–8000 cells/mL in M. edulis (Riisgård et al. 2011). These simulated experimental conditions produced four acclimation regimes: Algae-fed cold (Ac), Fasted cold (Fc), Algae-fed hot (Ah), Fasted hot (Fh), and eight sub-groups; Ac_Low-tide, Ac_High-tide, Fc_Low-tide, Fc_High-tide, Ah_Low-tide, Ah_High-tide, Fh_Low-tide, Fh_High-tide. (Fig. 1.)

Extreme heat-shock common treatment after acclimation regime period

All mussel treatment groups experienced a common treatment for 2 days after 12 days of respective acclimation regimes. During low tide, we exposed all mussels to an extreme heat-shock. The heat shock was induced by increasing the air temperature from 15 °C to 32 °C at a rate of ~ 2.8 °C/hr., then held at 32 °C for 6 h. We submerged all treatment baskets in the same tank during high tide and fed the mussels live algae (Fig. 1).

Fig. 1. Experimental design showing temperature and tides of hot tanks (non-isothermal) and cold (isothermal) tanks. Each temperature regime included two food levels (algae-fed and fasted). The red lines represent the ambient temperatures peaking at 23 °C during aerial exposure in the hot tank while temperatures between low and high-tide in the cold tank were isothermic (15 °C). The blue lines signify tide level (high or low) which were on a 12 h:12 h tidal cycle with low-tide starting at hr. 0/24 and high-tide starting at hr. 12 of a 24 h clock. Yellow and black ribbons represent a 12 h:12 h. day night cycle starting at hr. 0/24. Mussels were acclimated for 12-days in four regimes: Algae-fed cold (Ac), Fasted cold (Fc), Algae-fed hot (Ah), Fasted hot (Fh). Black vertical lines represent 24-hour intervals. Dissections occurred at the end of low and high tides (purple lines). Following acclimation, the remaining mussels were transferred to a two-day common treatment. The extreme heat-shock common treatment exposed mussels to tide, food, and light conditions similar to the 12-day acclimation algae-fed regime. Peak low-tide temperature was 32 °C and feeding occurred at the beginning of high-tide periods. Dissections occurred at the end of low-tide and across the high-tide interval.

Dissections

We dissected the digestive glands of five mussels per treatment group on day 12 at the end of the 12-hour low-tide (hr. 12) and halfway (hr. 18) into the 12-hour high tide. This formed eight dissection groups representing the unique acclimation subgroups: Ac_Low-tide, Ac_High-tide, Fc_Low-tide, Fc_High-tide, Ah_Low-tide, Ah_High-tide, Fh_Low-tide, Fh_High-tide. We also dissected at the end of low tide during the extreme heat-shock (12 hr.) and at three time points after submergence (15 hr., 18 hr., 24 hr.). The low-tide and subsequent high-tide samples comprised the extreme heat-shock regime. The extreme heat-shock regime included 16 groups. The group nomenclature includes the prefix (E) that denotes the two-day extreme acclimation, the original acclimation experience (Ac, Fc, Ah, Fh), time of sampling, and tidal height at the time of sampling: [E_Ac_12_Low-tide, E_Ac_15_High-tide, E_Ac_18_High-tide, E_Ac_24_High-tide], [E_Fc_12_Low-tide, E_Fc_15_High-tide, E_Fc_18_High-tide, E_Fc_24_High-tide], [E_Ah_12_Low-tide, E_Ac_15_High-tide, E_Ah_18_High-tide, E_Ah_24_High-tide], [E_Fh_12_Low-tide, E_Fh_15_High-tide, E_Fh_18_High-tide, E_Fh_24_High-tide] (see Fig. 1). We dissected 5–7 mussels per treatment group to collect samples from the extreme heat-shock regime. Dissections started at the end of the second acute heat-shock (low tide, day 14; hour 12), then again 3, 6, and 12 hours into high tide (hrs. 15, 18, and 24, respectively).

We weighed each closed mussel at the beginning of the dissection. We cut the adductor muscle to open the mussel, pouring off the excess fluid within. We quickly dissected out the entire digestive gland, immediately placing it into a chilled 1.5 mL tube. Using a fresh 18G needle and syringe to homogenize the digestive gland tissue, we manually transferred 29–473 mg of tissue into a separate 2 mL round-bottomed vial for later RNA extraction (DG_rna_). The residual tissue in the vial (DG_enzyme_) was used for enzyme activity assays. We promptly froze the samples in separate vials on dry ice, then stored tissue samples at −80 °C.

Digestive gland mass

Vials designated to contain digestive gland material were labeled and weighed before the dissections. This enabled us to weigh the wet mass of deep-frozen tissue to ± 0.1 mg in its storage vial, thereby minimizing heating above − 75 °C. We calculated the total digestive gland mass by including both tissue fractions.

Digestive enzyme assays

We homogenized the DG_enzyme_ samples per German et al. (2015) in 7 volumes of 25mM maleic acid buffer (pH 6.5), hereafter referred to as “buffer”. We used a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) with a 12 mm generator set to 1100–3000 rpm for 3 × 30 s, with 30 s between pulses to homogenize the entire DG_enzyme_ then centrifuged the homogenate for 2 min at 9400 x g, 4 °C. We flash-froze 100–500 µl aliquots of the supernatant using dry ice and stored them at −80 °C until just before use in digestive enzyme assays.

