Introduction

Bivalves are critical to estuarine function in terms of stimulation of microphytobenthic productivity through their release of ammonium and carbon dioxide as well as pelletization of fine particulate matter in addition to their importance as a critical link in energy conversion between primary producers and a variety of predators such as fish, birds, and crabs as well as humans1. One of the predicted consequences of global climate change is an increase in the occurrence of extreme climatological events such as droughts or extreme rainfalls that modify coastal salinity2. Estuaries, as transitional zones, will experience greatly those salinity fluctuations3. For bivalves, salinity is particularly problematic because it drives a reduction in activity and energy acquisition while also increases energy demand for maintaining cell volume and avoiding osmotic shock4,5,6,7. The maintenance of cell volume is achieved mainly by regulation of the nitrogen metabolism, which also increases oxydative metabolism demands and therefore mobilization of reserves resulting in elevated oxygen consumption rates4,5,6,7. Given the pairing of this higher metabolic demand with reductions of energy input from food because of reduced feeding activity4,5,6,7, the net effect of energy trade-offs at the individual level can be severe and translate to mass mortality events8,9 or indirectly affect population dynamics by altering growth performance, reproductive output or immune function4,5,6,7,10.

The cockle Cerastoderma edule is one of the most abundant bivalve species in tidal flats where it can comprise 60% of benthos biomass11 being critical to ecosystem function in addition to supporting several traditional fisheries12,13,14,15. This short-lived species is known to have high spatial and temporal variability10 but during the last decades cockle stocks have shown a progressive declining trend12,13,16,17,18,19. Declines of stocks are mostly due to mass mortality episodes and recruitment failures; both provoked by climate-related events8,9,12,16,17,19,20,21,22,23,24. The literature suggests that mass mortality events in cockles are often linked to torrential rains8,9,17,19,21,22 with the magnitude of a mortality event depending on the intensity and duration of the flooding episodes maintaining salinity levels below the lethal physiological threshold of the species (10–12.5)4,5,7,8,25,26. In fact, an increase in the frequency of flooding events in estuaries has been related to decrements in biomass of C. edule and to reductions in the fishery yields8,19,21,22. Timing of torrential rains can also be critical. The decrease of recruitment observed in cockles associated with delays on settlement through spring and summer has been related mostly to impairments with food sources (phytoplankton blooms) and seasonal peaks of predator abundance10,16,20,27, but sudden drops of salinity during the settlement season could also cause recruitment failure driven perhaps by differing physiological thresholds of early developmental stages. Ontogenetic changes of environmental requirements are common in species, especially for those which occupy distinct habitats during developmental stages28,29,30,31,32,33,34,35,36,37. A critical aspect of the life history of C. edule is that initial settlement occurs in the high intertidal apparently to minimize competition with adults and reduce predation risk38,39,40. After a period of growth the juveniles undergo a post-settlement migration via byssal thread drifting into the lower intertidal where environmental conditions are more stable41,42. C. edule lose their drifting and climbing abilities when they reach sizes ≈6 mm,which has been associated with the degeneration of the byssal glands43. Morphological and habitat differences of thread drifters support their consideration as a distinct developmental stage, which may have specific physiological requirements. Stage-specific requirements may contribute to episodic recruitment failures, as well as the spatial distribution of the species in different estuaries13,44. In order to reverse the decline of the stocks, management strategies (delimiting nursery areas, seeding and fishing grounds, etc) need to identify the spatio-temporal availability of suitable habitats for each developmental stage, which will be highly determined by their particular environmental constrictions13,45.

This study aims to identify stage-specific salinity requirements related to the loss of drifting capability after settlement and how that affects their physiological response to stress episodes of different duration. With that purpose, we designed a laboratory experiment where thread drifters and sedentary settlers were exposed to a wide range of salinities with reference to the following question while measuring the components of scope for growth (respiration, excretion and clearance rate):

Do the thread drifters and sedentary settlers differ in their response to acute (2 days of exposure) and acclimated (7 days of exposure) response to saline stress? One might expect thread drifters to be less susceptible to acute stress because they need to cope with a highly variable environment at the high-intertidal. On the other hand, one might expect the sedentary settlers to have a better acclimated response because they have lost their migratory capability so they need to develop better acclimated responses.

Material and Methods

Experimental setup

Laboratory spawns of adult cockles yielded larvae, which were raised until metamorphosis. Spat were maintained at salinity 35 ± 0.5 for 2–3 months and then divided into two size classes according to their drifting capability (<6 mm)43: thread drifters (D; L: 3.74 ± 0.67 mm; n = 224) and sedentary settlers (S; L: 9.66 ± 1.31 mm; n = 84) before transferring to the salinity treatments to simulate a sudden drop in salinity caused by a torrential rain.

