Ecophysiological adaptations to variable salinity environments in the crab Hemigrapsus crenulatus from the Southeastern Pacific coast: Sodium regulation, respiration and excretion

    Ecophysiological adaptations to variable salinity environments in the crab Hemigrapsus crenulatus from the Southeastern Pacific coast: Sodium regulation, respiration and excretion ´ Angel Urz´ua, Mauricio A. Urbina PII: DOI: Reference:

S1095-6433(17)30118-6 doi:10.1016/j.cbpa.2017.05.010 CBA 10231

To appear in:

Comparative Biochemistry and Physiology, Part A

Received date: Revised date: Accepted date:

13 February 2017 19 May 2017 22 May 2017

´ Please cite this article as: Urz´ ua, Angel, Urbina, Mauricio A., Ecophysiological adaptations to variable salinity environments in the crab Hemigrapsus crenulatus from the Southeastern Pacific coast: Sodium regulation, respiration and excretion, Comparative Biochemistry and Physiology, Part A (2017), doi:10.1016/j.cbpa.2017.05.010

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

RI

PT

Ecophysiological adaptations to variable salinity environments in the crab Hemigrapsus crenulatus from the Southeastern Pacific coast: Sodium regulation, respiration and excretion

SC

Ángel Urzúaa,b* & Mauricio A. Urbinac*

a

NU

Departamento de Ecología, Facultad de Ciencias, Universidad Católica de la Santísima Concepción, Casilla 297, Concepción, Chile b

MA

Centro de Investigación en Biodiversidad y Ambientes sustentables (CIBAS). Universidad Católica de la Santísima Concepción. Chile c

TE

D

Departamento de Zoología, Facultad de Ciencias Naturales y Oceanografía, Universidad de Concepción, Casilla 160-C, Concepción, Chile

AC CE P

Running title: Ecophysiological responses to salinity variation in benthic stages of Hemigrapsus crenulatus ms. has 29 pages, 4 figures, 2 tables

* Corresponding authors: Dr. Ángel Urzúa Departamento de Ecología, Facultad de Ciencias, Universidad Católica Ssma. Concepción, Alonso de Ribera 2850 Concepción, Chile. Tel: +56 41 2345265; Fax: +56 41 2345251 [email protected] Dr. Mauricio Urbina Departamento de Zoología, Facultad de Ciencias Naturales y Oceanografía, Universidad de Concepción, Casilla 160-C, Concepción, Chile [email protected]

1

ACCEPTED MANUSCRIPT Abstract The estuarine crab Hemigrapsus crenulatus is a key benthic species of estuarine and intertidal ecosystems of the South Pacific, habitats that experience wide fluctuations in

PT

salinity. The physiological strategies that allow this crab to thrive under variable salinities, and how they change during the benthic stages of their life cycle, were evaluated under

RI

laboratory conditions. Oxygen consumption, ammonia excretion and the regulatory

SC

capacity of Na+ through the normal range of environmental salinities (i.e. 5, 10, 15, 20, 25, 30) were evaluated in three size classes, ranging from juveniles to adults. In all sizes, the

NU

oxygen consumption, ammonia excretion and regulatory capacity of Na+ decreased as salinity increased, with the highest values at 5 and the lowest values at 30 salinity. Bigger

MA

crabs showed a higher capacity to regulate Na+, as well as higher respiration and excretion rates compared to smaller crabs, suggesting that they are better equipped to exploit areas of the estuary with low salinity. Regardless of its size, H. crenulatus is a strong hyper

D

regulator in diluted media (i.e. 5-20) while a conformer at salinities higher than 20. The

TE

regulatory capacity of Na+ was positively related with oxygen consumption and ammonia excretion rates. These relationships between sodium regulation, respiration and excretion

AC CE P

are interpreted as adaptive physiological mechanisms that allow H. crenulatus to maintain the osmotic and bioenergetic balance over a wide range of environmental salinities.

Key words: crustaceans, life cycle, benthic stages, salinity, ormoregulation, respiration, excretion, bioenergetic.

1. Introduction

In the open ocean, total salinity and ionic composition are relatively constant and only small fluctuations result from seasonal ice melt in Polar Regions, evaporation in the equatorial zones, and rainfall in the surface layers (Kalle, 1971). By contrast, coastal and estuarine waters do not show such physical stability. In these regions large salinity fluctuations occur across a range of different spatial and temporal scales. Since the maintenance of internal homeostasis (i.e. osmotic and ionic) is crucial for metabolic and physiological functioning, salinity plays a key role in the survival and development of

2

ACCEPTED MANUSCRIPT species inhabiting these coastal and estuarine zones (Kinne, 1971; Anger, 2003; Urbina et al., 2010; Urbina and Glover, 2015). Therefore, by affecting individuals at a behavioural, molecular, physiological, and biochemical level, salinity is also considered an important

PT

driver at both a population and ecosystem level (Newell, 1976; Luquet et al., 1998; Anger, 2003; Cieluch et al., 2004; Urbina et al., 2010).

RI

Osmoregulation is achieved by both active and passive mechanisms of ion transport (e.g.

SC

Na+, K+, Cl-, Ca2+). The key enzyme, directly or indirectly responsible for driving most ion and water movement is the Na+, K+-ATPase (NKA), which plays a central role in active

NU

osmoregulation (Torres et al., 2007; Lucu et al., 2008; Faleiros et al., 2010; McNamara and Faria, 2012). Depending on the concentration of the external medium (i.e. hypo or

MA

hyperosmotic), active absorption/excretion of ions counteracts the loss/gain of salt through the cellular membrane (Lucu, 1993; Lucu and Towle, 2003). The difference in osmotic concentration (i.e. osmolality) between the hemolymph and the external media indicates the

D

ability of an animal to osmoregulate, or its osmoregulatory capacity (Cieluch et al., 2007;

TE

Charmantier and Anger, 2011). A low osmoregulatory capacity implies that an animal has no ability to regulate its internal osmolality, thus it is said to osmoconform. Conversely, a

AC CE P

high osmoregulatory capacity implies that an animal has the ability to regulate its internal osmolality, irrespective of that of the external medium (i.e. osmoregulates). The osmoregulatory capacity also provides information about the direction of the response to osmotic stress, with positive values indicating hyper-osmoregulation and negative values indicating hypo-osmoregulation (Péqueux, 1995; Charmantier, 1998; Charmantier and Charmantier-Daures, 2001).

The ability to osmoregulate, however, comes at a cost. Active mechanisms to maintain osmotic balance, such as the one driven by NKA, consume ATP which fuels the pumping of ions against the concentration gradient. Therefore, ion regulation is also closely linked to other physiological processes (e.g. respiration and ammonia excretion), affecting both the metabolism and energy budget of an organism (Schmidt-Nielsen, 1997). Aquatic invertebrates are shown to change their metabolism in response to variations in salinity (Kinne 1971). Euryhaline species, for example, increase oxygen consumption at low salinities, while showing a decrease at elevated salinities. Such a response has therefore

3

ACCEPTED MANUSCRIPT been attributed to the additional energy requirements associated with osmoregulation (Kinne, 1971; Schmidt-Nielsen, 1997). Changes in the rates of ammonia excretion have also been attributed to osmoregulatory

PT

processes. The most common and accepted view is that changes in ammonia excretion reflect the changes in the catalysis/synthesis of amino acids, which are liberated to the

RI

extracellular compartment as a mechanism to maintain osmotic balance at low salinities

SC

(Larsen et al., 2014). Another potential mechanism in which salinity is involved in ammonia excretion and ion regulation is through the indirect coupling between NH4+ efflux

NU

and Na+ uptake (Weihrauch et al., 2004b), as well as through ion exchange during Na+ regulation in the hemolymph (Weihrauch et al., 2002). Therefore any change in the NKA

MA

activity, as a result of an osmoregulatory challenge, may also affect ammonia excretion rates.