We measured amylase activity using volume-optimized Somogyi-Nelson glucose assays, adapted from protocols outlined by German et al. (2015) and Wehrle et al. (2020). The assay used 1% potato starch (Sigma S-2630) substrate dissolved in a buffer containing 1 mM CaCl_2_. This starch solution was combined with pre-diluted homogenate in a 1:1 ratio and used in a 100 µL end-point assay performed in triplicate. (To minimize pipetting errors associated with small volumes, we pre-diluted freshly thawed homogenate with buffer at a 1:49 v/v ratio.) We included two homogenate-substrate control blanks for each assay. The starch substrate was added to the control blank after the enzyme reaction had stopped. After 30 min of incubation at 15 °C (additional replicates incubated at 23–32 °C to match the temperature at tissue collection) we stopped the assay using a pre-combined mixture of 200 ul Somogyi-Nelson Reagent A and 20 ul 1 N NaOH. We followed previous protocols for quantifying glucose concentration post-termination, plating the contents of each assay or blank vial in triplicate in flat-bottomed 96-well microplates. Using a BioTek Synergy H1 Hybrid spectrophotometer (BioTek/Agilent, Winooski, VT, USA), we measured absorbance at 650 nm, checked data quality (see supplemental materials), and used glucose standard curves to convert absorbance to activity in nmol glucose liberated per minute per gram of tissue (U).

Additionally, we analyzed the thermal performance of amylase activity at variable incubation temperatures in 5 °C increments (15–45 °C). Using four randomly selected individuals (E_Ac_12_Low-tide, Ah_12_Low-tide, Ac_18_High-tide, Fh_12_Low-tide), we assessed ex-vivo enzyme kinetics. For this, we performed amylase assays, as described above, at each of the seven incubation temperatures.

Gene expression

We used glass-fiber filters to extract RNA from the DG_rna_ samples with a TRIzol protocol (Qiagen; Germantown, MD) and glass-fiber filters. Using 0.75 µg RNA in a reverse transcription reaction following manufacturer instructions (iScript cDNA Synthesis Kit; BioRad, Richmond, CA), we synthesized cDNA to use as a template in qPCR reactions. The qPCR reactions used 1.5µL template at a 10-fold dilution, 1.5µL 5mM primers for α-amylase (AMY), heat-shock protein 73 (HSPA8), or α-tubulin (TUBA1A); reference gene; (Gracey et al. 2008) and SYBR green (iTaq; BioRad) in a Bio-Rad CFX thermocycler programmed for 40 cycles: 95 °C denaturing (5 min), 95 °C denaturing (0:10 min) 60 °C annealing (0:10 min), and 72 °C extension (0:10 min), with a temp melt curve each cycle. We designed the primers for the M. californianus AMY gene by aligning the digestive amylase enzyme sequences of highly annotated genomes against that of the M. californianus genome (GenBank accession number JAKFGE000000000). BLAST aligned with NCBI gene ID: 127726036. The primer pair sequences are as follows; AMY (L-AACTGTCGTCTGGTGTCCTT, R-TAGCTTGTAGATCACCGGGC); HSPA8. (L-AGACGCCGATATACAGTGGG, R-CGACAGTACAATGAGGCAGC); TUB1A (L-TCCAAGACACGGCAAATACA, R-TTGAAACCAGTTGGACACCA). We calculated gene expression (DDCt) fold-change with standardization by mean-centering and autoscaling (Willems et al. 2008). We considered algae-fed mussels exposed to cold aerial temperatures (15 °C) at low tide to be the controlled condition. (Note that per experimental design, these mussels had not experienced the extreme heat shock.)

Analyses

We compared the means of amylase enzyme activity, digestive gland mass, and gene expression fold-change across acclimation treatments, before and after acute heat stress, and at low and high tides via ANOVA with a = 0.05. For all analyses, we used enzyme activity assayed at 15 °C analyses, except for the repeated-measures ANOVA of the effect of assay temperature on enzyme activity. Each acclimation group had an associated unique environmental profile (subgroup), thus allowing comparisons of enzyme activities and transcript abundances both within and among groups. We compared acclimation groups at low and high tides to responses to common treatments E_12_Low-tide and E_18_High-tide. We also compared post-acute-heat-shock amylase activity and gene expression across all time-points with ANOVA using regime and time as main factors. Time points hr. 12 (low tide) and hr. 15 (high tide) were analyzed for the post-acute-heat-shock gene expression patterns of HSPA8. We removed enzyme activity outliers that fell outside > 1.5 times the interquartile range before performing the ANOVA. We performed Fisher’s LSD pairwise tests and controlled Type I error rate using Benjamini-Hochberg (BH) FDR p-value adjustments in line with comparing a large number of groups-16 subgroups. A Tukey’s test was performed for HSPA8 in line with comparing a limited number of groups being compared. Additionally, we performed principal component analyses (PCAs) with amylase activity, amylase gene expression, and digestive gland mass from each individual as variables. We conducted our analyses in both SPSS and R (v. 4.3.2–4.4.2).

