INTRODUCTION
Transition of aquatic organisms to terrestrial systems is one of the most important landmarks in the evolutionary history of animals (Truchot, Reference Truchot1990). In this context, intertidal animals are of great use in understanding this process, due to the fact that they are constantly exposed to both aquatic and semi-terrestrial conditions by tidal cycles. This process generates ecological, anatomical and physiological necessities that some groups, such as decapod crustaceans have been able to respond to (Morris, Reference Morris2002; Greenaway, Reference Greenaway2003; Lardies et al., Reference Lardies, Muñoz, Paschke and Bozinovic2011).
Exposure to the air produces a decrease in the ability of aquatic organisms to capture O2 efficiently, and many crustaceans have developed different strategies that allow them to overcome these limitations, for example annexed respiratory structures such as decalcified locomotive appendages in some intertidal species (Stillman, Reference Stillman2000; Vargas et al., Reference Vargas, Lagos, Contreras and Cáceres2010), the ability to maintain bimodal respiration (Henry, Reference Henry1994; Greenaway, Reference Greenaway2003; Lardies et al., Reference Lardies, Muñoz, Paschke and Bozinovic2011), or, as in low latitudes, pulmonary structures that render them completely terrestrial (Farrelly & Greenaway, Reference Farrelly and Greenaway1994; Halperin et al., Reference Halperin, Ansaldo, Pellerano and Luquet2000). In this last group, studies regarding the effect of air exposure have been carried out to measure parameters such as desiccation, hypoxia and temperature (Pellegrino, Reference Pellegrino1984; Jensen & Armstron, Reference Jensen and Armstrong1991; Hofmann & Somero, Reference Hofmann and Somero1995; Full Reference O'Mahoney and Full1984; O’Mahoney & Lagos et al., Reference Lagos, Muñoz, Contreras and Cáceres2011).
In marine aquatic crustaceans, the collapse of gills during emersion periods challenges both O2 capture and CO2 excretion, producing acidification of haemolymph, and therefore, an increase in enzymes such as lactate dehydrogenase (LDH). This enzyme catalyses the conversion of lactate, and is involved in the final step of anaerobic glicolysis. It is an efficient indicator of physiological stress due to environmental conditions (Varley & Greenaway, Reference Varley and Greenaway1992; Astete-Espinoza & Cáceres Reference Astete-Espinoza and Cáceres2000; Morris, Reference Morris2002). This alteration in the acid–base balance is regulated by the ‘strong ion differences’ (SID) (Varley & Greenaway, Reference Varley and Greenaway1992; Luquet & Ansaldo, Reference Luquet and Ansaldo1997), which produce a change in pH that activates homeostatic mechanisms (Henry & Wheatly, Reference Henry and Wheatly1992; Wheatly & Henry, Reference Wheatly and Henry1992; Lagos & Cáceres, Reference Lagos and Cáceres2008; Vargas et al., Reference Vargas, Lagos, Contreras and Cáceres2010).
Crustaceans also carry out active regulation of acid–base balance by ionic exchange with the environment through the gills (Cl− for HCO3− and Na+ for H+), but crabs exposed to the air cannot perform this regulation. It has been suggested that some intertidal organisms could be using dissolution of exoskeleton CaCO3 in to HCO3− and Ca2+ as a compensatory mechanism during air exposure; CaCO3 is highly soluble and sensitive to pH changes and its dissolution can be quantified through changes in the concentration of Ca2+ in the haemolymph (Henry et al., Reference Henry, Kormanik, Smatresk and Cameron1981; Henry & Wheatly, Reference Henry and Wheatly1992; Luquet & Ansaldo, Reference Luquet and Ansaldo1997; Henry, Reference Henry2001). This phenomenon has been observed in molluscs and porcellanid crustaceans, where the concentration of Ca2+ in the exoskeleton decreases in relation to an increase in haemolymph during air exposure of more than two hours (Henry et al., Reference Henry, Kormanik, Smatresk and Cameron1981; Lagos & Cáceres, Reference Lagos and Cáceres2008; Montecinos et al., Reference Montecinos, Cisterna, Cáceres and Saldías2009; Vargas et al., Reference Vargas, Lagos, Contreras and Cáceres2010).