Since osmoregulation and growth are both processes that require energy (Schmidt-Nielsen,

D

1997), osmoregulation will affect the energy budget of aquatic organisms (Mu et al., 2005;

TE

Hulathduwa et al., 2006; Urbina et al., 2010) and ultimately might determine the energy available for growth (Urbina and Glover, 2015). For example crabs and shrimps exposed to

AC CE P

suboptimal salinities present an increase in the production of ammonia (Rosas et al., 1999, 2002; Urbina et al., 2010), as well as an increase in metabolic rate (Aguilar et al., 1998; Urbina et al., 2010), which leads to an alteration in their growth potential (Guerin and Stickle 1997a, 1997b; Gillikin et al., 2004; Urbina et al., 2010). Crustaceans are one of the marine invertebrate taxa that are able to inhabit coastal regions and tidal estuaries across the globe, and are thus able to occupy habitats with low and/or fluctuating salinities (Anger, 1995; Charmantier and Anger, 2011). However, despite this distribution most marine crustaceans are osmoconformers (Péqueux, 1995), lacking the mechanisms to compensate big changes in salinity. It is therefore only a select group of crustaceans that are able to occupy environments with variable salinity, most of them osmoregulating (Thurman et al., 2010; Charmantier and Anger, 2011; de Faria et al., 2011). Members of the superfamily Grapsoidea include marine, brackish and terrestrial species, and this provides the ideal opportunity to understand the processes and physiological adaptations involved in the colonization of variable salinity environments in a single group. Hemigrapsus crenulatus (Milne Edwards, 1837) (Decapoda, Varunidae) belongs to this

4

ACCEPTED MANUSCRIPT superfamily and is a key species in brackish water ecosystem from the South eastern Pacific and New Zealand (Retamal, 1981). Due to its high abundance in estuarine environments (Hicks, 1974; Pulgar et al., 1995; Seneviratna and Taylor, 2006; Riquelme-Bugueño, 2006),

PT

H. crenulatus is not only considered an euryhaline crab, but it has also recently been shown that adults are able to maintain a positive scope for growth in a relatively-wide range of

RI

salinities (i.e. 5-33) by altering food consumption (Urbina et al., 2010). Despite H.

SC

crenulatus being an excellent osmoregulator, this has only been demonstrated in adult organisms.

NU

Since ecophysiological adaptation is a key factor to determine the capacity of aquatic invertebrates to inhabit environments with variable salinity (Kinne, 1971; Willmer et al.,

MA

2000), it is expected that the osmoregulatory capacity demonstrated in H. crenulatus is a key factor explaining its high abundance and dominance in estuarine ecosystems in the Southern Pacific. We, therefore, hypothesize that this high osmoregulatory capacity will not

D

only be present in adults, but it will also be present over a wider range of size classes (e.g.

TE

from juveniles to adults). The main objective of this study was therefore to investigate the regulatory capacity of Na+, and its relationship with other fundamental physiological

AC CE P

processes (e.g. respiration and ammonia excretion), across a wide range of size classes throughout the benthic development of H. crenulatus.

2. Material and methods

2.1. Manteinance, experimental design and sampling Juveniles and adults of Hemigrapsus crenulatus (n = 100) were captured by hand net from the Rio Cariquilida, which connects to Maullin Estuary (41°37’S, 73°35’W), southern Chile. They were then transported in cool boxes to the Laboratorio de Ecofisiología de Crustáceos of the Universidad Austral de Chile (Pelluco, Puerto Montt). Once in the laboratory, only crabs (n = 72) at inter-moult stage (C4), following the methods and classification of molt cycle described by Skinner (1962) and Drach & Tchernigovtzeff (1967), were selected for the experiments. Crabs were kept for 2 days under conditions similar to those observed at the collection site with respect to temperature and salinity (12 ºC and a salinity of 10). Carapace width, as the greatest distance across the carapace, of

5

ACCEPTED MANUSCRIPT each crab was measured with Vernier callipers and they were classified in three size ranges, each containing 24 crabs, Size I (small) = 11.2–17.6 mm; Size II (medium) = 18.5–23.1 mm and Size III (large) = 24–34.4 mm. Four crabs from each size class were randomly

PT

selected and individually placed in 5 l aquaria with aerated water at different salinities (5, 10, 15, 20, 25 and 30) (4 individual’s aquaria x 6 salinities x 3 sizes = 72 crabs).

RI

Experimental salinities were obtained by dilution of filtered (1 µm) natural sea water

SC

(salinity 33) with distilled water. Salinity was checked daily with a portable conductivity meter YSI model 30/10 FT. Constant flow from a recirculation system fed the aquaria with

NU

aerated water at the corresponding salinity at 12 ± 1 ºC. Photoperiod was maintained 12h:12h day: night cycle. The range of experimental salinities was based on the daily

MA

natural salinity oscillations occuring in the Cariquilda estuary during an annual cycle (7-30, Westermeier et al., 1993; Urbina et al., 2010). The crabs were fed daily with fresh pieces of mussel (Mytilus sp.), which are also highly abundant at the collection site, with food and

D

water being changed daily. The physiological variables (i.e. oxygen consumption, ammonia

TE

excretion, regulatory capacity of sodium (Na+)) were quantified on each crab in a steady state (post-absorbtive, contanst temperature, salinity and photoperiod; Urbina et al., 2010)

AC CE P

and under routine metabolism (Willmer et al., 2000), after 7 days of acclimatization to the experimental salinities. Once the physiological measurements were completed (see below), crabs were freeze-dried for 48 h in a vacuum dryer (Operon FDU-7012), and the dry weight was quantified to the nearest 0.01mg using a precision balance (Precisa model 120A), with crabs from each size range weighing on average: Size I = 0.50 ± 0.12 g; Size II = 1.35 ± 0.21 g and Size III = 2.50 ± 0.45 g.

2.2. Oxygen consumption (R) Oxygen consumption was determined by closed respirometry, using an optical oxygen probe and meter (Precision Sensing System-PreSens ® model Microx TX3). Crabs were individually placed in airtight chambers of 1 l volume, filled with clean water at their respective salinities (12 °C), and oxygen concentration was determined at the beginning and at the end of the incubation period. Owing to the different sizes, size I crabs were incubated for 4 h, size II crabs for 3 h, and size III crabs were incubated for 2 h. Oxygen level never fell below 80% saturation during the incubations and the oxygen probe was

6

ACCEPTED MANUSCRIPT calibrated daily using a saturated solution of Sodium sulphite (NO3 S2) (0% oxygen saturation) and water after intense bubbling (100% oxygen saturation) (Paschke et al.,

PT

2010).

2.3. Excretion rate of ammonia (N)

RI

Crabs were incubated in 250 ml of filtered (0.2 μm) and sterilized (UV) water at the

SC

corresponding salinity. The incubation periods were 4 h for size I, 3.5 h for size II and 3 h for size III. The concentrations of ammonium-N were then quantified in both initial and

NU

final samples using the method described by Koroleff and Grasshoff (1983), modified for microplates. At the peak of the colour reaction, plates were read at 640 nm using a plate

MA

reader (Thermo Scientific Multiskan TM).

2.4. Regulatory capacity of sodium (RC-Na+)

D

The regulatory capacity of sodium was determined as the difference between the sodium

TE

concentration in the crab’s hemolymph and the external medium (water) (see Charmantier et al., 2002). Crabs were removed from their tanks, placed on ice and a 35 μl hemolymph

AC CE P

sample was quickly withdrawn. A 21G needle, attached to a pre-cooled hypodermic syringe, was inserted at the base of the third walking leg. Samples were then centrifuged at 10,000 g for 10 min at 6 °C, in a refrigerated centrifuge (Boeco model U32-R). Samples were kept on ice during intermediate steps. The concentration of sodium in each sample was then measured in the supernatant using a Na+ ion-selective sensor (ORION model 9811BN) at 12 ºC.

2.5. O:N ratio The ratio of oxygen consumed to nitrogen excreted, used as a proxy of metabolic substrate for energy production (Mayzaud and Conover, 1988), was calculated in atomic equivalents according to the method described by Widdows (1985).