Results

Amylase digestive enzyme activity across regimes

We tested the performance of amylase activity and gene expression in mussels across different air temperatures and food levels following their acclimation to optimal, single-stressor (hot or fasted), or multi-stressor (hot and fasted) tidal conditions, producing four treatment groups: Algae-fed cold, Fasted cold, Algae-fed hot, Fasted hot. There were no differences in amylase activity among these four regimes when subgroups were pooled and analyzed by FDR. However, comparisons of standard deviations among acclimation regimes revealed that amylase activity of cold-acclimated groups (SD_Ac_= 2.92 U, SD_Fc_ = 2.56 U) was more variable than that of hot-acclimated mussels (SD_Ah_= 1.26 U, SD_Fh_= 2.17 U). When only comparing low-tide groups among regimes, Ah_Low-tide and Fc_Low-tide had significantly higher (p < 0.05) amylase activities (7.38 U and 7.02 U respectively) than Fh_Low-tide (2.25 U). Algae-fed cold mussels at low tide had intermediate enzyme activity (5.22 U) that was not different from the other regimes.

Amylase activity experimentally assayed at acclimation temperatures the mussels experienced (15 °C, 23 °C, or 32 °C) revealed similar regime comparison trends to when assay tests were all incubated at 15 °C (Fig. S-1). However, at 32 °C, activity was 69% higher than when the same animal samples were assayed at 15 °C. This temperature effect was most pronounced in the fasted mussels (repeated-measures ANOVA, p < 0.001).

Intra-regime acclimation analyses

Single-stressor (Ah or Fc) groups trended towards greater differences in mean activity between low and high tide than did optimal and multi-stressed (Ac and Fh) groups (Fig. 2). Enzyme activity in the algae-fed hot acclimation group (Ah) during low tide had more than twice the average activity of Ah high tide (p < 0.01). This activity increase jumped > 20% when assayed at 23 °C. The Fc low tide acclimation group’s mean activity appeared the second highest of all experimental treatment groups and had nearly twice that of Fc high tide, but not significantly different (p = 0.10). Alternative to the trends observed in Ah, Fc, and Ah, the Fh group appeared to slightly increase (non-significantly) amylase enzyme activity from low to high tide (Fig. 2).

Comparisons between acclimation versus extreme heat-shock periods

With all four moderate acclimation-regime groups pooled, amylase activity was generally 1.5x higher than the extreme heat-shock acclimation (Fig. 2). In mussels that had been fasted, the extreme heat-shock treatment and the resumption of feeding appeared to depress low-tide amylase activity. Fasted Cold Group Performance: Compared to the low tide fasted acclimation group (Fc_Low-tide), the mussels in extreme heat-shock during low tide (E_Fc_12_Low-tide) and subsequent recovery at high tide (E_Fc_18_High-tide) had 2- and 7-fold lower amylase activities, respectively (p < 0.05). Together, the enzyme activity data suggest a negative effect from the extreme low tide heat on the fasted cold-acclimated group. However, when assay temperature was 32 °C for the extreme heat-shock during low tide the results showed a recovery of the apparent negative effects of high heat. There was not a difference between low tide values pre and post extreme heat-shock (Fig. S-1). Fasted Hot Group Performance: Unlike fasted cold comparisons, the mean amylase activity of fasted hot-acclimated mussels, after extreme heat-shock (E_Fh_18_High-tide), was not significantly different than its fasted hot-acclimation counterparts, possibly related to its lower acclimation low tide enzyme activity compared to other regimes. Hence, physiological perturbation was evident in fasted hot-acclimated mussels before exposure to extreme heat-shock, which did not further negatively impact their amylase activities. Algae-Fed Cold Group Performance: Algae-Fed cold mussels after extreme heat-shock (E_Ac_18_High-tide) were only marginally negatively affected by extreme heat stress. Average activity in E_Ac_18 High-tide was 40% lower than Ac_Low-tide, but not statistically different. Algae-Fed Hot Group Performance: Algae-fed hot-acclimated mussels underperformed after extreme heat-shock (E_Ah 18_High-tide and E_Ah_12_Low-tide) by showing 3- and 2-times lower amylase activity, respectively, than algae-fed hot-acclimated mussels at low tide (p < 0.01). However, similar to the fasted cold group, recovery can occur from increases in activity resulting from kinetics (as determined from the higher assay temperature test) (Fig. S-1).

Amylase gene expression across regimes

Only one pair of conditions among the sixteen subgroups revealed a difference in amylase gene expression (p = 0.02). Analysis of performance after extreme heat stress revealed that gene expression of E_Fh_18_High-tide was > 5x higher than in acclimation group Fh_Low-tide, but neither subgroup differed from Fh_High-tide (Fig. 2). Gene expression in hot-regime mussels trended ≈30% higher than the cold-regime groups and appeared rhythmic as low tide gene expression means were consistently, but non-significantly, lower than those of high tide.