Nevertheless, the effects of air exposure are not the same in the different environments that these organisms inhabit, due to differences caused by humidity and/or temperature. For example, inhabiting lower latitudes is associated with higher physiological costs than higher latitudes (Lardies et al., Reference Lardies, Muñoz, Paschke and Bozinovic2011). This is because different populations of the same species, distributed along a wide latitudinal range, live under condition gradients that may vary significantly, producing phenotypic variations at different levels of environmental pressure (Lardies & Castilla, Reference Lardies and Castilla2001; Ricklefs & Wikelski, Reference Ricklefs and Wikelski2002; Mizera & Meszéna, Reference Mizera and Meszéna2003; Lardies & Bozinovic, Reference Lardies and Bozinovic2008). This phenotypic plasticity may cause effects on metabolism, body size (Laptikhovsky, Reference Laptikhovsky2006; Lardies & Bozinovic, Reference Lardies and Bozinovic2006; Lardies et al., Reference Lardies, Bacigalupe and Arias2010; Monaco et al., Reference Monaco, Brokordt and Gaymer2010) and also the ability to capture O2 in a bimodal manner, from both water and air (Lardies et al., Reference Lardies, Muñoz, Paschke and Bozinovic2011). Nevertheless, variation of acid–base balance during air exposure has not been studied using geographical gradients to see if this phenomenon behaves in an analogous way to the metabolism of individuals.
Our goals are: (1) to evaluate spatial variation of acid–base balance; and (2) to evaluate how acid–base regulation may compromise respiration. We will use the intertidal crab Cyclograpsus cinereus (Decapoda: Grapsidae) as a study model, in a latitudinal gradient of 2000 km along the Chilean coastline. This crustacean lives under boulders of the supratidal zone and spends most of the tide cycle out of the water (Bahamonde & López, Reference Bahamonde and Lopez1969). We will estimate acid–base equilibrium during emersion periods of six populations of C. cinereus using variables associated with respiratory physiology: pH, Ca2+, Cl− and haemolymphatic lactate dehydrogenase (LDH) enzyme activity.
MATERIALS AND METHODS
Study sites
Samples of Cyclograpsus cinereus adult male crabs, in intermolt stage C (Moriyasu & Mallet, Reference Moriyasu and Mallet1986; Luquet & Ansaldo, Reference Luquet and Ansaldo1997), were collected by hand in rocky intertidal zones at six localities along the Chilean coastline during spring months: Arica (18°28′S 70°18′W), Antofagasta (23°38′S 70°24′W), La Serena (29°54′S 71°15′W), El Quisco (33°23′S 71°4′W), Caleta Lenga (36°45′S 70°10′W) and Valdivia (39°48′S 73°14′W). The localities used correspond to zones that are subject to different environmental temperatures (see Figure 1) (for more details on superficial ocean and air temperatures, see Table 1). At each site, approximately 100 individuals were collected and transported in thermally isolated containers to the Animal Physiology Laboratory of Universidad Católica de la Santísima Concepción. In the laboratory, samples from all populations were acclimatized in seawater tanks at 13 ± 0.5°C, with salt levels of 30 psu for a period of 96 h (Chen & Chia, Reference Chen and Chia1997), before the experimental trials.
Fig. 1. Location of six study sites for individuals of Cyclograpsus cinereus collected along the coast of Chile. Sites were grouped to the following geographical regions: Arica–Antofagasta, northern region; La Serena–El Quisco, central region; and Lenga–Valdivia, southern region.
Table 1. Latitudinal gradient information of the six study sites along the Chilean coast. Sea surface temperatures (SST) and air temperature (AT) (data from SHOA, 2006 and FAO, 1985, respectively). Temperature is presented as the annual average, with annual average minimum and maximum temperatures in parentheses.