2.6. Statistical analyses The statistical analyses were performed using the program STATISTICA 8 (StatSoft) and SigmaPlot 12 (Systat Software) employing standard methods (Sokal and Rohlf, 1995;

7

ACCEPTED MANUSCRIPT Quinn and Keough, 2002) with 95% confidence levels (p < 0.05). All the physiological rates of H. crenulatus were expressed per individual and per hour for each of the experimental salinity conditions. The relationships between external salinity and

PT

physiological parameters (respiration, ammonia excretion and regulatory capacity of sodium) for the three size ranges of H. crenulatus, were fitted to linear (y = y0 + ax) for R,

RI

N, and O:N, and polynomial regressions (y = y0 + ax + bx2 + cx3) for RC-Na+, based on

SC

their best fit (r2) (Quinn and Keough, 2002). Normal distribution and homogeneity of variance were tested with Kolmogorov–Smirnov and Levene median tests, respectively.

NU

When data did not meet parametric assumptions, a square root transformation was used (only for sodium concentration in the hemolymph). The effect of crab size on the

MA

respiration, ammonia excretion and sodium regulatory capacity was checked using a test for equality and comparisons of slopes of regression analyses (Sokal and Rohlf, 1995).

TE

D

3. Results

No moults and no mortalities were recorded at any of the experimental salinities or sizes

AC CE P

tested during the whole duration of the experiments.

3.1. Oxygen consumption (R)

Respiration rate increased as salinity decreased in all sizes of the estuarine crab (p < 0.001; Fig. 1A; Table 1). Irrespective of the size, crabs exposed to low salinity (5) consumed on average ~ 2.5 fold more oxygen than those crabs exposed to high salinity (30) (Fig. 1A). For example, individuals of small size (size I) consumed about 0.51 ± 0.03 mg O2 h-1 ind-1 at 5 salinity compared to 0.14 ± 0.04 mg O2 h-1 ind-1 at 30 salinity (Fig. 1A,). Crabs of intermediate size (size II) showed average oxygen consumption values of 0.93 ± 0.06 mg O2 h-1 ind-1 at 5 salinty compared to 0.35 ± 0.08 mg O2 h-1 ind-1 at 30 salinity, while individuals of large size (size III) consumed about of 1.54 ± 0.05 mg O2 h-1 ind-1 at 5 salinity compared to 0.54 ± 0.03 mg O2 h-1 ind-1 at 30 salinity (Fig. 1A). The oxygen consumption was significantly different between sizes (p < 0.001, Table 2).

8

ACCEPTED MANUSCRIPT

3.2. Ammonia excretion rate (E) The excretion rate of ammonia-N of H. crenulatus followed the same pattern as oxygen

PT

consumption, increasing as salinity decreased (p < 0.001; Fig. 1B, Table 1). In all sizes studied the excretion rate was ~3.5 fold higher at 5 than at 30 (Fig. 1B). For example, at 5

RI

salinity small crabs (size I) averaged ammonia excreation values of 0.50 ± 0.13 mg 10-2 N

SC

h-1 ind-1 compared to 0.15 ± 0.04 mg 10-2 N h-1 ind-1 at 30 salinity (Fig. 1B). Medium crabs (size II) reached values about of 0.95 ± 0.4 mg 10-2 N h-1 ind-1 at 5 salinity, compared to

NU

0.36 ± 0.10 mg 10-2 N h-1 ind-1 at 30 salinity (Fig. 1B). Finally, large crabs (size III) showed average values of 1.42 ± 0.57 mg 10-2 N h-1 ind-1 at 5 salinity compared to values of 0.56 ±

MA

0.32 mg 10-2 N h-1 ind-1 at 30 salinity (Fig. 1B). Similar to the pattern observed with oxygen consumption, the excretion rate was significantly different between sizes (p <

D

0.001, Fig. 2B, Table 2).

TE

3.3. Na+ in the haemolymph and external medium The sodium concentration in the haemolymph of the three crab size classes was

AC CE P

significantly different to the external sodium concentration at salinities lower or equal to 20 (p < 0.001, Fig. 2A, Table 1). In comparison to the isosmotic line, the sodium content in the haemolymph was significantly higher in salinities lower than or equal to 20 (p < 0.05, Fig. 2A). For example at 5 salinity haemolymph sodium concentration was 375 ± 31 mM Na+, compared to values of 86 ± 5 mM Na+of the external medium. Consistently at 20 salinity haemolymph sodium concentration was 400 ± 32 mM Na+ compared to values of 365 ± 21 mM Na+ of the external medium. However, between 25 and 30 salinity the concentration of Na+ in the haemolymph was similar or slightly lower to that of the external medium (p = 0.178, Fig. 2A). For example at 25 salinity haemolymph Na+ averaged 420 ± 25 mM Na+ compared to 450 ± 26 mM Na+ in the external medium. The same pattern was evident at 30, with average values of 510 ± 18 mM Na+ in haemolymph and 540 ± 30 mM Na+ in the external medium (p = 0.192). In all sizes studied, H. crenulatus behaves as a Na+ regulator in salinities between 5 and < 20, while it behaves as a Na+ conformer between 20 and 30 (Fig. 2A).

9

ACCEPTED MANUSCRIPT

3.4. Regulatory Capacity of sodium (RC-Na+) In the three size ranges the regulatory capacity for Na+ increased as salinity decreased (p <

PT

0.001, Fig. 2B, Table 1). For example, for size III crabs positive RC-Na+ values were calculated in low and intermediate salinities, 194 ± 30, 178 ± 15, 146 ± 13 and 79 ± 28 at 5,

RI

10, 15 and 20 respectively (Fig. 2B). However, at high salinities RC-Na+ values were

SC

lower, 38 ± 21 and 58 ±16 mM of Na+ at 20 and 30 respectively (Fig. 2B). The analyses of slope comparisons indicate that bigger crabs had higher Na+ regulatory capacity than

NU

smaller crabs (Table 2).

MA

3.5 O:N ratio

Although both oxygen consumption and ammonia excretion followed a similar pattern as salinity decreased, the O:N ratio in the three crab sizes increased as salinity decreased (p <

D

0.01, Fig. 3, Table 1). Maximum values were measured between 5 and 15 (62.42 ± 1.73,

TE

79.10 ± 1.43, and 88.68 ± 1.44; sizes I, II, and III, respectively), intermediate values at 15 (53.6 ± 1.61; 73.6 ± 0.84, 81.2 ± 0.78; sizes I, II, III respectively) and the lowest values

AC CE P

were calculated between 20 and 30 (48.5 ± 2.1, 55.5 ± 1.61, 59.86 ± 0.93; size I, II, III, respectively) (Fig. 3). In comparison to smaller crabs, larger individuals showed higher values of O:N (88-60 vs. 62-48; size III vs. I, respectively) (p < 0.01, Fig. 3, Table 2). 3.6. Relationships among RC-Na+, R and E The relationships between regulatory capacity of sodium, and oxygen consumption and ammonia excretion rate were positive and highly significant (p < 0.001; Fig. 4; Table 1). In H. crenulatus (size III), the oxygen consumption and excretion rate increased as sodium regulatory capacity increased, showing maximum values of 1.42 ± 0.2 mg O2 ind-1 h-1 and 1.30 ± 0.3 mg 10-2 N h-1 ind-1 at the higher regulatory capacities (i.e. 170 and 190 mM of Na+) (Fig.4 A,B). Likewise, oxygen consumption 0.19 ± 0.09 mg O2 ind-1 h-1 and ammonia excretion values 0.14 ± 0.08 mg 10-2 N ind-1 h-1 were lowest when the regulatory capacity was also low (i.e. 20-40 mM of Na+) (Fig.4 A,B).

10

ACCEPTED MANUSCRIPT

PT

4. Discussion

Our results have shown that all benthic sizes of H. crenulatus (11.2-34.4 mm) tested in the

RI

present study were able to tolerate salinities ranging between 5-30. This is attributed to an increased sodium (and likely other ions as well) regulatory capacity at low salinities.

SC

Sodium regulatory capacity scaled as crab size increased, suggesting that the ability to tolerate low salinities increases during benthic development of this species.