PCA analysis across regimes

PCA’s revealed relationships between activity, gene expression, and digestive gland mass (Fig. S-2). Amylase activity and gene expression was anticorrelated in 3 of the 4 regimes, algae-fed hot, fasted cold, fasted hot, while gene expression was anticorrelated with digestive gland mass in the optimal regime. Enzyme activity and digestive gland mass were highly correlated in single-stressed regimes, algae-fed hot and fasted cold. However, activity had weak or no correlation in the optimal regime, algae-fed cold. Similarly, digestive gland mass had little correlation with amylase measurements for the multistress regime, fasted hot.

Amylase activity and gene expression during submergence

The general trends for amylase activity and gene expression depict a unique pattern reflective of the experimental conditions. For enzyme activity, both algae-fed acclimated groups (Ac and Ah) presented a similar temporal trend after experiencing acute heat-shock at 32 °C (Fig. 3). Both treatments exhibited peak enzyme activity at hr. 12, collected at the end of low tide, which dropped 3 hours (hr. 15) into high tide before rising again (hr. 18) to conclude with the lowest point of activity at the end of high tide (hr. 24). The fasted cold (Fc) treatment reflected a similar enzyme activity pattern: decreased activity at the end of high tide (hr. 24). However, before that, the enzyme activity held steady from the end of low tide (hr. 12) to 3 hours (hr. 15) into high tide reflecting significantly different expression from the three other regimes. More uniquely, the fasted hot (Fh) treatment reflected high enzyme activity at the end of low tide (hr. 12), before progressively declining at hrs. 15 and 18. The amylase activity for this group peaked at hr. 24, the end of high tide (just before the expected onset of low tide) (p < 0.05).

While 3 of the 4 treatments (algae-fed cold (Ac), algae-fed hot (Ah) and fasted cold (Fc)) had very similar patterns of amylase gene expression temporally, the fasted hot (Fh) group, representing a bioenergetic crunch, had a unique trend in gene expression. In mussels from the optimal condition treatment (Ac) only, we observed a minor (but non-significant) increase in gene expression 3 hours (hr. 15) into high tide, after acute heat-shock. Additionally, the algae-fed cold (Ac) group had the highest peak expression at the end of high tide (hr. 24), followed by algae-fed hot (Ah) and fasted cold (Fc) groups. On the other hand, fasted hot (Fh) was in sync with the single-stress treatments, algae-fed hot (Ah) and fasted cold (Fc), having low expression at the end of low tide (hr. 12) and 3 hours (hr. 15) into high tide. However, the Fh group reflected peak expression earlier than the other treatments at 6 hours (hr. 18) into high tide (p < 0.05).

Heat-shock protein 73 gene expression following acute Heat-shock

We measured heat-shock protein 73 (HSPA8) gene expression during the low tide acute heat-shock (hr. 12) and at the beginning of the subsequent high tide (hr. 15) (p < 0.05; Fig. 4). Tukey’s test revealed that the fasted hot group (Fh) had higher HSPA8 expression at low tide (hr. 12) than at high tide (hr. 15). Indeed, compared to other groups, the Fh group showed the highest gene expression during the heat-shock and the lowest expression during submergence (NS). Mussels previously acclimated to elevated heat generally showed early peak expression of heat-shock protein 73, compared to the cold acclimated-regime mussels, which reached peak HSPA8 expression during submergence. On average, the gene expression in the hot acclimated-regime groups during low tide was ≈ 2 times higher than that of the cold acclimated-regime groups. High tide brought the reverse: the average gene expression of cold acclimated-regime mussels had gene expression ≈ 2 times higher than the average gene expression of hot acclimated-regime mussels during high tide.

Temperature breakpoint assay

Thermal kinetics data revealed similar changes in response to increasing heat from 15 °C to 40 °C among all four individuals we measured. Enzyme activity generally increased with rising temperatures (Fig. S-3). One of the two (Ac) mussels measured had nearly doubled amylase activity from 35 °C to 40 °C, which then decreased to a similar level as the other samples at 45 °C. The other three samples had an average Q_10_ of 1.45 across the complete temperature range, closely matching the average Q_10_ of 1.42 for 51 samples measured at both 15 °C and 23 °C for other parts of this study.

Digestive gland mass

Digestive gland mass responded differently to heat-shock and recovery depending on whether the mussels had been acclimated to an algae diet or had been fasted (p < 0.001). At the end of high tide after the heat-shock (hr. 24), the algae-fed groups had lower digestive gland masses than the fasted groups. In the algae-fed cold acclimated group, digestive gland mass increased at hr. 15 and reached its lowest at the end of the high tide (hr. 24). Both heat-acclimated mussel groups (Ah and Fh) showed a sharp decrease in digestive gland mass during the low-tide heat-shock, however the Fh group rebounded by the end of high tide. All the while, the fasted cold group (Fc) showed a stable and comparatively high digestive gland mass over time.

