Reproduction reduces HSP70 expression capacity in Argopecten purpuratus scallops subject to hypoxia and heat stress

In scallops, gonad production is highly demanding energetically, and reproduction usually occurs during spring−summer, a period of strong environmental changes. The synthesis of heat-shock proteins (HSPs) is a major mechanism of stress tolerance in animals, including scallops, and HSP expression contributes considerably to cellular energy demand. Therefore, reproductive investment may limit the availability of energy (in terms of ATP) for the expression of HSP in organisms exposed to environmental stress. We evaluated the stress response capacity of adult Argopecten purpuratus scallops to high temperature and hypoxia. Stress response capacity was assessed through gene expression (for temperature stress) and protein induction of 70 kD HSP at 3 reproductive stages: immature, mature and spawned. We also evaluated the effect of reproductive status on the cellular ATP provisioning capacity through citrate synthase activity. Immature scallops exposed to thermal stress showed 1.3and 1.5-fold increases in hsp70 mRNA and HSP70 protein levels, respectively, and those exposed to hypoxia doubled their level of HSP70 compared to non-stressed immature scallops. However, following gonad maturation and spawning, hsp70 mRNA increased by only 0.49and 0.65-fold, respectively, after thermal stress and HSP70 protein levels of scallops exposed to thermal and hypoxia stressors did not differ from those of nonstressed animals. In parallel, citrate synthase showed its highest level in immature scallops, declined with gonad maturation, and was lowest in spawned scallops. These results suggest that reproductive investment reduces the stress response capacity of A. purpuratus and that mature and spawned scallops could be more vulnerable to environmental stressors than immature individuals.