NU

Estuarine environments are characterized by abrupt and marked variations in salinity at tidal, daily, weekly, yearly and seasonal time scale. These fluctuations generate a strong

MA

selective pressure over the organisms living in such variable environments (Kinne, 1971; Schmidt-Nielsen, 1997). The salinity tolerance of an animal is achieved by a combination

D

of mechanisms at behavioural (e.g. avoidance), physiological (e.g. volume, water and ion regulation, metabolism of amino acids, NKA activity (Péqueux, 1995; Urbina and Glover,

TE

2015), and molecular levels (e.g. NKA isoforms, upregulation, transcription, and placement (McNamara and Faria, 2012; Urbina et al., 2013)). Our results showed that H. crenulatus

AC CE P

actively regulates hemolymph Na+ in salinities between 5 and 20, but it does not at salinities greater than 25. At salinities higher than 20, hemolymph Na+ in H. crenulatus conforms to that of the external medium. A similar response has been described in individuals from the same species from New Zealand (Hicks, 1973; Taylor and Seneviratna, 2005), in another crab species (Panopeus herbstii: Blasco and Forward, 1988; Carcinus maenas: Siebers et al., 1983, Cieluch et al., 2004; Neosarmatium meinerti, Neosarmatium smithi: Gillikin et al., 2004; Neohelice granulata: Bianchini et al., 2008). The regulatory capacity for sodium (RC-Na+) increased as salinity decreased, in all sizes tested. However, the slope or strength of this relationship varied depending on crab size. In the present study, bigger crabs showed an enhanced RC-Na+ compared to small crabs, which is advantageous for tolerating low salinities. Whether or not RC-Na+ in small crabs is compromised due to a lower capacity for Na+ uptake or a higher Na+ diffusion (efflux) at low salinities is still unknown. These findings suggest that tolerance to low salinity increases as a crab develops. In fact, our results are the first to provide a physiological explanation for the size-salinity dependant distribution commonly reported in this species 11

ACCEPTED MANUSCRIPT natural habitat (i.e. estuaries). For example, several studies in the Cariquilda and Maullin estuaries have reported that large H. crenulatus (e.g. adults) are commonly found in low salinity estuarine areas, while small H. crenulatus (e.g. juveniles) are usually found in areas

PT

of the estuary where salinity is higher and more stable (Grandjean, 1985; Urzúa, 2005). Our results suggest that RC-Na+, and osmoregulatory ability in general, increases during the

RI

benthic development of H. crenulatus allowing them to colonize and exploit estuarine areas

SC

of lower and fluctuating salinities. Therefore, we suggest that early benthic stages (i.e. megalopa and juvenile) of H. crenulatus rely more on behavioural strategies to avoid low

NU

salinities, but as they grow, the enhancement of the osmoregulatory capacity would allow them to rely more on physiological strategies to colonize areas of low salinity.

MA

This size-salinity dependent distribution has been previously suggested to be the result of different size-dependant tolerances to salinity change, originating from the development of osmoregulatory structures and the activity of some key transporters (Na+/K+-ATPase)

D

(Charmantier et al., 2002; Gillikin et al., 2004; Taylor and Seneviratna, 2005; Cieluch et al.,

TE

2007), as a mechanism to avoid cannibalism from bigger con-specifics (Iribarne et al., 1997; Luppi et al., 2002; Bas et al., 2005), or a combination of both (Charmantier, 1998;

AC CE P

Posey et al., 2005; Anger et al., 2008; Pardo et al., 2011). The crucial role that NKA plays in acclimation to different salinities has been widely suggested in crustacean species. In the posterior gills of Carcinus maenas, for example, a 4fold increase in the NKA activity has been reported after transfer to diluted seawater (from 32 to 10 salinity; acclimation period: 3 weeks). This response has been, in part, attributed to NKA synthesis in order to meet this new osmotic challenge (Lucu and Devescovi, 1999). Similarly, in the intertidal/estuarine crabs Hemigrapsus nudus, Leptograpsus variegatus and Neohelice granulata, the NKA activity increased 2-fold after exposure to 50% diluted seawater (Corotto and Holliday, 1996; Cooper and Morris, 1997; Castilho et al., 2001; respectively). Therefore, although we did not directly assess branchial NKA activity after exposure to different salinities, the observed Na+ hyper-regulation at low salinities is likely the result of an enhanced NKA activity (and likely in other transporters as well). Osmoregulation is energy consuming (Péqueux, 1995; Schmidt-Nielsen, 1997), which may have consequences for growth and energy budget (Romano and Zeng, 2006; Urbina et al., 2010; Ye Le et al., 2009). The marked increases in both respiratory and ammonia excretion

12

ACCEPTED MANUSCRIPT rates of H. crenulatus at low salinities support such a change in the energy budget of this species. Oxygen consumption increased in all H. crenulatus sizes studies at low salinity. This could be signalling an increase in the ATP demand as a result of an increased NKA

PT

activity under such salinity conditions. This physiological response has been previously documented in the same species (Urbina et al., 2010), species from the same genus

RI

(Hemigrapsus spp, Corotto and Holliday, 1996; Hemigrapsus takanoi, Shinji et al., 2009),

SC

other estuarine crabs (Neohelice (Chasmagnathus) granulata, Castilho et al., 2001; Uca spp, Thurman, 2003), seawater shrimps (Macrobrachium tenellum, Aguilar et al., 1998)

NU

and also freshwater shrimps (Macrobrachium tuxtlaense, Ordiano et al., 2005). In decapod crustaceans, the moulting is also an energy consuming process, reflected in a

MA

increase in metabolic rate (Skinner 1962). Moulting also involves sevaral other extensive physiological changes in crabs, which could further be modulated by key environmental factors such as temperature, photoperiod and salinity (Skinner 1985). In H. crenulatus, how

D

these factors (salinity in special) influence moulting, and the extent of the physiological

TE

changes during moulting in have not been studies to date. Therefore, in the present study is important to mention that only intermoult crabs were used, and our results do not include

AC CE P

potencial effects of moulting on the responses to salinity reported here. In decapod crustaceans, an increase in the urine excretion rate at low salinities has been proposed to aid the regulation of water volume (Greenaway, 1991). Under low salinity conditions, urine would be more diluted in order to retain ions such as Na+, K+, Ca2+, Cl-, in the hemolymph (Weihrauch et al., 2004a). Our results support this idea, as ammonia excretion rate (i.e. as a proxy for urine excretion) increased at low salinities in the three sizes evaluated. Similar results have been found in other coastal /estuarine crustaceans (Weihrauch et al., 2001; Diaz et al., 2004; Shinji et al., 2009; Masui et al., 2009) and also in the same species (Urbina et al., 2010). In addition, the increased ammonia excretion rate in dilute media is associated directly with the exchange and regulation of Na+ in the hemolymph and consistently with the participation of the NKA (Weihrauch et al., 2004b), where a higher excretory activity at low salinities stimulates and increases the activity of specific ion pumps like the Na+/K+ by 90% (Towle and Kays, 1986; Masui et al., 2002; Weihrauch et al., 2002) and V-ATPases (Tsai and Lin, 2007) that drive ion uptake and underpin NH4+ excretion (Weihrauch et al., 2004b). This idea is further supported by a

13

ACCEPTED MANUSCRIPT recent study on an amphidromous fish, where a positive relationship was found between NKA activity and ammonia excretion across salinities ranging from 0 to 43 (n = 60) (Urbina and Glover, 2015).

PT

The maintenance of homeostasis over a wide range of salinities imposes a considerable energy demand. Therefore a tight relationship must exist between the mechanisms used to

RI

overcome salinity changes and how those mechanisms are fuelled (e.g. energy budget)

SC

(Greenaway, 1988; Rosas et al., 2001; Urbina et al., 2010; Urbina and Glover, 2015). As mentioned above, juveniles or small H. crenulatus individuals are able to live in estuaries

NU

by utilising a behavioural strategy and only inhabiting areas were salinity is high and/or more stable. Similar findings have been reported in other estuarine crabs (Stickle et al.,

MA

2007; Pardo et al., 2011). However, as larger H. crenulatus are exposed to lower and more drastic changes in salinity, they must fuel the extra costs associated with osmoregulation. Increases in the ingestion rate at low salinities have previously been documented in H.