Fig. 2. Digestive enzyme activity (A) and relative gene expression (B) in digestive glands of M. californianus, (N = 4–7). Mussels were acclimated for 12 days to four regimes: Algae-fed cold (Ac), Fasted cold (Fc), Algae-fed hot (Ah), Fasted hot (Fh). All mussels were subsequently subjected to a two-day common extreme (E) heat-shock treatment. The tidal sequence was 12 h:12 h; low: high-tide. High- and low-tide temperatures in the cold regime were isothermic (15 °C). Peak aerial temperature in hot regime was 23 °C. The common treatment aerial temperate peaked at 32 °C. Box plots are blue and red signifying the cold and hot acclimation regimes prior to all sampling. Horizontal lines are the median. Boxes with asterisks represent subgroups significantly different than each regime’s Low-tide group (serving as a control). The details of the similarities and contrasts are described in the results section. Fig. 3. Amylase mean activity and relative gene expression in digestive glands of M. californianus (N=4-7) after an extreme heat-shock of 32 °C. Low tide is represented as hr. 12. Submerged samples were taken at hr. 15, 18, and 24. Asterisks suggest that at least one group is significantly different at the corresponding time-point. Error bars represent 1±S.E. from the mean. Fig. 4. Heat-shock protein 73 (HSPA8) relative gene expression during the low tide extreme heat-shock (hr. 12) and three hours into the subsequent high tide (hr. 15) in digestive glands of M. californianus (N = 5). Mussels were previously acclimated to fed (A) or unfed (F) conditions in combination of either cold (c; 15 °C) or warm (h; 23 °C) air during low-tide. Error bars represent 1 ± S.E. from the mean.

Discussion

Mussels face unprecedented environmental challenges due to intense present and future heat waves in the eastern Pacific Ocean (Masanja et al. 2023). The current study investigated the function of the digestive gland in mussels under moderate and extreme environmental conditions. We acclimated the Pacific intertidal mussel M. californianus to laboratory tidal simulations that exposed half the collected mussels to moderate aerial convective heat-stress environments while the other half experienced optimal temperatures. When submerged, half of each temperature group fasted and the other half were immersed in suspended phytoplankton. From these four regimes we measured amylase activity, ex-vivo, at cool (15 °C) assay incubation temperatures (i.e., no enhancement of activity by assay thermal kinetics). We predicted that algae-fed mussels would show decreased amylase activity across tides when acclimated to moderate daily aerial heat stress– +8 °C to a peak temperature of 23 °C compared to optimal thermal conditions (15 °C)– to conserve energy during the ATP demanding heat-shock recovery processes. We also hypothesized that starvation would trigger relatively higher levels of amylase activity to compensate for lack of energy intake—exhibiting energy-balancing.

Differences between algae-fed hot and cold entrained mussels: Remarkably, the data collected did not support the prediction of energy conservation in well-fed mussels regularly exposed to heat stress. Although amylase activities during low tide were not different between fed heat-exposed mussels (Ah) vs. fed cold-acclimated (Ac) mussels due to the high variance in the latter group, the mean of the fed heat exposed group appeared nearly a third higher, suggesting an energy-balancing strategy. The low-tide heat exposed mussels also showed higher activity than its complimentary high-tide group. In comparison, the cold acclimated mussels did not show differences between low and high tide. Induced transcription/translation of amylase genes to mitigate the costs of an activated HSR could explain the elevated activity as seen in oysters in submerged conditions (Moal et al. 1989), although we did not see such patterns concurrently within individuals in the current tidal-related study. It is very likely that there is a time lag between an increase in gene transcription and a corresponding increase in synthesis of the protein it encodes when mussels are experiencing tidal fluctuations.

Well-fed mussels likely relied on their energy reserves from robust feeding to fuel metabolism during heat stress or recovery. In this context previous studies in Mytilus found that under moderate aerial heat stress, the expression of heat-shock genes and proteins correlates with elevated ATP hydrolysis (Chan et al. 2023; Connor and Gracey 2020; Péden et al. 2016; Tomanek and Zuzow 2010). Elevated heat can have a negative impact on glycogen stores in mussels (Lesser 2016) further supporting the inference of high ATP usage following aerial heat stress. Additionally, previous observations of robust gene and protein expression of mitogen activated protein kinase (MAPK) signaling proteins during heat stress have clearly defined the environmental sensitivity of the HSR in mussels and other marine invertebrates (Du et al. 2024; Gourgou et al. 2010; Wang et al. 2025). Lastly, amylase enzyme thermal kinetics (tested via assay temperatures matched with respective acclimation temperatures) revealed potentially greater compensation for the energetic trade-off between growth and repair. The results indicated that mean amylase activities were higher in warmed assays versus the same sample assayed in cold conditions.

Seiderer and Newell (1979) observed a positive relationship between amylase activity and assay temperature in genetically distant mussel species, Chromytilus meridionalis, in agreement with our observations of the temperature dependence of amylase activity. Remarkably, they found that activity was consistently higher in well-fed, heat-acclimated mussels (in water) compared to cooler-water entrained individuals, regardless of the assay incubation temperature. The authors postulated that thermal enhancement in heat-acclimated C. meridionalis is an adaptation to offset metabolic losses (i.e., energy) related to high summer temperatures in nature. Additionally, Liu et al. (2023) found that amylase activity increased concomitantly with rising water temperatures in Pearl Oysters (Pinctada fucata martensii), suggesting that intertidal bivalves share this conserved phenotype. More importantly, previous research found that bivalves close their valves during warm-water stress, resulting in a decline in their filtering and feeding rates (Anestis et al. 2007). Therefore, amylase activity in warm-water acclimated mussels may be an interaction of variable amylase synthesis and feeding behavior together. Notably, thermal biology studies performed in air, such as the current study, isolate temperature effects from feeding effects on digestive enzyme activity in sessile intertidal mussels. However, a large volume of studies of amylase in aerially exposed mussels are required to confirm that moderate heat stress can stimulate their digestive function, provided that they are optimally fed.