Two replicated beakers (1 L) per Salinity treatment (8 levels: 3, 5, 10, 15, 20, 25, 30 and 35), Size (2 levels: D and S) and time of exposure (2 and 7 days) were maintained in an orthogonal design in a controlled temperature room at 14 °C. No sand was included in the beakers since preliminary studies reported no differences in survival or pumping rates with/without sediment in laboratory experiments25,46. Density per beaker was adjusted to those determined in preliminary experiments to give replicable results for rate measurements (250 and 45 indiv/beaker for D and S, respectively). Water was changed daily before feeding (1% of dry weight of microalgae per cockle live weight) with a mixture of Isochrysis galbana (TISO), Tetraselmis suecica, Chaetoceros gracilis and Rodomonas lens (1:1:1:1).

Physiological performance metrics (respiration, ammonium excretion and clearance rates) were measured on day 2 and 7 of the experiment, considering day 2 as acute stress response and day 7 as acclimated stress response.

Physiological measurements

Each replicate was divided into two pseudo-replicates to measure physiological performance of the individuals during acute and acclimation response to salinity treatments.

Clearance Rate

Clearance rate (CR; Lh−1) was calculated on pools of cockles (40 and 15 ind. for D and S Size treatments, respectively) in a static system. Two plastic beakers of 100 ml with filtered seawater (50 µm) were used per replicate. A pool of individuals was placed in each beaker and after 30 minutes of acclimation, ≈400000 cells ml−1 of I. galbana were added while aeration was kept high to maintain suspension of the microalgae. During basal measurements at salinity 35 a sample of 10 ml was taken from the centre of the beaker with a pipette, every 5 minutes during 30 minutes to establish the period of time with maximum CRs for each size class (15 and 10 minutes for D and S, respectively). These periods of time were used to take CR samples throughout the rest of the experiment. Two beakers without cockles, but with the same microalgal density were also sampled at the beginning and at the end of each run of measurements to subtract the difference on particles caused by sedimentation of microalgae to the concentration obtained at the experimental beakers. Samples were fixed with lugol to avoid degradation until processing with a counter coulter (Beckman Coulter Multisizer 3) to calculate particle concentration.

CR was calculated with the following equation47:

$${\rm{CR}}={\rm{v}}(\mathrm{ln}\,{{\rm{c}}}_{0}-\,\mathrm{ln}\,{{\rm{c}}}_{1})/{\rm{t}}$$

where v is the volume of seawater in the beaker and c0 and c1 are the cell concentrations at the beginning and at the end of the time interval (t). CR was also corrected by the number of individuals in the pool.

Respiration Rate

Oxygen consumption rate (R; mgO2 h−1) was calculated on pools of cockles (75 and 15 individuals for D and S, respectively) using closed respirometers attached to dissolved oxygen probes (Hach Lange LDO101). Two cylindrical respirometers of 150 ml filled with aerated 50 µm-filtered seawater at 14 °C were used per replicate. A pool of individuals was placed in the respirometer while seawater was carefully moved with a magnetic stirrer. Dissolved oxygen concentration was recorded every 30 seconds until it declined ≈20% from the initial value. R was calculated using the slope of the relationship between oxygen concentration and time elapsed, and corrected by the chamber volume and the number of individuals in the pool. Two empty respirometers were monitored simultaneously with each run to subtract the difference on oxygen concentration caused by electrode drift, bacterial respiration, etc. from the values obtained at the experimental respirometers.

Ammonium Excretion

Ammonium excretion rate (ER; mg NH4-N h−1) was calculated on pools of cockles (40 and 5 individuals for D and S, respectively) after CR measurements. One chamber of 20 ml filled with 50 µm filtered seawater was used per replicate. A pool of individuals was placed in the chamber and, after 1.5–2.5 hours, a sample of 10 ml was collected and ammonium concentration calculated by the phenolhypochlorite method48. Two empty chambers were used per run as controls to subtract other ammonium sources from the values obtained at the experimental chambers and correct ERs which were also divided by the number of individuals in the pool.

O:N index

O:N index was calculated as the ratio between oxygen atoms consumed per atom of nitrogen excreted, using their atomic equivalents over the R and ER values previously calculated. O:N represents the balance between carbohydrates and lipids versus protein catabolism. Balanced catabolism would oscillate between 50 and 6049 while values below 30–20 are indicative of stress6,26,49 and pure protein catabolism will reach values between 3 and 1349.

Standardization of physiological rates

All the physiological rates were standardised to 0.1 g DWtissue (DWs = 0.1) using the formula:

$${{\rm{Y}}}_{{\rm{s}}}={{\rm{Y}}}_{{\rm{ob}}}\,\ast \,{({{\rm{DW}}}_{{\rm{s}}}/{{\rm{DW}}}_{{\rm{ob}}})}^{{\rm{b}}}$$

where Ys is the standardized rate for a selected weight (DWs), Yob is the observed physiological rate for an animal of a particular weight (DWob), and b is the weight exponent for the physiological rate to weight allometry. We employed b = 0.49 for CR and b = 0.77 for R and ER50.