D

crenulatus (Urbina et al., 2010), and suggested as the mechanism for fuelling these extra

TE

osmoregulatory costs. Changes in the energy substrate being metabolized have also been suggested as a mechanism for fuelling osmoregulation at salinity extremes (Rosas et al.,

AC CE P

1999; Wang et al., 2004). The increased O:N ratio observed at low salinities might suggest changes in the metabolic substrate used to to fuel these extra osmoregulatory costs. Support for this idea also comes from a recent study in the amphidromous and euryhaline fish Galaxias maculatus, which increases its reliance in protein metabolism at both salinity extremes (0.6 and 43) (Urbina and Glover, 2015). Findings from the present study suggest that the preference for using different energy substrates change in a size-dependant fashion. Regardless of the salinity, it seems that smaller individuals utilize a greater proportion of protein than larger animals (O:N ratios of 48.5 ± 2.1 vs. 59.86 ± 0.93). Rivers are well known to carry considerable amounts of terrestrial items, which could be transported to the estuaries, and therefore become potential food items. Whether or not the availability of organic matter from terrestrial origin influences the preference to metabolize bigger proportions of carbohydrates and lipids in bigger crabs needs further research. Hemigrapsus crenulatus could use a combination of different compensatory mechanisms, allowing this species to thrive in habitats that vary in salinity (i.e. estuaries). This suggests that the characteristics demonstrated in H. crenulatus would be found in other species

14

ACCEPTED MANUSCRIPT transitioning between brackish and freshwater habitats over an evolutionary timescale (McNamara and Faria, 2012). The colonization of inland waters has evolved in several lineages within the super family Grapsoidea, resulting in several estuarine, semi-terrestrial

PT

species (Schubart et al., 2000). Although the members of this super family are abundant in various estuarine and intertidal environments, only a few species have lost the connection

RI

with the marine environment (Schubart et al., 2000). This is not the case for H. crenulatus,

SC

which despite its ability to tolerate low salinities, has not been able to colonize fresh water environments. Poor tolerance and low survival have been reported in H. crenulatus exposed

NU

to freshwater (Hicks, 1979; Corotto and Holliday, 1996), limiting its distribution only to estuaries and intertidal zones (Riquelme-Bugueño, 2006; Balboa et al., 2009; Diaz-

MA

Jaramillo et al., 2013). After hatching, larval stages of H. crenulatus are carried to the sea where they develop. Then, after metamorphosis to first juvenile they start their trip back up the estuary. This feature of its life cycle presents a natural and gradual acclimation to low

D

salinities as juveniles move to the upper part of the estuary. It is hypothesized that this

TE

mechanism is more energy efficient compared to a more dramatic settlement under different saline conditions, and as such might explain the high abundances of this estuarine

AC CE P

crab. This gradual estuarine migration has also been suggested as energy efficient in some fish species (Urbina and Glover, 2015). Some features of this life cycle are also characteristics in other semi-terrestrial members of the family (Schubart et al., 2000, Schubart and Diesel, 1998, 1999; Anger, 2001; Augusto et al., 2007). Our results show that H. crenulatus is able to tolerate a wide range of environmental salinities by an enhanced osmoregulatory capacity associated with the tight regulation of basic physiological processes (oxygen consumption and ammonia excretion). A switch in the metabolic substrate used at low salinities seems to contribute to fuel the extra osmoregulatory costs. These physiological and bioenergetic mechanisms are, however, not fully developed in juveniles, forcing young H. crenulatus to rely on behavioural strategies to live in habitats of changing salinities of the South Pacific. Considering the wide distribution of H. crenulatus in the South Pacific (Retamal and Moyano, 2010) and New Zealand (McLay et al. 2011), and that recent studies suggest that osmoregulatory capacity could vary with temperature, latitude, and between populations (Thurman, 2005; GonzalezOrtegon, 2006; Charmantier and Anger, 2011), future research should explore potential for

15

ACCEPTED MANUSCRIPT intra-specific variability in the remarkable osmoregulatory ability of H. crenulatus within its ecological and latitudinal distribution.

PT

Acknowledgments. The authors thank Dr. Robert Ellis for chequing the language of this manuscript and to the anonimous reviewers for their constructive criticism. Financial

RI

support was provided by the CONICYT to A.U. (PAI: 79130025; FONDECYT: 11140213)

SC

and to MU (FONDECYT: 11160019). We also thank to Dr. Kurt Paschke and Juan Pablo Cumillaf for their support during the experiments. The experiments comply with the current

NU

Chilean animal care and manipulation legislation.

MA

References

AC CE P

TE

D

Aguilar, M., Díaz, F., Bückle, L.F., 1998. The effect of salinity on oxygen consumption and osmoregulation of Macrobrachium tenellum. Mar. Freshw. Behav. Phy. 31, 105-113. Anger, K., 1995. The conquest of freshwater and land by marine crabs: adaptations in lifehistory patterns and larval bioenergetics. J. Exp. Mar. Biol. Ecol. 193, 119-145. Anger, K., 2001. The Biology of Decapod Crustacean Larvae. Lisse, The Netherlands, A.A. Balkema. Anger, K., 2003. Salinity as a key parameter in the larval biology of decapod crustaceans. Inv. Rep. Dev. 43, 29-45. Anger, K., Spivak, E., Luppi, T., Bas, C., Ismael, D., 2008. Larval salinity tolerance of the South American salt-marsh crab, Neohelice (Chasmagnathus) granulata: physiological constraints to estuarine retention, export and reimmigration. Helgoland. Mar. Res. 62, 93-102. Augusto, A., Greene, L.J., Laure, H.J., McNamara, J.C., 2007. Adaptive shifts in osmoregulatory strategy and the invasion of freshwater by brachyuran crabs: evidence from Dilocarcinus pagei (Trichodactylidae). J. Exp. Zool Part A. 307, 688698. Balboa, L., Hinojosa, A., Riquelme, C., Rodriguez, S., Bustos, J., George-Nascimento, M., 2009. Alloxenic Distribution of Cystacanths of Two Profilicollis Species in Sympatric Crustacean Hosts in Chile. J. Parasitol. 95, 1205-1208. Bas, C., Luppi, T., Spivak, E., 2005. Population structure of the South American estuarine crab, Chasmagnathus granulatus (Brachyura: Varunidae) near the southern limit of its geographical distribution: comparison with northern populations. Hydrobiologia. 537, 217-228. Bianchini, A., Lauer, M.A., Nery, L.E.A., Colares, E.P., Monserrat, J.M., dos Santos Filho., E.A., 2008. Biochemical and physiological adaptations in the estuarine crab Neohelice granulata during salinity acclimation. Comp. Biochem. Phys. A. 151, 423436. Blasco, E., R. B. Forward, R.B., 1988. Osmoregulation of the xanthid crab, Panopeus herbstii. Comp. Biochem. Physiol. 90, 135-139.