Laboratory analysis
To evaluate the effect of air exposure on the measured variables, crabs were subjected to different air exposure times: 0 (completely submerged organisms), 6, 15, 60, 120, 240 and 360 min (Luquet & Ansaldo, Reference Luquet and Ansaldo1997). Zero minutes of air exposure is considered control treatment. Three to five individuals for each treatment were placed in individual 250 ml containers with damp sand (using seawater) to avoid desiccation. Containers were then placed inside a refrigerated chamber in a PolyScience® (PolyScience PPO7R-20, PolyScience, USA) thermal bath for the rest of the experiment, defined by the exposure time, at the same temperature as the one used for acclimatization.
At the end of each experimental period, individual samples of haemolymph were collected by cephalothoraxic puncture using tuberculin syringes (1 ml). Immediately after extraction, samples were processed and the following parameters measured: (a) pH (NBS scale) using a pHmeter (HANNA® model 1332; Hanna Instruments S.L.; Spain) (b) Ca2+ concentration in haemolymph using the method described by Moorehead & Biggs (Reference Moorehead and Biggs1974); (c) Cl− concentration in haemolymph using the method described by Schales & Schales (Reference Schales and Schales1941); and (d) lactate dehydrogenase (LDH) activity using the method described by Klin (Reference Klin1970).
Statistical analysis
The sample localities were grouped into three geographic zones, because the nearest locality pairs did not present significant differences among the variable responses and phenotypic traits analysed (one way ANOVA; P > 0.001). The three biogeographic regions were: Arica and Antofagasta represent the northern region; La Serena and El Quisco represent the central region; and Caleta Lenga and Valdivia represent the southern region.
Before statistical analysis, variance homogeneity for the data were tested using the Hartley Fmax test (Sokal & Rohlf, Reference Sokal and Rohlf1997). To evaluate if there is any significant difference between each exposure time for each variable measured, an ANCOVA was used for each zone, using zone and air exposure as factors and body mass as a covariable. The statistically significant level considered was 0.05 (Zar, Reference Zar1996). Differences between groups (a posteriori comparison) were evaluated using a Tukey HSD test (Sokal & Rohlf, Reference Sokal and Rohlf1997). All analyses were carried out using the STATISTICA® program, v.6.0 (StatSoft, USA) for Windows® operating system. Data are presented as a mean plus or minus standard error (X ± EE).
RESULTS
Mean value for body mass of sampled organisms for each zone was 0.25 ± 0.008 g for the north zone, 0.688 ± 0.033 g for the central zone and 1.130 ± 0.109 g for the south zone. The body mass of the three zones varied significantly (F1,303 = 327.18; P < 0.05) (Tukey a posteriori P < 0.05).
Haemolymph parameters
Mean values for pH were independent from body mass of the organisms (F1,71 = 1.43; P = 0.235). The pH was significantly different between different air exposure times and geographic zones. Initial times were significantly lower in pH values than final air exposure times (F5,71 = 15.99; P < 0.05), northern organisms separated from central and southern organisms (F2,71 = 215.069; P < 0.05) (Tukey a posteriori P < 0.05). The highest variations were observed in the north zone, where the lowest value was found in the control group (6.54 ± 0.04) and the highest value was found after 120 min of air exposure (7.14 ± 0,12) (Tukey a posteriori P < 0.05) (Figure 2A). No significant interaction was observed between exposition time and locality (F10,71 = 1.29; P = 0.254). In individuals from the northern zone, the lowest pH values were found in organisms under immersion (air exposure time of 0) with a subsequent alkalinization that stabilized after 60 min of air exposure (see Figure 2A).
Fig. 2. Haemolymphatic values measured in the intertidal crab Cyclograpsus cinereus over several times of air exposure in north (Arica and Antofagasta), central (La Serena and El Quisco) and south (Lenga and Valdivia) zones of Chilean coast (mean ± SE): (A) variations in haemolymphatic pH; (B) Ca2+ haemolymphatic concentrations; (C) Cl− haemolymphatic concentrations. Significant differences among regions are highlighted by asterisks (for statistics see Results). Zero minutes indicate control treatment (no air exposure).
Mean values for haemolymphatic Ca2+ were determined independently from the body mass of organisms (F1,89 = 0.06; P = 0.801). Within this parameter an interaction between locality and time was observed. Organisms from the northern zone after 60 min of air exposure presented the highest values in regard to the other experimental groups (Figure 2B) (Tukey a posteriori P < 0.001). The lowest values for Ca2+ were obtained at the initial time in comparison to other exposition times (Tukey a posteriori P < 0.001).