INTRODUCTION
In most organisms, reproduction requires high levels of energy investment.The amount of energy invested and the stage during which this energy is needed depend, in part, on the organism's reproductive strategy.In broadcast-spawning mollusks such as scallops, the majority of energy investment into gamete production occurs during gonad maturation (Sastry 1968, Barber & Blake 1983, Martínez 1991).In scallops, this process occurs even when food is limited, and as much as 50% of the energy reserves stored in somatic tissues can be mobilized to support reproductive maturation (Martínez & Mettifogo 1998, Lodeiros et al. 2001, Brokordt & Guderley 2004).The mobilization of energy reserves and re-channeling of consumed energy to support gonad maturation limit the amount of energy available or decrease the metabolic capacity to support other vital processes.For example, in mollusks, reproductive investment has been shown to decrease the amount of energy available for growth (Iglesias & Navarro 1991), increase maintenance metabolic demands (Kraffe et al. 2008), decrease aerobic metabolic capacity (Brokordt et al. 2000a,b) and decrease swimming ability and escape response capacity (Brokordt et al. 2000a,b, 2003, 2006, Kraffe et al. 2008).Additionally, in several bivalve species (e.g.mussels, scallops and oysters), periods of reproductive activity and mass mortality have been observed to coincide (Tremblay et al. 1998, Xiao et al. 2005, Samain et al. 2007), possibly due to the presence of several stressful environmental factors.In several bivalve species, reproductive maturation occurs during the spring−summer season, characterized by strong environmental fluctuations in conditions such as temperature and oxygen level (Cheney et al. 2000, Tomaru et al. 2001, Cabello et al. 2002, Xiao et al. 2005, Li et al. 2007, Zhang et al. 2010).
When environmental conditions exceed an organism's ability to adapt physiologically, this produces physiological stress.Organisms have evolved several physiological responses to tolerate environmental stress.Among cellular stress responses, one of the most important is the production of stress proteins, commonly known as heat-shock proteins (HSPs) (Morris et al. 2013).HSPs are present in all organisms; they are among the most abundant soluble proteins in the body and are induced by most stressors (Calderwood 2007).HSPs are molecular chaperones that decrease the aggregation of unfolded proteins, assist in protein refolding, and facilitate the channeling of irreversibly denatured proteins towards pro teo lytic degradation (Parsell & Lindquist 1993).Among the HSPs, the HSP70 family (so denoted due to its 70 kD mass) is the most abundant, and, in many organisms, is considered the most important (Feder & Hofmann 1999, Sørensen 2010).HSP70 activity augments tolerance to several stressful conditions, in clud ing extreme temperature, hypoxia, UV radiation, and the presence of toxins; it also participates in the immune response (reviewed by Feder & Hofmann 1999, Calderwood 2007).Under 'non-stress' conditions, HSPs also play an important role in protein biogenesis by preventing the premature folding and aggregation of emerging polypeptides (Frydman et al. 1994, Hartl & Hayer-Hartl 2002).Therefore, HSPs not only increase the organism's capacity to tolerate stress conditions but also enhance the efficiency of protein synthesis.However, gene expression and protein synthesis processes, as well as the chaperoning activity of these proteins, generate high cellular demands for energy in terms of ATP (Hofmann & Somero 1995, Somero 2002, Sharma et al. 2010).Therefore, we propose that upon exposure to a stress factor, the observed level of induced HSP70 may reflect not only the level of molecular damage, but also the capacity to express this protein, which could be energetically limited or affected by the physiological status of the animals.There is some evidence that supports this idea; for example, a study made in blood cells from rainbow trout showed that the in vitro inhibition of energetic metabolic pathways decreased ATP by 79%, and this reduced hsp70 mRNA levels after heat shock (Currie et al. 1999).Also, Mizrahi et al. (2011) observed a negative correlation between endogenous levels of the HSP70 isoform of the foot tissue and albumen gland mass (larger albumen glands indicative of more mature animals) of land snails (Shincterochila cariosa and S. zonata).Finally, we have recently shown that the marine snail Concholepas concholepas under reduced nutritional status showed lower levels of HSP70 induction upon exposure to stress factors during low tide compared with snails in good nutritional status (Jeno & Brokordt 2014).
As both reproduction and the synthesis of stress proteins are energetically expensive, there could be an energetic compromise between these 2 processes.We hypothesized that reproductive investment may limit the availability of energy (in terms of ATP) for HSP70 expression in organisms exposed to environmental stress.To test this hypothesis, we compared the hsp70 mRNA levels and HSP70 abundance among Argopecten purpuratus scallops at different reproductive stages (immature, mature and spawned) exposed to 2 different stress factors (hypo xia and high temperature).Hsp70 mRNA levels were measured only upon exposure to thermal stress.In parallel, we evaluated the effect of reproductive status on the aerobic metabolic capacity of these scallops, measured through citrate synthase (CS) enzymatic activity.Through the activity of this key en zyme we aimed to assess the effect of reproduction upon one of the main ATP provisioning metabolic path ways.We used A. purpuratus as a model because as a broadcast spawner this species has previously been shown to have high reproductive investment (Martínez 1991, Martínez & Mettifogo 1998, Martínez et al. 2000).Also, most natural beds and cultures of A. purpuratus are located in bays near upwelling zones and are thus exposed to frequent environmental changes, especially during the reproductive period (Zhang et al. 2010, CEAZA Oceanographic Monitoring System unpubl. data).

Animal procurement and holding conditions
Adult Argopecten purpuratus (70−80 mm shell height) with immature (n = 60) and mature (n = 120) gonads were obtained from aquaculture centers lo cated in Coquimbo, northern Chile (30°16' S, 71°35' W), during summer 2011.Summer is the most active reproductive season for A. purpuratus at Coquimbo, and because reproduction in this species is somewhat asynchronous (Cantillanez et al. 2005), it was possible to find animals at different stages of maturation.Reproductive stage was initially determined using a visual scale following Disalvo et al. (1984) and Martínez & Pérez (2003), where immature individuals show flaccid and pale gonads, and mature individuals show intensely coloured, turgid gonads.In the case of spawned scallops, individuals were chosen from animals that were induced to spawn and subsequently showed empty gonads.Visual observations were thereafter verified via gonad mass measurements, as described below.The scallops were transported to the Universidad Católica del Norte's laboratory in Coquimbo.In order to reduce stress arising from the transport process, individuals were acclimated to laboratory conditions for 4 d in 1000 l tanks supplied with filtered, aerated, running seawater (~18°C) and fed a diet of 50% Isochrysis galbana and 50% Chaetoceros calcitrans.Following acclimation, 60 mature scallops were stimulated to spawn by adding excess microalgae.