16

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Castilho, P.C., Martins, I.A., Bianchini, A., 2001. Gill Na+, K+-ATPase and osmoregulation in the estuarine crab, Chasmagnathus granulata Dana, 1851 (Decapoda, Grapsidae). J. Exp. Mar. Biol. Ecol. 256, 215-227. Cieluch, U., Anger, K., Aujoulat, F., Buchholz, F., Charmantier-Daures, M., Charmantier, G., 2004. Ontogeny of osmoregulatory structures and functions in the green crab, Carcinus maenas (Crustacea, Decapoda). J. Exp. Biol. 207, 325-336. Cieluch, U., Anger, K., Charmantier-Daures, M., Charmantier, G., 2007. Osmoregulation and immunolocalization of Na+/K+-ATPase during the ontogeny of the mitten crab Eriocheir sinensis (Decapoda, Grapsoidea). Mar. Ecol. Prog. Ser. 329, 169-178. Cooper, A.R., Morris, S., 1997. Osmotic and ionic regulation by Leptograpsus variegatus during hyposaline exposure and in response to emersion. J. Exp. Mar. Biol. Ecol. 214, 263-282. Corotto, F.S., Holliday C.W., 1996. Branchial Na, K-ATPase and osmoregulation in the purple shore crab, Hemigrapsus nudus (Dana). Comp. Biochem. Physiol. 113A, 361368. Charmantier, G., 1998. Ontogeny of osmoregulation in crustaceans: a review. Invertebr. Reprod. Dev. 33, 177-190. Charmantier, G., Charmantier-Daures, M., 2001. Ontogeny of osmoregulation in crustaceans: the embryonic phase. Am. Zool. 41, 1078–1089. Charmantier, G., Giménez, L., Charmantier-Daures, M., Anger, K., 2002. Ontogeny of osmoregulation, physiological plasticity, and larval export strategy in the grapsid crab Chasmagnathus granulata (Crustacea, Decapoda). Mar. Ecol. Prog. Ser. 229, 185194. Charmantier, G., Anger K., 2011. Ontogeny of osmoregulatory patterns in the South American shrimp Macrobrachium amazonicum: Loss of hypo-regulation in a landlocked population indicates phylogenetic separation from estuarine ancestors. J. Exp. Mar. Biol. Ecol. 396, 89-98. Díaz, F., Re, A.D., Sierra, E., Diaz-Iglesias, E., 2004. Effects of temperature and salinity fluctuation on the oxygen consumption, ammonium excretion and osmoregulation of the blue shrimp Litopenaeus stylirostris (Stimpson). J. Shellfish. Res. 23, 903-910. Díaz-Jaramillo, M., Socowsky, R., Pardo, L.M., Monserrat, J.M., Barra, R., 2013. Biochemical responses and physiological status in the crab Hemigrapsus crenulatus (Crustacea, Varunidae) from high anthropogenically-impacted estuary (Lenga, southcentral Chile). Mar. Environ. Res. 83, 73-81. Drach, P. and C. Tchernigovtzeff (1967). "Sur la méthode de détermination des stades d’intermue et son application générale aux crustacés." Vie Et Milieu Serie A Biologie Marine 18: 595-609. de Faria, S. d., Augusto, A., McNamara, J., 2011. Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea. J. Comp. Physiol. B. 181, 175186. Faleiros, R.O., Goldman, M.H.S., Furriel R.P.M., McNamara, J.C., 2010. Differential adjustment in gill Na+/K+- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae). J. Exp. Biol. 213, 38943905.

17

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Gillikin, D.P., De Wachter, B., Tack, J.F., 2004. Physiological responses of two ecologically important Kenyan mangrove crabs exposed to altered salinity regimes. J. Exp. Mar. Biol. Ecol. 301, 93-10. Gonzalez-Ortegon, E., Pacual, E., Cuesta, J.A., Drake, P., 2006. Field distribution and osmoregulatory capacity of shrimps in a temperate European estuary (SW Spain). Estuar. Coast. Shelf. S. 67, 293-302. Grandjean, M.M. 1985. Efecto de la temperatura sobre el balance energético y el crecimiento en Hemigrapsus crenulatus (Milne-Edwards, 1837) bajo condiciones de laboratorio. Tesis de magíster. Universidad Austral de Chile, Valdivia, Chile. Greenaway, P. 1988. Ion and water balance. Biology of the Land crabs. W. W. Burggren and B. R. McMahon. New York, Cambridge University Press: 211-248. Greenaway, P. 1991. Nitrogenous excretion in aquatic and terrestrial crustaceans. Mem. Queensland. Mus. 31: 215-227. Guerin, J.L., Stickle, W.B., 1997. Effect of salinity on survival and bioenergetics of juvenile lesser blue crabs, Callinectes similis. Mar. Biol. 129, 63-69. Guerin, J.L., Stickle W.B., 1997. A comparative study of two sympatric species within the genus Callinectes: osmoregulation, long-term acclimation to salinity and the effects of salinity on growth and moulting. J. Exp. Mar. Biol. Ecol. 218, 165-186. Hulathduwa, Y., Stickle, W., Brown, K., 2007. The effect of salinity on survival, bioenergetics and predation risk in the mud crabs Panopeus simpsoni and Eurypanopeus depressus. Mar. Biol. 152, 363-370. Hicks, G.R.F., 1974. Combined effects of temperature and salinity on Hemigrapsus edwardsi (Hilgendorf) and H. crenulatus (Milne Edwards) from Wellington Harbour, New Zealand. J. Exp. Mar. Biol. Ecol. 13, 1-14. Iribarne, O., Bortolus, A., Botto, F., 1997. Between-habitat differences in burrow characteristics and trophic modes in the southwestern Atlantic burrowing crab Chasmagnathus granulata. Mar. Ecol. Prog. Ser. 155, 137-145. Kalle, K., 1971. Salinity. General Introduction. In: Marine Ecology, Vol. I (2), Chap. 4. O. Kinne (Ed.). John Wiley & Sons Ltd. The University Press, Glasgow. 683-995. Kinne, O., 1971. Salinity. Marine Ecology. A comprehensive, integrated treatise on life in oceans and coastal waters. O. Kinne. London, Wiley. 1: 683-1244. Koroleff, F., Grasshoff, K., 1983. Determination of nutrients. Determination of ammonia. In Grasshoff K., Ehrhardt M. and Kremling K. (eds) Methods for seawater analysis. Weinheim: Verlag Chemie GmbH, pp. 150–157. Larsen, E.H., Deaton, L., Onken, H., O´Donnell, M., Grosell, M., Dantzler, W.H., Weihrauch., D., 2014. Osmoregulation and Excretion. Compr. Physiol. 4, 405-573. Lucu, C., 1993. Ion transport in the gill epithelium of aquatic Crustacea. J. Exp. Zool. 265, 378-386. Lucu, C., Devescovi, M., 1999. Osmoregulation and branchial Na+, K+-ATPase in the lobster Homarus gammarus acclimated to dilute seawater. J. Exp. Mar. Biol. Ecol. 234: 291-304. Lucu, C., Towle, D.W., 2003. Na+/K+-ATPase in gills of aquatic crustacea. Comp. Biochem. Physiol. A. 135, 195-214. Lucu, C., Pavicic, J., Ivankovic, D., Pavicic-Hamer, D., Najdek, M., 2008. Changes in Na+/K+-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation. Comp. Biochem. Physiol. A. 149, 362-372. 18