Differences between fasted hot and cold entrained mussels: We predicted that fasted mussels would engage in energy-balancing to mitigate severely impacted energy budgets following starvation. Amylase activity in fasted cold-acclimated regime mussels during low tide trended marginally higher (25%) than activity during low tide in fed cold-acclimated mussels. This discovery is inconsistent with the adaptive modulation hypothesis, which states that there should be a positive correlation between food consumption and digestive processes (Karasov and Douglas 2013). A field study of M. californianus similarly revealed higher-than-predicted cellulase activity, supporting the conceptual model of energy compensation (Connor et al. 2016). Mussels high on the shore have fewer opportunities to feed and encounter more significant periods of heat stress than mussels low on the shore. However, the high-shore mussels showed higher cellulase activity than their low-shore counterparts. Importantly, their study revealed that amylase activity did not vary between high- and low-shore mussels. Although mussels high on the shore were nutrient restricted, they were not starving or chronically heat-stressed at the time of sampling like the mussels of our single stressor acclimation regimes. Additionally, the samples were collected in July which encompassed mussels that had recently experienced early morning low tides and early afternoon high tides, relieving them of extensive midday heat stress. Alternative to Connor et al’s 2016 observations of mussels in nature, Moal (1989) found that amylase activity was higher in oysters transplanted to the high shore versus those placed low on the shore. Simultaneously, the adenylate energy charge (an index of a cell’s energy status) was similar between the differentially elevated acclimatized groups, although energy intake was lower in high shore groups. This observation is consistent with the concept that oysters high on the shore overproduced amylase to compensate, at least partially, for energy deficits occurring during interruptions in feeding due to aerial exposure. Additionally, previous studies revealed increased assimilation efficiency under conditions of low food supply, a functional response in agreement with energy-balancing during environmental stress (Thompson and Bayne 1972).

Although fasted cold mussels in this study showed apparent energy-balancing via elevated amylase activity, this pattern was not present in fasted hot mussels. The fasted hot mussels showed signs of decreasing amylase activity at low tide compared to activity patterns across all other acclimation-regime groups. Therefore, the combined heat stress and fasting (top-down and bottom-up stress, respectively) may have severely impacted this group’s energy budgets—possibly to the point of adenylate energy molecule costs for synthesizing amylase outweighed the energy gained from increased starch digestion and assimilation. Mussels also expend energy on feeding, reflected as an elevated metabolic rate (i.e., specific dynamic action) (Secor 2009). The total energy spent to assimilate nutrients could have contributed to the observed conservation strategy (Bayne and Scullard 1977; Widdows and Hawkins 1989).

The findings from the current study are partially consistent with the results reported by Pham et al. (2023) and Chan et al. (2023). Both of these studies found reduced amylase activity in mussels acclimated to aerial heat stress. For example, Pham et al. (2023) also acclimated M. californianus to a 12 h: 12 h emersion: immersion cycle, with a + 9 °C; 26 °C daily heat event, observing lower enzyme activities across low and high tides in warm-acclimated mussels versus those subjected to cold conditions. The tides and techniques were similar to the current study, except for the heat stress profile, acclimation time, and food level/quality. The mussels in Pham et al’s study were likely malnourished to some degree (due to lower-quality food then the present study) while enduring chronic stress, resulting in the activation of a conservation strategy. Similarly, Chan et al. (2023) showed depressions in amylase activities during low tide in heat-acclimated mussels (8:16 h emersion: immersion; +10 °C; 25 °C) versus mussels acclimated to cold low tide conditions. Interestingly, while Pham et al.’s heated mussels had relatively variable amylase activity, both Chan et al. (2023) and the current study found greater variance in cold-acclimated mussels. Differences amongst studies highlight flexible feeding and digestion traits that likely optimize energy acquisition (reviewed in Willows 1992). Lower carbohydrase activity has been revealed in other ectothermic taxa following acclimation to elevated thermal conditions. For example, Frederick and colleagues (2022) discovered lowered amylase activity in one of two abalone species following a moderate heat-shock. Similarly, Du and colleagues (2024) found lower amylase activity in fish acclimated to a sustained increase in temperature. The underlying mechanisms for these observations are complex therefore deserving meticulous investigation.