Experimental design
We first performed the thermal stress trial.Fifteen scallops from each reproductive stage (immature, mature and spawned) were subjected to a rapid temperature increase from 18 to 24°C and then maintained at 24°C for 6 h.This increase of temperature was previously tested in a preliminary experiment to ensure a significant stress response in the scallops.An additional 15 scallops from each reproductive stage were maintained at baseline temperature (18°C) over the same 6 h and served as controls (i.e.unstressed scallops).
For the hypoxia stress trial, 15 scallops from each reproductive stage were subjected to a rapid decrease in seawater oxygen content from saturation levels (~8.0 mg O 2 l −1 ) to hypoxic conditions (2.0− 1.5 mg O 2 l −1 ) by adding gaseous nitrogen.Scallops were maintained under this hypoxic condition for 6 h.
As controls (i.e.unstressed scallops), an additional 15 scallops from each reproductive stage were maintained under baseline oxygen conditions over the same 6 h.During this trial, scallops were maintained at 18°C.
Hypoxia treatments were based upon the oxygen fluctuations measured in Tongoy Bay during the spring− summer season by the CEAZA Oceanographic Monitoring System.Therefore, both the hypoxia level (2.0−1.5 mg O 2 l −1 ) and the duration of the hypoxia cycles followed real environmental conditions to which the scallops are exposed.
Following each experiment, individuals were measured and their gonads were dissected and weighed.Each individual's gills were then extracted, deep frozen in liquid nitrogen and stored at −80°C for later HSP70 quantification and CS enzymatic activity determination.Gill tissues from each scallop exposed to thermal stress trials were stored in RNAlater (Ambion) at −20°C until processing for hsp70 gene transcription determination.
In preliminary assays, we evaluated HSP70 levels after stress exposure in different tissues: muscle, mantle and gills.We did not measure HSP70 in gonads or digestive glands because these are very unstable tissues, i.e. they experience large changes in short time periods associated with reproductive status or food availability, respectively.The preliminary assays showed that after stress, HSP70 levels increased 1.5 times in muscle tissue, 2.0 times in mantle tissue and 3 times in gill tissue, compared with their respective control tissue from unstressed scallops.Based on these results, we performed the complete study in the gill tissue, which represents a large proportion of the scallops' soft tissues.Because we were looking for the association be tween HSP70 induction capacity and energetic metabolism capacity, CS was also measured in gill tissue.

Total RNA extraction, cDNA synthesis and mRNA transcription analysis with quantitative real-time PCR
Total RNA was isolated from the gill tissues using Trizol reagent (Invitrogen) according to the manufacturer's instructions.RQ1 RNase-Free DNase (Promega) was used to eliminate DNA contamination.Equal amounts of RNA from 3 individuals per treatment were pooled (thus the 15 individuals per treatment became n = 5 per treatment).Each RNA pool was reverse transcribed using the Revertid H Minus First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's instructions.We used 200 ng of total RNA for the real-time PCR analysis.Hsp70 gene transcription was performed using specific A. purpuratus primers (hsp70F 5'GAG GCC GTC GCC TAT GGT GC3'; hsp70R 5'GCG GTC TCG ATA-CCC AGG GAC A3') (GenBank accession number FJ 839890), for which PCR efficiency was previously verified through the standard curve method.The designed primers were selected from a conserved region that does not discriminate between genes encoding for different hsp70 isoforms and thus between constitutive and inducible ones.However, in preliminary studies using these primers, hsp70 mRNA showed a strong increase after stress, which indicates that we are measuring the hsp70 inducible isoforms.
All real-time PCR reactions were performed in triplicate in a 20 µl reaction mixture containing 5 µl cDNA, 0.2 mM of each primer, SYBR Green qPCR master mix 2X (Fermentas) and 50 nM RoX solution.Real-time PCR reactions were run in a StepOne Plus Real-Time PCR System (Applied Biosystems) using the comparative ΔΔC T method (Livak & Schmittgen 2001) and EF1α (GenBank accession number ES469321.1)as an endogen control.In preliminary studies, the stability of this endogenous gene was tested for our species and tissues.Initial denaturing time was 10 min at 95°C, followed by 40 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, with a ramp rate of the melt curve of 95°C (15 s), 55°C (15 s) and 95°C (15 s).