Mean values for haemolymphatic Cl− were independent from body mass of organisms (F1,89 = 0.33; P = 0.563). Concentrations on haemolymphatic Cl− differed significantly between the three geographic zones, with the highest values observed in the southern zone (F2,89 = 13.41; P < 0.001) (Tukey a posteriori P < 0.05). No differences were observed between exposition times or for geographic and time interaction (F5,89 = 0.54; P = 0.74 for time factor and F10,89 = 0.69; P = 0.72 for factor interaction) (Figure 2C).
Enzyme activity
Values for LDH enzyme activity were independent from body mass of individuals. (F1,71 = 0.008; P = 0.929). The LDH enzyme activity in haemolymph was significantly different between analysed geographic zones (F2,71 = 41.58; P < 0.001). Among central and south zones was found the highest LDH activity, nevertheless these zones did not show significant differences (Tukey a posteriori P > 0.05). In contrast, the lowest values were found in the north zone (see Figure 3), (Tukey a posteriori P < 0.05). No significant differences were found between air exposure times or for the geographic zone and time interaction (F5,71 = 0.740; P = 0.590 for time and ANCOVA F10,71 = 1.18; P = 0.720 for factor interaction, respectively). The LDH and other parameters show differences which were associated with the origin of the individuals, with a clear separation between organisms from lower latitudes with those of intermediate and higher latitudes.
Fig. 3. Haemolymphatic values of LDH activity measured in the intertidal crab Cyclograpsus cinereus over several times of air exposure in north (Arica and Antofagasta), central (La Serena and El Quisco) and south (Lenga and Valdivia) zones of Chilean coast (mean ± SE). Significant differences among regions are highlighted by asterisks (for statistics see Results). Zero minutes indicate control treatment (no air exposure).
DISCUSSION
Many environmental factors can be selective agents for organisms, and temperature is one of the most important ones, as it affects processes that range from the molecular to the ecosystem level (Fischer & Fiedler, Reference Fischer and Fiedler2002; Pörtner et al., Reference Pörtner, Bennett, Bozinovic, Clarke, Lardies, Lenski, Lucassen, Pelster, Shiemer and Stillman2006). The clear differential pattern found for Cyclograpsus cinereus between the north and central–south regions (Table 1), indicated that temperature, both aquatic and air, may act as a key selective agent that marks physiological respiratory differences for this species in both zones (Camus, Reference Camus2001; Lardies et al., Reference Lardies, Bacigalupe and Arias2010, Reference Lardies, Muñoz, Paschke and Bozinovic2011; Lagos et al., Reference Lagos, Muñoz, Contreras and Cáceres2011). In this study, the effect of air exposure on acid–base balance was evaluated in different populations of C. cinereus subjected to different climatic temperature regimes (see Thiel et al., Reference Thiel, Macaya, Acuña, Arntz, Bastías, Brokordt, Camus, Castilla, Castro, Cortés, Dumont, Escribano, Fernández, Lancelotti, Gajardo, Gaymer, Gómez, González, González, Haye, Illanes, Iriarte, Luna-Jorquera, Luxoro, Manríquez, Marín, Muñoz, Navarrete, Pérez, Poulin, Sellanes, Sepúlveda, Stotz, Tala, Thomas, Vargas, Vásquez and Vega2007). Different patterns were found in respiratory parameters between populations of the northern region and those from the central–south regions which are consistent with patterns reported for this species in other physiological and life history traits (Lardies et al., Reference Lardies, Bacigalupe and Arias2010, Reference Lardies, Muñoz, Paschke and Bozinovic2011).
Variations in haemolymphatic pH for C. cinereus clearly separated the northern population from the central and southern ones. In individuals from the northern region, the lowest pH values were found in organisms under immersion (air exposure time of 0) with a subsequent alkalinization that stabilized after 60 min of air exposure (see Figure 2A). This indicates that this population has a high ability to maintain adequate acid–base regulation during air respiration periods (Henry et al., Reference Henry, Kormanik, Smatresk and Cameron1981). This physiological response has been described in crustaceans such as Petrolisthes laevigatus (Decapoda: Porcellan) and Cardisoma carnifex (Decapoda: Gecarcinidae), which suffer an increase in PCO2 and a decrease in pH during immersion in comparison to emersion periods (Morris & Adamezewska, Reference Morris and Adamezwska1996; Lagos & Cáceres, Reference Lagos and Cáceres2008).