In summary, the mussels analyzed in the present study maintained robust levels of amylase activity when exposed to daily tidal conditions that included relatively optimal food supply and temperature (15 °C) (algae-fed cold). These conditions may have facilitated a steady-state digestion of starch in line with optimal filtration of dissolved algae within the tank system (Riisgård et al. 2011). Amylase activity was also substantial in mussels acclimated to either algae-fed hot or fasted cold (i.e., either a single top-down or bottom-up stressor). However, compared to other groups at low tide, the mussels acclimated to combined starvation and aerial heat (multi-stressed) had moderately impacted amylase activity at low tide. This finding is consistent with Fitzgerald et al. (2012), which revealed lower growth and condition index in mussels exposed to combined aerial heat and low-food stress compared to these stressors measured in isolation. Relatedly, Schneider et al. (2010) revealed higher mortality in warm-underfed acclimated mussels versus those subjected to optimal environmental treatments (high food, cold, submerged). From a broader context, this is a potential mechanism of the temperature-size rule, which states that there is a negative relationship between temperature and the size of organisms (Hoefnagel and Verberk 2015).

The extreme aerial heat-shock of + 17 °C; 32 °C, occurring after regime acclimation, negatively impacted fasted mussels versus those that were fed. Previous studies examining mussels under extreme heat stress in water also show decreased amylase activity (Khan et al. 2020; Shang et al. 2022). Surprisingly, well-fed cold mussels presented more resistance to extreme heat stress (highest variance among other extreme acclimated groups). This observation suggests a potential amelioration of the trade-off between growth and heat-stress recovery (Thaler et al. 2012). A noted feat for the well-fed cold-acclimated mussels, given that they did not experience prior thermal threats that could have primed their heat-shock recovery systems in advance. In a previous study, Rankins and colleagues (2024) subjected M. californianus mussels naïve to thermal stress to two days of tidally driven intervals of aerial convective heat exposure, finding that fasted aerially heat-shocked (+ 10 °C) mussels had higher amylase activities than algae-fed heat-stressed mussels. These findings suggest that fasting during submergence may prime the digestive gland to sustain subsequent acute multi-stress (heat + hypoxia) in the short term—a type of cross-protection (Hilker et al. 2016). However, our results suggest fasting across tidal cycles may not enhance mussels’ ability to digest starch when exposed to subsequent heat stress. Lastly, mussels acclimated to moderate heat displayed an earlier peak HSP gene expression response to acute extreme heat in comparison to cold acclimated mussels. Earlier responses to stress, following previous stress acclimation, are a common feature of heat hardening (Hilker et al. 2016). Heat hardening has been previously observed in several taxa of bivalves (Dong et al. 2023; Dunphy et al. 2018; Georgoulis et al. 2021).

There is a thermal breakpoint beyond which an enzyme’s activity declines, imposing limitations on functionality. To this end, lowered enzyme activity in bivalves following extreme heat stress greater than 30 °C has been validated in other studies (Khan et al. 2020; Xu et al. 2023). Mussels digest their food extracellularly and intracellularly, the latter of which uses an endolysosomal system that is particularly sensitive to the negative impacts of heat stress (Múgica et al. 2015). The precise combination of air-exposure, heat, and food quality/quantity driving amylase activity and other enteric responses in mussels remains elusive.

Cycles of digestive behavior, modulated by exogenous and endogenous forces, are depicted in both terrestrial and marine animals. Light/dark cues are known to entrain tissues and organisms across a wide range of taxa and conditions (Hastings 1997; Reppert and Weaver 2002), including bivalves under subtidal conditions (Mat et al. 2014). If photoperiod cues have strong ties to digestion, then amylase activity in the combined starvation and heat exposed acclimation group could be sustained rather than decreased, in agreement with the other regimes. In this context, we found that low tide generally induced higher amylase activity than high tide, suggesting a rhythm. These findings were similar to those of Pham et al. (2023), which subjected mussels to a similar tide regime. Alternatively, in the environmentally compressed (fasted hot) mussels, the pattern was the opposite, with relatively low amylase activity at low tide and high activity at high tide. Mussels feed at high tide hence, amylase produced during low tide may be anticipatory in agreement with a study by Moal et al. (2000) which showed that amylase activity in oysters was highest before the start of feeding periods. Theoretically, exposure to air or the associated functional hypoxia could be sensed by mussels initiating the anticipatory response, whilst the level of stored energy could modulate the intensity of amylase activity. Functional hypoxia and energy availability likely shape amylase regulation and may underlie the observed energy-balancing strategy during low tide. Acclimation to heat and starvation simultaneously could have eliminated the anticipatory strategy because mussels enter a survival mode. Conversely, Langton (1977) revealed that amylase activity in the digestive gland of M. edulis correlated with the tide. Since temperature conditions were moderate during sampling, Langton (1977) isolated the effects of twice-daily spring tides and reported that peak amylase activity occurred near the end of high tide and descended until the end of low tide. Effectively providing evidence of a lag between the tide-height transition and peak activity. The lag may have originated from gene expression regulatory networks, translation processes, or proteolytic cleavage of stored amylase zymogen. Along this line, Huvet et al. (2003) also validated oscillations in digestive enzyme activity that correlated with feeding time in oyster. Finally, endogenous circadian controls may drive digestion in bivalves (Zaldibar et al. 2004).