Extraction and quantification of total protein for HSP70 determination
Total protein was quantified for 0.03 g of gill from each individual.Gill tissue was homogenized in 150 µl of homogenization buffer (32 mM Tris-HCl at pH 7.5, 2% SDS, 1 mM EDTA, 1 mM Pefabloc and 1 mM protease inhibitor cocktail).The homogenate was incubated for 5 min at 100°C, then resuspended in 100 µl of homogenization buffer and re-incubated at 100°C for 5 min.The homogenate was centrifuged at 10 600 × g for 20 min.Total protein was quantified in an aliquot of the supernatant with a Micro-BCA kit using a microplate spectrophotometer EPOCH (BioTek).

Quantification of HSP70 protein levels
HSP70 was measured in the gill tissue of each individual by enzyme-linked immunosorbent assay (ELISA), which was validated in previous assays by comparing ELISA results with immunoprobing of western blots from western blot analyses (using the antibodies described below), which showed only one band at the level of 70 kD HSP.Total protein (30 µg ml −1 ) was diluted in 0.05 M carbonatebicarbonate buffer at pH 9.6, and 50 µl of sample per well was incubated in an ELISA plate overnight at 4°C with 3 blanks (containing buffer only).The plate was then washed twice with phosphatebuffered saline (PBS) (200 µl per well).Next, 200 µl of blocking buffer (PBS + 5% skim milk) was added to each well and incubated for 2 h.The wells were washed again with PBS.Subsequently, 100 µl of the primary antibody -polyclonal mono-specific antiepitope that recognize the inducible and constitutive forms of HSP70 specific for Argopecten purpuratus, developed in immunized mice with a synthetic peptide epitope (Group of Im muno logical Markers on Aquatic Organisms, Catho lic University of Valparaiso) -diluted 1:400 in blocking buffer + 0.05% Tween-20 was added to each well, and the plate was incubated overnight at 4°C.The plate was then washed 4 times with PBS, incubated with goat antimouse IgG (Thermo Scientific) secondary antibody, diluted in blocking buffer + 0.05% Tween-20 for 2 h at 25°C, and washed again 4 times with PBS.Next, 100 µl of substrate solution (10 mg o-phenylenediamine dihydrochloride in 25 ml of 0.05 M citrate phosphate buffer) was added, followed by incubation of the plate for 30 min at 25°C.Finally, the plate was read at 450 nm in a microplate spectrophotometer.The absorbance of the sample was corrected by the mean absorbance of the blanks.HSP70 levels were expressed relative to the respective protein level of the unstressed immature group.

CS activity
We homogenized the samples of gill tissue on ice (n = 7 per reproductive status), in 10 volumes of 50 mM imidazole-HCl, 2 mM EDTA-Na 2 , 5 mM EGTA, 1 mM dithiothreitol and 0.1% Triton X-100, pH 6.6.The homogenates were centrifuged at 4°C for 15 min at 600 ×g.We measured enzyme activity at controlled room temperature (20°C) using a UV/Vis spectrophotometer (Cary 50 Bio, Varian).Conditions for enzyme assays were adapted from conditions used by Brokordt et al. (2000a) for Chlamys islandica, as follows (all concentrations in mmol l −1 ): TRIS-HCl 75, oxaloacetate 0.3 (omitted for the control), DTNB 0.1 and acetyl CoA 0.2, pH 8.0.Enzyme activity was examined by following the absorbance changes at 412 nm to detect the transfer of sulphydryl groups from CoASH to DTNB.The extinction coefficient used for DTNB was 13.6 cm −1 µmol −1 .All assays were run in duplicate and the specific activities are expressed in international units (µmol of substrate converted to product per minute) per gram of gill mass.

Statistical analyses
To evaluate the effect of reproductive stage on hsp70 mRNA levels and HSP70 abundance in A. purpuratus exposed to thermal and hypoxia stress factors, we performed 2-way ANOVAs for each stress factor.Model predictors were reproductive stage (with 3 levels: immature, mature and spawned) and presence/absence of the stressor (high temperature or hypoxia).To evaluate the effect of reproductive stage on CS enzyme activity, we performed a 1-way ANOVA.For both ANOVAs, normality of the dependent variable was tested using the Shapiro-Wilks test (SAS Institute 1999) and homogeneity of variances using Levene's test (Snedecor & Cochran 1989) to verify that the data met model assumptions.A posteriori tests for specific differences were conducted via the multiple pairwise comparisons least-square means (Lenth & Hervé 2015), with significance evaluated at p ≤ 0.05.