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Luppi, T. A., Spivak, E.D., Anger, K., Valero, J.L., 2002. Patterns and processes of Chasmagnathus granulata and Cyrtograpsus angulatus (Brachyura: Grapsidae) recruitment in Mar Chiquita Coastal Lagoon, Argentina. Estuar. Coast. Shelf. S. 55, 287-297. Luquet, C.M., Cervino, C.O., Ansaldo, M., Carrera Pereyra, V., Kocmur, S., Dezi, R.E., 1998. Physiological response to emersion in the amphibious crab Chasmagnathus granulata Dana (Decapoda Grapsidae): biochemical and ventilatory adaptations. Comp. Biochem. Physiol. A. 121, 385-393. Masui, D. C., Furriel, R.P.M., McNamara, J.C., Mantelatto, F.L.M., Leone, F.A., 2002. Modulation by ammonium ions of gill microsomal (Na+, K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion. Comp. Biochem. Physiol. C. 132, 471-482. Masui, D.C., Mantelatto, F.L.M., McNamara, J.C., Furriel, R.P.M., Leone, F., 2009. Na+, K+-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion. Comp. Biochem. Physiol. A. 153, 141-148. Mayzaud, P., Conover, R.J., 1988. O:N atomic ratio as a tool to describe zooplankton metabolism. Mar. Ecol. Prog. Ser. 45, 289-302. McLay, C.L., Hinnendael, F., Lavery, S., Riquelme-Bugueño, R., 2011. Morphological and molecular comparison of Hemigrapsus crenulatus (milne edwards, 1837) (brachyura: varunidae) from New Zealand and Chile: was miss rathbun right?" J. Crustacean. Biol. 31, 582-589. McNamara, J.C., Faria, C.S., 2012. Evolution of osmoregulatory patterns and gill ion transport mechanisms in the decapod Crustacea: a review. J. Comp. Physiol. B. 182, 997-1014. Mu, Y.C., Wang, F., Dong, S.L., Huang, G.Q., Dong, S.S., 2005. Effects of salinity fluctuation pattern on growth and energy budget of juvenile shrimp Fenneropenaeus chinensis. J. Shellfish. Res. 24, 1217-1221. Newell, R. C. (1976). Adaptation to Environment: Essays on the Physiology of Marine Animals. London, Butterworths. Pardo, L. M., Gonzalez, K., Fuentes, J.P., Paschke, K., Chaparro, O., 2011. Survival and behavioral responses of juvenile crabs of Cancer edwardsii to severe hyposalinity events triggered by increased runoff at an estuarine nursery ground. J. Exp. Mar. Biol. Ecol. 404, 33-39. Paschke, K., Cumillaf, J., Loyola, S., Gebauer, P., Urbina, M., Chimal, M.A., Pascual, C., Rosas, C., 2010. Effect of dissolved oxygen level on respiratory metabolism, nutritional physiology, and immune condition of southern king crab Lithodes santolla (Molina, 1782) (Decapoda, Lithodidae). Mar. Biol. 157, 7-18. Péqueux, A., 1995. Osmotic regulation in crustaceans. J. Crust. Biol. 15, 1-60. Posey, M.H., Alphin, T.D., Harwell, H., Allen, B., 2005. Importance of low salinity areas for juvenile blue crabs, Callinectes sapidus Rathbun, in river-dominated estuaries of southeastern United States. J. Exp. Mar. Biol. Ecol. 319, 81-100. Pulgar, J., Aldana, M., Vergara, E., George-Nascimento, M., 1995. Behavior of the estuarine crab Hemigrapsus crenulatus (Milne-Edwards 1837) in relation to the parasitism by the acanthocephalan Profilicollis antarcticus (Zdzitowiecki 1985) in southern Chile. Rev. Chil. Hist. Nat. 68, 439-450. Quinn, G.P., Keough M.J., 2002. Experimental Design and Data Analysis for Biologists. 19

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Cambridge CB2 2RU, U.K., Cambridge University Press. Retamal, M.A., 1981. Catalogo ilustrado de los Crustáceos Decápodos de Chile. Gayana. Zoología. Universidad de Concepción, Chile. 44, 1-110. Retamal, M.A., Moyano, H.I., 2010. Zoogeografía de los crustáceos decápodos chilenos marinos y dulceacuícolas. Lat. Am. J. Aquat. Res. 38, 302 - 328. Riquelme-Bugueño, R., 2006. Incidence patterns of limb autotomy in the estuarine crab, Hemigrapsus crenulatus (H. Milne Edwards, 1837) (Brachyura, Grapsoidea) from a temperate estuary in the eastern South Pacific. Crustaceana. 79, 925-932. Romano, N., Zeng C., 2006. The effects of salinity on the survival, growth and haemolymph osmolality of early juvenile blue swimmer crabs, Portunus pelagicus. Aquaculture 260, 151-162. Rosas, C., Martínez, E., Gaxiola, G., Brito, R., Sanchez, A., Soto, L.A., 1999. The effect of dissolved oxygen and salinity on oxygen consumption, ammonia excretion and osmotic pressure of Penaeus setiferus (Linnaeus) juveniles. J. Exp. Mar. Biol. Ecol. 234, 41-57. Rosas, C., Cuzon, G., Gaxiola, G., Pascual, C., Taboada, G., Arena, L., van Wormhoudt, A., 2002. An energetic and conceptual model of the physiological role of dietary carbohydrates and salinity on Litopenaeus vannamei juveniles. J. Exp. Mar. Biol. Ecol. 268, 47-67. Schmidt-Nielsen, K., 1997. Animal Physiology: adaptation and environment. Cambridge, UK, Cambridge University Press. Schubart, C.D., Diesel, R., Hedges, S.B., 1998. Rapid evolution to terrestrial life in Jamaican crabs. Nature. 393, 363-365. Schubart, C.D., Diesel, R., 1999. Osmoregulation and the transition from marine to freshwater and terrestrial life: a comparative study of Jamaican crabs of the genus Sesarma. Arch. Hydrobiol. 145, 331-347. Schubart, C.D., Cuesta, J.A., Diesel, R., Felder, D.L., 2000. Molecular phylogeny, taxonomy, and evolution of nonmarine lineages within the American grapsoid crabs (Crustacea: Brachyura). Mol. Phylogenet. Evol. 15, 179-190. Seneviratna, D., Taylor H.H., 2006. Ontogeny of osmoregulation in embryos of intertidal crabs (Hemigrapsus sexdentatus and H. crenulatus, Grapsidae, Brachyura): putative involvement of the embryonic dorsal organ. J. Exp. Biol. 209, 1487-1501. Shinji, J., Strussmann, C.A., Wilder, M.N., Watanabe, S., 2009. Short-term responses of the adults of the common Japanese intertidal crab, Hemigrapsus takanoi (Decapoda: Brachyura: Grapsoidea) at different salinities: osmoregulation, oxygen consumption, and ammonia excretion. J. Crustacean. Biol. 29, 269-272. Siebers, D., Winkle, A., Leweck, K., Madian, A., 1983. Regulation of sodium in the shore crab Carcinus maenas, adapted to environments of constant and changing salinities. Helgolander Meeresun. 36, 303-312. Skinner, D. M. (1962). "The structure and metabolism of a crustacean integumentary tissue during a molt cycle." Biol. Bull. mar. biol. Lab. Woods Hole 123, 635-647. Skinner, D. M. (1985). "Interacting factors in the control of the Crustacean molt cycle." Amer. Zool. 25, 275-284. Sokal, R.R., Rohlf, F.J., 1995. Biometry. The principles and practice of statistics in Biological Research. San Francisco, Freeman. Stickle, W.B., Wyler, H.J., Dietz, T.H., 2007. Effects of salinity on the juvenile crab physiology and agonistic interactions between two species of blue crabs, Callinectes 20