Our time series data revealed variations in enzyme activity in submerged mussels. When submerged, mussels exhibit individual variation in valve closure from minutes to hours (Gracey and Connor 2016). Subtidal valve movements not only control feeding but also correlate with metabolic state (Gracey and Connor 2016). Similar to valve closure in air, subtidal valve closure induces metabolic depression, providing an external food sensor-driven mechanism to align the level of food presentation with fuel usage. Collectively, previous studies suggest that valve opening, filtration, and ingestion positively correlate with digestive enzyme activity (Albentosa et al. 2012; Connor et al. 2016; Riisgård et al. 2011), highlighting a potential environmental-physiological logic for the hourly changes in amylase activity we observed during submergence. Our gene expression data partially explained the variability in corresponding amylase enzyme activity. For the fasted hot group, peak amylase gene expression occurred midway through high tide, in step with peak enzyme activity occurring at the end of high tide. Submergence slightly muted the enzyme activity of the other groups, consistent with corresponding gene expression not rising until the end of low tide. This temporal pattern might explain the high enzyme activity occurring at low tide in optimally conditioned (algae-fed, cold-acclimated) and single-stressed (heat-stressed or fasted) groups.

Temperature-modulated mechanisms inducing variable amylase activity may be explained by changes in isoform usage, conformational alterations, enzyme denaturation, and differential gene expression. In line with discovery of complex interactions, our PCA analysis integrated the effects of gene expression, amylase activity, and digestive gland weight on data variance. It revealed that gene expression and mass contributed more to the overall variance of the hot regime acclimation, along with extreme heat-shock data. Alternatively, activity appeared to drive variance of the data in cold acclimated mussels and their extreme heat-shock performance rather than gene expression or digestive gland weight. Variance may also be contributed to differential enzyme kinetics between mussel groups. In this context, the ex vivo thermal kinetics assay assessing variable incubation temperatures revealed that enzyme activity increased across the experimental range (15–45 °C) for all tested groups. Notably, the only individual that displayed a thermal breakpoint in the temperature range we tested had already experienced the extreme heat-shock. Even so, that breakpoint was above the extreme heat-shock temperature. Whether repeated extreme heat-shocks could shift the breakpoint of amylase has yet to be assessed. Still, amylase in M. californianus appears to resist denaturing under moderate to extreme thermal stress, which may be a result of selective forces. Alternatively, Brock (1986) reported amylase from M. edulis with ex vivo melting temperatures of 24 to 28 °C, which is lower than what we report for M. californianus. However, the selection pressures acting on these two Mytilus species may be quite different; the historic geographic latitudinal range of M. edulis in North America spans the Arctic to North Carolina in the Atlantic (Smith and Dall 1889), whereas M. californianus resides at lower (warmer) latitudes in the Pacific.

Summary

Mytilus californianus is an ecologically essential species within the coastal habitats of the northeast Pacific Ocean. As a sessile organism within the intertidal zone, it must display a comprehensive range of phenotypic flexibility across biological functions, including feeding and digestion. Feeding rhythmicity is a common feature of bivalves driven by exogenous and endogenous cues. In this study, we acclimated mussels to four simulated tidal environments, developing distinct treatment groups. The treatment groups include optimal conditions (algae-fed cold), two suboptimal conditions (fasted cold and algae-fed hot), and one extreme condition (fasted hot). Enzyme activity across all experimental conditions during the acclimation period was higher during low tide compared to high tide, except multi-stress conditions (fasted hot). This suggests anticipation of feeding upon the return of high tide.

Energy-balancing involves variables such as ingestion, feeding costs, and respiration, which previous studies have correlated with intertidal environmental factors like aerial exposure, temperature, and food concentration in seawater. Therefore, it is a logical inference that mussels are neurologically sensing these environmental conditions. Hence, mussels must adjust energy balance-related endogenous control systems after each tidal transition. To this end, Albentosa et al. (2012) found positive relationships among food abundance, scope for growth (energy balance), and amylase activity in submerged mussels. Here, we propose a conceptual model that relates amylase activity with scope of growth with tide level differentiation. Under moderate environmental conditions, we postulate that increased amylase activity at the end of low tide potentially elevates growth if met with starch polymers to consume at the commencement of high tide or if heat-shock repair costs are not elevated (i.e., energy-balancing) (Fig. 5). From this perspective, regarding the effects of temperature and food on enzyme function, marine heatwaves across the globe are predicted to become more common in the future which may overcome compensatory responses (Jacox et al. 2022). Broad conservation implications emerge from elucidating the thermal digestive physiology of a key foundation species in the context of nutrient availability (Menge et al. 2019). It is paramount to investigate the dynamics between thermal digestive physiology and fluctuating nutrient availability.

Fig. 5. Mussels within the intertidal zone display indeterminate growth. Scope for growth (SFG) predicts potential growth based upon the equation Assimilation-(Respiration+Excretion). Albentosa et al. (2012) predicted a positive relationship between SFG and amylase enzyme activity in submerged mussels. Mussels acclimated or acclimatized to chronic but not extreme heat or starving, negatively effects growth potential in mussels. We predict that mussels mitigate the costs of these single stressors by increasing digestive performance during low tide—increasingly displaying energy-balancing power with decreasing SFG. The difference between low and high tide becomes less extreme toward optimal thermal conditions and food presentation.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1