Changes in gonad mass and metabolic capacity with reproductive status
The mean gonad mass was approximately 170% greater in mature Argopecten purpuratus scallops than in immature scallops, indicating substantial reproductive investment (Fig. 1).After spawning, the mean gonad mass was similar to that of immature animals.
CS activity varied with A. purpuratus reproductive status (Fig. 2).This enzyme showed its highest levels in immature scallops, tended to decline with gonadal maturation, and attained its lowest level in spawned scallops (Fig. 2).CS activity was significantly different between immature and spawned scallops (p = 0.044).

Hsp70 mRNA levels and HSP70 abundance in scallops of different reproductive status following different stressors
Hsp70 mRNA relative levels increased significantly after exposure to thermal stress in scallops with each of the different reproductive statuses (Table 1, Fig. 3).However, in immature scallops, this increase in hsp70 mRNA levels was more than 2-fold higher than in mature and spawned scallops.Immature stressed scallops showed 130% more hsp70 mRNA than unstressed scallops, whereas stressed mature individuals showed 49% and spawned scallops 65% more hsp70 mRNA than unstressed scallops.Following exposure to thermal or hypoxia stress, immature A. purpuratus had significantly higher HSP70 protein levels (1.5-and 2-fold higher, respectively) than non-stressed immature scallops (Table 1, Fig. 4).However, following gonad maturation and spawning, there were not statistically significant differences (p > 0.05) in the HSP70 levels between scallops exposed and not exposed to thermal or hypoxia stress.Spawned scallops ex posed to thermal stress and hypoxia had slightly higher levels of HSP70 relative to control animals, but this difference was not significant (Table 1, Fig. 4).