Variation in Ca2+ concentrations were larger for individuals of the north region, which is associated with the ability to use CaCO3 reservoirs from the soluble exoskeleton to obtain Ca2+ and HCO3− when pH lowers, thus maintaining internal acid–base balance (Henry et al., Reference Henry, Kormanik, Smatresk and Cameron1981; Gunthorpe et al., Reference Gunthorpe, Sikes and Wheeler1990; Lagos & Cáceres, Reference Lagos and Cáceres2008; Montecinos et al., Reference Montecinos, Cisterna, Cáceres and Saldías2009; Vargas et al., Reference Vargas, Lagos, Contreras and Cáceres2010). On the other hand, absence of variations in Ca2+ in populations in the central and south regions do not indicate any mobilization of exoskeletal CaCO3, at least under the conditions of this study. One of the described mechanisms that crustaceans use for acid–alkali regulation is modification of strong ion difference (SID), which is the difference between the sum of strong cations and anions, mainly Cl− and Na+ (Randall et al., Reference Randall, Burggren and French2001). In crustaceans such as Chasmagnathus granulata, a large increase in Cl− has been observed within the first 2 h of air exposure. In this case, the high SID of this time frame indicated a high capacity to regulate this variable that regulates pH (Luquet & Ansaldo, Reference Luquet and Ansaldo1997). The lack of variability of haemolymphatic Cl− concentration in air exposure is an indicator of the fine adjustement of Cyclograpsus cinereus to semi-terrestrial habitats; however, the higher concentration of Cl−, along with null conversion of Ca2+ variation in organisms from high latitudes might indicate that C. cinereus uses different strategies in different geographic regions. This means that populations from high latitudes regulate pH more efficiently by using a SID mechanism, whereas those from lower latitudes would dissolve CaCO3 from the exoskeleton. This shows a higher independence of these organisms in aquatic environments, because haemolymphatic Ca2+ concentrations occur in terrestrial crustaceans where acid–alkali regulation using gills is not a feasible option (Henry et al., Reference Henry, Kormanik, Smatresk and Cameron1981; Innes et al., Reference Innes, Forster, Jones, Marsden and Taylor1986; Lagos & Cáceres, Reference Lagos and Cáceres2008).
Since C. cinereus is a poikilotherm, its metabolism is conditioned to environmental temperature, which follows a decreasing pattern towards colder areas (see Table 1 and Thiel et al., Reference Thiel, Macaya, Acuña, Arntz, Bastías, Brokordt, Camus, Castilla, Castro, Cortés, Dumont, Escribano, Fernández, Lancelotti, Gajardo, Gaymer, Gómez, González, González, Haye, Illanes, Iriarte, Luna-Jorquera, Luxoro, Manríquez, Marín, Muñoz, Navarrete, Pérez, Poulin, Sellanes, Sepúlveda, Stotz, Tala, Thomas, Vargas, Vásquez and Vega2007), meaning that populations from high latitudes have a lower metabolic rate than those of lower latitudes (Vernberg, Reference Vernberg1959; Osovitz & Hofmann, Reference Osovitz and Hofmann2007; Lardies et al., Reference Lardies, Muñoz, Paschke and Bozinovic2011; Whiteley et al., Reference Whiteley, Rastrick, Lunt and Rock2011). The higher response capacity in low latitude populations is reflected in the fact that they have a lower LDH activity, showing that under emersion conditions they do not have to resort to alternative anaerobic energy capture, which is necessary in intermediate and higher latitudes in general. In populations from high latitudes, where LDH activity is higher, this may be a sign that under the same conditions, these populations must resort to anaerobic energy capture due to their lower capacity to maintain effective air respiration. This is in contrast to higher latitude populations, which have the ability to breathe directly from the air (Lardies et al., Reference Lardies, Muñoz, Paschke and Bozinovic2011), as has been described also for Cyclograpsus lavauxi (Innes et al., Reference Innes, Forster, Jones, Marsden and Taylor1986; Waldron et al., Reference Waldron, Taylor and Foster1986).