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

sapidus and C. similis from coastal Louisiana. J. Exp. Mar. Biol. Ecol. 352, 361-370. Taylor, H.H., Seneviratna D., 2005. Ontogeny of salinity tolerance and hyperosmoregulation by embryos of the intertidal crabs Hemigrapsus edwardsii and Hemigrapsus crenulatus (Decapoda, Grapsidae): Survival of acute hyposaline exposure. Comp. Biochem. Phys. A. 140, 495-505. Thurman, C.L., 2003. Osmoregulation by six species of fiddler crabs (Uca) from the Mississippi delta area in the northern Gulf of Mexico. J. Exp. Mar. Biol. Ecol. 291, 233-253. Thurman, C.L., 2005. A comparison of osmoregulation among subtropical fiddler crabs (Uca) from southern Florida and California. B. Mar. Sci. 77, 83-100. Thurman, C., Hanna, J., Bennett, C., 2010. Ecophenotypic physiology: osmoregulation by fiddler crabs (Uca spp.) From the northern caribbean in relation to ecological distribution. Mar. Freshw. Behav. Phy. 43, 339-356. Torres, G., Charmantier-Daures, M., Chifflet, S., Anger, K., 2007. Effects of long-term exposure to different salinities on the location and activity of Na+-K+-ATPase in the gills of juvenile mitten crab, Eriocheir sinensis. Comp. Biochem. Phys. A. 147, 460465. Towle, D.W., Kays, W.T., 1986. Basolateral localization of Na+ + K+-ATPase in gill epithelium of two osmoregulating crabs, Callinectes sapidus and Carcinus maenas. J. Exp. Zool. 239, 311-318. Tsai, J.R., Lin, H.C., 2007. V-type H+-ATPase and Na+, K+-ATPase in the gills of 13 euryhaline crabs during salinity acclimation. J. Exp. Biol. 210, 620-627. Urbina, M., Paschke, K., Gebauer, P., Chaparro, O.R., 2010. Physiological energetics of the estuarine crab Hemigrapsus crenulatus (Crustacea: Decapoda: Varunidae): responses to different salinity levels. J. Mar. Biol. Assoc. UK. 90, 267-273. Urbina M.A., Schulte P.M., Bystriansky, J., Glover C.N., 2013. Differential expression of Na+, K+-ATPase a -1 isoforms during seawater acclimation in the amphidromous galaxiid fish Galaxias maculatus. J. Comp. Physiol. B. 183, 345-357. Urbina, M.A., Glover C.N., 2015. Effect of salinity on osmoregulation, metabolism and nitrogen excretion in the amphidromous fish, inanga (Galaxias maculatus). J. Exp. Mar. Biol. Ecol. 473, 7–15. Urzúa, Á., 2005. Efecto de la salinidad sobre la osmorregulación y bioenergética en Hemigrapsus crenulatus (Milne-Edwards, 1837) (Decapoda: Grapsidae). Diplom Thesis. Universidad Católica de la Santísima Concepción. Chile. Wang, X.G., Ma, S., Dong, S.L., Cao, M., 2004. Effects of salinity and dietary carbohydrate levels on growth and energy budget of juvenile Litopenaeus vannamei. J. Shellfish. Res. 23, 231-236. Weihrauch, D., Ziegler, A., Siebers, D., Towle, D.W., 2001. Molecular characterization of V-type H+-ATPase (B-subunit) in gills of euryhaline crabs and its physiological role in osmoregulatory ion uptake. J. Exp. Biol. 204, 25-37. Weihrauch, W., Ziegler, A., Siebers, D., Towle, D.W., 2002. Active ammonia excretion across the gills of the green shore crab Carcinus maenas: participation of Na+/K+ATPase, V-type H+ ATPase and functional microtubules. J. Exp. Biol. 205, 27652775. Weihrauch, D., Morris, S., Towle, D.W., 2004. Ammonia excretion in aquatic and terrestrial crabs. J. Exp. Biol. 207, 4491-4504. Weihrauch, D., McNamara, J.C., Towle, D.W., Onken, H., 2004. Ion-motive ATPases and 21

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

active, transbranchial NaCl uptake in the red freshwater crab, Dilocarcinus pagei (Decapoda, Trichodactylidae). J. Exp. Biol. 207, 4623-4631. Westermeier, R., Gomez, I., Rivera, P., 1993. Suspended farming of Gracilaria chilensis (Rhodophyta, Gigartinales) at Cariquilda River, Maullín, Chile. Aquaculture. 113, 215–229. Widdows, J., 1985. Physiological procedures. In: Bayne, B.C., Brown, D.A., Burns, K., Ivanovici, A., Livingtone, D.R., Lowe, D.M., Moore, M.N., Steabbing, A.R.D., Widdows, J. (Eds.), The Effects of Stress and Pollution on Marine Animals. Praeger Publishers, New York, pp. 161–178. Willmer, P., Stone, G., Johnston, I., 2000. Environmental physiology of animals. 1st edition. Oxford, UK: Blackwell Science Ltd. Ye, L., Jiang, S., Zhu, X., Yang, Q., Wen, W., Wu, K., 2009. Effects of salinity on growth and energy budget of juvenile Penaeus monodon. Aquaculture 290, 140-144.

22

ACCEPTED MANUSCRIPT Legends of figures and tables Fig 1. Ecophysiological responses of different sizes of Hemigrapsus crenulatus to different

PT

salinities, A) oxygen consumption and B) excretion rate at different salinities. Mean values

RI

± SD, for regression analyses see Table 1.

Fig 2. Ionoregulatory responses at different salinities of Hemigrapsus crenulatus at

SC

different sizes, A) Na+ in the haemolymph and the external medium; B) regulatory capacity

NU

of sodium. Mean values ± SD, for regression analyses see Table 1. Fig 3. O:N ratio calculated for different sizes of Hemigrapsus crenulatus at different

MA

external salinities. Mean values ± SD, for regression analyses see Table 1. Fig 4. Hemigrapsus crenulatus, different sizes. Physiological relationships: A) oxygen

D

consumption vs. regulatory capacity of sodium (RC-Na+); B) excretion rate vs. RC-Na;

TE

Mean values ± SD, for regression analyses see Table 1. Table 1. Hemigrapsus crenulatus, different sizes. Regression analyses of physiological

AC CE P

parameters at different salinities: R = respiration, E = excretion, Na+ = sodium in haemolymph, RC-Na+ = regulatory capacity of sodium; y0 = intercept; a, b, c = slopes; r2 = determination coefficient; p = significance level; nd = no data available. Table 2. Hemigrapsus crenulatus, different sizes. Test for equality slopes of regression analyses of physiological parameters. Asterisks denote statistically different slopes (a, b, c, see table 1).

23

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Fig 1.

24

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Fig 2.

25

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Fig 3.

26

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Fig 4.

27

ACCEPTED MANUSCRIPT Table 1

Na+

PT

RI

MA

(RC-Na+)

y0 a b nd 0.588 -0.0172* nd 0.981 -0.0257* nd 1.668 -0.0410* nd 0.00601 -0.000182* nd 0.0108 -0.000302* nd 0.0154 -0.000397* 154.766 31.925* -1.794* 230.870 27.937* -1.818 239.910 38.416* -2.569* 32.361 18.314* -1.470* 99.799 16.639* -1.646* 117.505 24.805* -2.245* nd 64.198 -0.8614* nd 82.729 -1.1895* nd 100.086 -1.4178* nd 0.092 0.386* nd 0.211 0.379* nd 0.290 0.535* nd 0.0804 0.404* nd 0.0967 0.516* nd 0.255 0.483*

SC

E

Size I II III I II III I II III I II III I II III I II III I II III

NU

Physiological parameters R

D

O:N ratio

AC CE P

E vs. RC-Na+

TE

R vs. RC-Na+

28

c nd nd nd nd nd nd 0.0356* 0.0388 0.0527* 0.0282* 0.0343 0.0454* nd nd nd nd nd nd nd nd nd

2

r 0.853 0.848 0.887 0.853 0.881 0.915 0.904 0.623 0.767 0.837 0.689 0.821 0.856 0.906 0.9126 0.795 0.694 0.813 0.751 0.796 0.845

p <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.002 <0.001 <0.001 <0.001 <0.001 0.005 0.0021 0.002 <0.001 <0.001 <0.001 <0.0001 <0.001 <0.001

ACCEPTED MANUSCRIPT

MS

F

p

5 2 2 49

0.826 2.349 0.0541 0.00628

131.549 374.064 8.620

PT

<0.001 <0.001 <0.001

5 2 2 46

0.846 1.747 0.0425 0.00639

132.336 273.185 6.644

<0.001 <0.001 <0.001

5 2 2 44

15.570 7.346 2.391 0.484

32.202 15.178 4.940

<0.001 <0.001 <0.01

5 2 2 44

11.863 7.352 2.898 0.484

24.514 15.193 5.987

<0.001 <0.001 <0.01

D

TE

AC CE P

NU

RI

df

MA

Physiological parameters R Salinity Size Among regressions Error E Salinity Size Among regressions Error + Na Salinity Size Among regressions Error RC-Na+ Salinity Size Among regressions Error O:N Salinity Size Among regressions Error R RC-Na+ Size Among regressions Error E RC-Na+ Size Among regressions Error

SC

Table 2

5 1684.102 844.944 <0.001 2 3140.781 1575.786 <0.001 2 15.230 7.641 <0.001 49 1.993 5 2 2 47

1.122 0.503 0.308 0.00418

268.421 <0.001 120.334 <0.001 74.684 <0.001

5 2 2 47

2.288 2.472 0.430 0.0202

113.267 <0.001 122.376 <0.001 21.287 0.007

29