DISCUSSION
Our results show that the capacity of mature and spawned Argopecten purpuratus to increase hsp70 mRNA and HSP70 levels following exposure to thermal and hypoxia stressors was markedly reduced relative to that of immature individuals.Interestingly, the reduction in HSP70 induction capacity after gonad maturation was similar for both stressors.In parallel to these reductions, we observed a decrease in the activity of the mitochondrial enzyme CS.CS is a key enzyme regulating the tricarboxylic acid cycle and thus the capacity for ATP generation via mitochondrial oxygen-dependent meta bolism of cells and/ or tissue (Storey 2004).Therefore, reduced hsp70 mRNA and HSP70 levels following reproductive processes could reflect an energetic compromise between the production of these proteins and gametogenic activity.
In several scallop species, available energy becomes limited during reproduction, as revealed by the sharp de crease in energy reserves following gona dal maturation and spawning (Martínez 1991, Martí nez et al. 2000, Brokordt & Guderley 2004).In A. pur pur atus from the same studied population, an approximately 50% de crease in adductor muscle carbo hydrate stores has been observed following gona dal maturation, persisting until after spawning (K.Brokordt unpubl.data).We have also observed reductions in the quality and oxidative capacity of muscle mitochondria following gonadal maturation and spawning in Chlamys islan dica scallops (Brokordt et al. 2000a).Decreased metabolic capacity of the anaerobic and aerobic metabolic pathways (through reduction of glycogen phosphorylase, pyru-  vate kinase, phosphofructokinase, octopine dehydrogenase and CS) following gona dal maturation and spawning has also been ob served in C. islandica (Brokordt et al. 2000a) and Eu vo la ziczac (Brokordt et al. 2000b).Moreover, maintenance metabolic require ments were higher after gonad maturation in Placopecten magellanicus (Kraffe et al. 2008).Therefore, the declines in energy availability and metabolic capacity along with the increased metabolic demands of gonadal maturation, which last until spawning, could limit the amount of energy available for the gene transcription and protein synthesis of HSP70, explaining our results.
The actual energetic cost of mounting a cellular stress response through HSPs is poorly understood.Stress proteins play an important role in energydependent processes, such as protein translocation into cellular organelles (Beckmann et al. 1990) and the binding and release of denatured proteins and short peptides to HSP70 (McKay et al. 1994, Mayer & Bukau 2005).Recently, it was observed that under in vitro conditions, one Hsp70 molecule consumed 5 ATPs to unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to its native state (Sharma et al. 2010).Thus, ATP is considered indispensable for correct HSP70 functioning (Morimoto 1993, Currie et al. 1999).Gene transcription and translation are also energetically expensive.Hsp70 mRNA transcription appears to be energetically limited in the red blood cells of rainbow trout subjected to extreme energetic stress, such as that occurring during combined heat shock and metabolic inhibition (i.e.decreased ATP by 79%; Currie et al. 1999).Although the exact amount of ATP necessary for HSP protein synthesis is unknown, the entirety of protein synthesis is estimated to cost between 25 and 40% (in mussels and fish, respectively) of an organism's total oxidative metabolism (Hawkins 1985, Lyndon et al. 1992).A study evaluating the cost of maintaining high HSP levels upon ex posure to long-term stress in the trout Oncorhynchus mykiss found that chronic stress (i.e.elevated HSP72 and HSP89) decreased metabolic condition (i.e.re duced muscle phosphocreatine, ATP and gly co gen) (Viant et al. 2003).Similarly, individual Dro so phila melanogaster that express additional copies of the hsp70 gene increase their metabolic rate by as much as 35% following exposure to heat stress (Hoekstra & Montooth 2013).We observed different effects of the reproduction on the hsp70 mRNA transcription and HSP70 protein levels in scallops exposed to thermal stress.After gonad maturation and spawning, hsp70 mRNA significantly increased (but less than before gonad maturation) in stressed scallops; however, there were no such increases in protein level in the same individuals.Although post-transcriptional regulation cannot be dismissed, these differences may reflect how the energetic restriction affected each of these processes.Given that we measured the expression of only one form of HSP70, and we cannot discard the potential existence of other forms of HSP70 in A. purpuratus, as have been reported for other bivalve species (Zhang et al. 2012), we may have evaluated only part of the response due to HSP70.Therefore, the effect of the energetic restriction may be even higher than observed in this study.Our results and the previous examples show that HSP synthesis and activity are energetically costly; however, the processes of degrading a misfolded protein and re-synthesizing another cost one thousand times more (Sharma et al. 2010).Therefore, this cellular stress response is considered a key physiological adaptation (Sharma et al. 2010, Morris et al. 2013).HSP70 levels have traditionally been used as biomarkers of stress; i.e. the result of individuals attempting to cope with the damage caused by environmental assaults (Sørensen 2010, Morris et al. 2013).However, considering the energetic cost of this cellular response, variation in HSP70 levels among individuals exposed to the same stressor could indicate variation in the capacity to mount this cellular stress response (Sørensen 2010).This latter approach, i.e.HSP70-induced levels as an indicator of stress response capacity, although suggested in the literature (Sørensen 2010), is supported by almost no empirical evidence.However, a study in 2 land snails has shown a negative correlation between the endogenous HSP70 isoform levels in foot tissue and albumen gland mass (indicative of reproductive maturation) (Mizrahi et al. 2011).These authors indicate that reduced levels of HSP70 isoforms, preceding egg laying, may reduce the snail's ability to cope with external stress (Mizrahi et al. 2011).Nevertheless, this study did not evaluate the changes in HSP induction in these snails upon exposure to stress.Additionally, in a recent study in the intertidal marine snail Concholepas concholepas, we found that individuals in poor energetic condition showed reduced HSP70 induction in response to hypoxia and/ or heat and cold thermal stress relative to individuals in good energetic condition (Jeno & Brokordt 2014).Finally, in Crassostrea gigas oysters, levels of the 72 and 69 kDa HSPs were lower in post-spawning than in pre-spawning individuals after being stimulated by heat shock, indicating that spawning reduced HSP synthesis (Li et al. 2007).Also, the post-spawning oysters had depleted glycogen stores and reduced adenylate energy charge compared with pre-spawning individuals, indicative of lower energy availability for metabolic activity (Li et al. 2007).These studies and the present study support the 'capacity approach' for observed HSP levels.
In life-history evolution, trade-offs represent the cost paid in the currency of fitness (i.e. the ability to produce fertile progeny), because an improvement in a fitness-related trait is associated with a detrimental change in another fitness-associated trait (Reznick 1985, Stearns 1989).One prominent trade-off is the cost of reproduction, in which current reproductive parental investment reduces parental longevity (e.g.reducing survival probability) or future reproductive capacity (Reznick 1985).Several energetic trade-offs between fitness-associated traits have been observed in mollusks.For example, in the cockle Cerastoderma edule, gonadal maturation has been associated with a decrease in the amount of energy available for growth (Iglesias & Navarro 1991), and for the scallops C. islandica (Brokordt et al. 2000a,b), A. purpuratus (Brokordt et al. 2006, Pérez et al. 2009) and Placopecten magellanicus (Kraffe et al. 2008), it is associated with a decrease in metabolic support for the escape response.In D. melanogaster and the abalone Haliotis discus hannai, induction of HSP70 has been associated with fitness (Krebs & Feder 1997, Sørensen et al. 2003, Cheng et al. 2006), because it increases tolerance to physiological stressors and may be critical for survival in some circumstances.Our results suggest that the observed reduction in HSP70 synthesis capacity following reproductive investment is a reproductive cost, as the decreased stress response capacity may limit future survival probability, and may reflect the existence of an evolutionary trade-off between these traits.
Energetic trade-offs between reproduction and other physiological functions can be sufficiently strong that mortality rates increase during postmaturation and spawning processes (Perdue et al. 1981, Barber & Blake 1983, Rocha et al. 2001).Mass mortality after maturation and spawning has been reported in several species of bivalves (Perdue et al. 1981, Barber & Blake 1983) and often coincides with strong environmental change (Cheney et al. 2000, Tomaru et al. 2001, Xiao et al. 2005).Argopecten purpuratus beds and artificial culture systems typically occur in bays near upwelling zones and are therefore ex posed to fluctuations in dissolved oxygen levels.Periods of decreased oxygen levels can persist for several days during the spring− summer season (Zhang et al. 2010, CEAZA Oceanographic Monitoring System unpubl. data).Similarly, increased mortality rates of mature A. purpuratus have been ob served in cultures in northern Chile (Bahía Inglesa and Tongoy Bay; Camanchaca Ostimar and INVERTEC growing companies, unpubl.data) and in natural beds in Peru (Cabello et al. 2002), most likely in association with periods of hypoxia.During the spring− summer season, scallops are not only more subject to hypoxia (< 2.0 mg O 2 l −1 ), but have also reached gonadal maturation and are initiating spawning.In culture, these processes are compounded with increased manipulation (e.g.sorting and cleaning of culture systems).Increased energy demands resulting from reproduction and environmental stressors, in combination with reduced HSP70 synthesis capacity, could partially explain the in crease in mortality rates observed in natural and cultured A. purpuratus populations.
In conclusion, our results suggest that reproductive investment may reduce the stress response capacity of A. purpuratus, such that mature and spawned scallops should be more vulnerable to environmental stress effects than immature individuals.The similar ob served effects of the 2 different types of stressor (hypoxia and heat stress, both of which occur fre quently in natural and cultured scallops populations) suggest a consistent pattern of response in this species.