In general, a latitudinal pattern has been reported in regard to the ability that crustaceans have to maintain adequate respiration under air conditions; pulmonated crustaceans and amphibians are found at low latitudes, and these characteristics tend to disappear towards higher latitudes. Several species of brachyuran and anomuran possess physiological modifications such as decreased gill area and the development of accessory respiratory structures such as lungs and highly vascularized gill chambers that allow them to occupy terrestrial and semi-terrestrial habitats, as well as the ability to survive prolonged emersion periods (Innes et al., Reference Innes, Forster, Jones, Marsden and Taylor1986; Waldron et al., Reference Waldron, Taylor and Foster1986; Farrely & Greenaway, Reference Farrelly and Greenaway1994; Stillman, Reference Stillman2000; Vargas et al., Reference Vargas, Lagos, Contreras and Cáceres2010).
Variation observed in acid-balance and, specifically, differences in LDH activity, in C. cinereus may be determined by acclimatization and/or adaptation (i.e. genetic basis). Studies carried out in the fish Fundulus heteroclitus indicate that there are differences in both concentration and activity of LDH enzyme, and also in the transcription speed in the LDH locus (Crawford & Powers, Reference Crawford and Powers1992). Populations inhabiting high latitudes have a high rate of LDH–RNA transcription, which results in a high enzyme concentration. This seems to be a genetically-controlled evolutionary adaptation mechanism, and not a direct acclimatization effect. This also has a larger effect on the enzyme, which is an important mechanism for adaptation affecting diverse physiological processes (Paynter et al., Reference Paynter, Dimichele and Powers1991; Schulte et al., Reference Schulte, Glémet, Fiebig and Powers2000). This should take place in C. cinereus, given the almost null response of the LDH enzyme activity in low latitude populations. When organisms are faced with temperature change, they may maintain their physiological rates using three different strategies: (a) quantitative, involving changes in substrate and enzyme concentration; (b) qualitative, where protein variants with different thermal properties are used; and (c) modulation, which changes the environment where the protein is to minimize temperature impact (Yang & Bielanoski, Reference Yang and Bielanoski2000; Hochachka & Somero, Reference Hochachka and Somero2002). Therefore, comparative studies between species or different populations, like this study, are useful to determine evolutionary adaptation; nevertheless, since these crustaceans are intertidal, acclimatization rates would be relatively quick (Willmer et al., Reference Willmer, Stone and Johnston2000) and individuals from all populations were placed in a common environment. Therefore, the difference in the parameter measures are most probably due to local adaptation and may correspond to heritable traits. Nevertheless, results from transgenerational studies are needed to confirm that this variability is due to genetic differences among populations.
Oceanic uptake of anthropogenic carbon dioxide (CO2) is altering the seawater chemistry of the oceans with consequences for organisms and ecosystems (Fabry et al., Reference Fabry, Seibel, Feely and Orr2008). High quantities of CO2 in oceans (i.e. hypercapnia) are producing a calcium carbonate saturation horizon to shoals in many regions, particularly in high latitudes (Gatusso & Hanson, Reference Gattuso and Hansson2011). Hypercapnia can impact marine organisms both via decreased calcium carbonate (CaCO3) saturation, which affects calcification rates, and via disturbance to acid–base physiology. Cyclograpsus cinereus show significant geographic variability in response to hypercapnia that indicates low buffering capacity in southern regions, the projected most-impacted population, and a ‘natural’ higher buffering capacity in low latitude populations (but see Pane & Barry, Reference Pane and Barry2007). Evidence suggests that there is now a critical need to test the physiological consequences of ocean acidification, while integrating into the analysis the variability among geographic populations.
COMPLIANCE
This study complies with current Chilean legislation regarding the collection and treatment of invertebrates.
FINANCIAL SUPPORT
This research was funded by FONDECYT (M.A.L. grant number 1110743) and ANILLO ACT-132.