Pools of organisms used for the physiological measurements for the sedentary size class were dissected to calculate the tissue dry weight (DWtissue). After dissecting the tissue from the shell, both were dried separately at 60 °C for 48 h and then weighed. Because of the small size of the thread drifters, dissections of animals from that age class were made under the microscope only on a subsample of 10 individuals covering a wide range of sizes from each replicate. Tissue and shell were dried separately and weighed to calculate allometric relationships between DWtotal and DWtissue per replicate using regression models on log-transformed weights. The rest of the pool was dried without dissecting to calculate DWtotal, and regressions calculated per each replicate were employed to extrapolate DWtissue for each pool.

Data Analysis

Generalized additive models (GAMs), as implemented in the mgcv library of R 3.3.2, were used to investigate the effect of Size and Salinity on the standardized physiological rates (R, CR and ER) and O:N index on day 2 (Acute stress) and day 7 (Acclimation). This analysis type is useful when the form of the response can be complex and it is difficult to pre-judge the various parametric options51. The significance of the interaction between Size and Salinity was evaluated by a likelihood-ratio test comparing Akaike Information Criterion of the model with and without interaction. Size was included as a factor (D and S) and Salinity was included as a smoothed term in the model, using thin plate regression splines and estimating for each size level when the interaction was significant. Model validation included the verification of homogeneity (lack of structure of the residuals) and normality (quantile−quantile plot of the residuals)52.

All the analysis were performed on R.3.3.2.53.

Data availability

Raw data has been made available through PANGAEA data repository (https://doi.pangaea.de/10.1594/PANGAEA.889440). The authors also compromise to make materials, other data and associated protocols available to the Editorial Board Members of Scientific Reports, referees contacted by the journal or readers without undue qualifications in material transfer agreements.

Results

Acute Response

After 2 days of exposure, both size levels (thread drifters and sedentary settlers) showed a marked reduction in activity at salinities below 15, with continuous valve closure and almost complete inhibition of R and ER (Fig. 1). At salinities >15 physiological rates progressively increased, although thread drifters and sedentary settlers showed some differences (Fig. 1).

For thread drifters CR was resumed at salinity 15 and it increased almost linearly with salinity until it reached a plateau at salinity 25 (Fig. 1A). Sedentary settlers reached higher CR values than drifters once pumping was resumed (Table 1; Fig. 1A,B), but the relationship between CR and salinity showed an on/off response with a feeding activation threshold at salinity 20 (Fig. 1B). Both size and salinity were significant but so was the interaction term, probably due to the differences in the shapes of the response curves.

With regard to R, the interaction term was not significant but the size and salinity terms were (Table 1; Fig. 1C,D), indicating higher metabolic rates for thread drifters. Oxygen consumption progressively increased with salinity attaining maximum R for both size classes at salinities 20–25 to slightly decrease again at higher salinity treatments (Table 1; Fig. 1C,D).

ER relationship with salinity also differed between size classes (Table 1: significant interaction term as well as size and salinity terms) peaking at different salinities for thread drifters and sedentary settlers with maximum ammonium excretion at salinity 15 and 25, respectively (Fig. 1E,F).

Thread drifters showed higher O:N values than sedentary settlers according with the higher R and lower ER observed (Table 1). For both size classes, a linear relationship was observed between O:N and salinity, reaching maximum values at the highest salinities (30–35; Fig. 1G,H).

Acclimated Response

Four days after the beginning of the experiment, all the treatments at salinities below 15 suffered 100% mortality in both size levels (thread drifters and sedentary settlers). Therefore, acclimated response (7th day of the experiment) was only measured for salinities >15.

During the acclimated response, thread drifters and sedentary settlers showed again different physiological responses with regard to salinity. For thread drifters no significant relationships between salinity and any of the physiological rates (CR, R and ER) were detected (Table 1; Fig. 2A,C,E), while the physiological responses of sedentary settlers were significant for CR, R and ER (Table 1; Fig. 2B,D,F). This contrasting response resulted in significant interaction terms for Salinity and Size for CR, R, and ER (Table 1).

CR of sedentary settlers was almost completely suppressed at salinity 15, while maximum values were recorded at intermediate salinities (20–25; Fig. 2B), reinforcing the presence of a feeding activation threshold at salinity 20. R and ER showed an inverse relationship with salinity (Fig. 2D,F), although oxygen consumption reached a plateau at salinity 25 while excretion continued decreasing following a linear relationship with salinity (e.d.f = 1; Table 1).

Thread drifters continued to maintain higher O:N values than sedentary settlers, according to the higher R and lower ER recorded (Table 1). Nonetheless, both size classes showed a similar pattern between O:N and salinity with maximum values again at the higher salinity treatments (Table 1; Fig. 2G,H).