Fig. 1 .Fig. 2 .
Fig. 1.Changes in gonad mass in Argopecten purpuratus scallops in 3 different reproductive stages (immature, mature and spawned).Immature and mature scallops were obtained from a cultured population.Spawning was induced in the laboratory.Values represent means ± SE (n = 60 per reproductive stage).Means sharing the same letter are not significantly different (p ≥ 0.05) from one another as indicated by a posteriori multiple comparisons (least-square means)

Fig. 3 .
Fig. 3. Relative Hsp70 gene transcription (mRNA) levels measured in gill tissue of Argopecten purpuratus scallops exposed (stressed) and not exposed (unstressed, controls) to thermal stress in 3 different reproductive stages (immature, mature and spawned).Values represent means ± SE (n = 5 replicates per condition; each replicate includes 3 individuals' total RNA).Means sharing the same letter are not significantly different (p ≥ 0.05) from one another as indicated by a posteriori multiple comparisons (least-square means)

Fig. 4 .
Fig. 4. Relative HSP70 protein levels measured in gill tissue of Argopecten purpuratus scallops exposed (stressed) and not exposed (unstressed, controls) to thermal and hypoxia stress in 3 different reproductive stages (immature, mature and spawned).Values represent means ± SE (n = 15 per condition).Means sharing the same letter are not significantly different (p ≥ 0.05) from one another as indicated by a posteriori multiple comparisons (least-square means)

Table 1 .
Results of 2-way ANOVAs comparing hsp70 gene transcription and protein induction levels between Argopecten purpuratus scallops with different reproductive status, exposed to stress by temperature or hypoxia.Reproductive status: immature, mature and spawned; temperature: 24°C (stress temperature) or 18°C (control temperature); oxy