Temperature-dependent activity in early life stages of the stone crab Paralomis granulosa (Decapoda, Anomura, Lithodidae): A role for ionic and magnesium regulation?

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Abstract

Marine brachyuran and anomuran crustaceans are completely absent from the extremely cold (− 1.8 °C) Antarctic continental shelf, but caridean shrimps are abundant. This has at least partly been attributed to low capacities for magnesium excretion in brachyuran and anomuran lithodid crabs ([Mg2+]HL = 20–50 mmol L 1) compared to caridean shrimp species ([Mg2+]HL = 5–12 mmol L 1). Magnesium has an anaesthetizing effect and reduces cold tolerance and activity of adult brachyuran crabs. We investigated whether the capacity for magnesium regulation is a factor that influences temperature-dependent activity of early ontogenetic stages of the Sub-Antarctic lithodid crab Paralomis granulosa. Ion composition (Na+, Mg2+, Ca2+, Cl, SO42−) was measured in haemolymph withdrawn from larval stages, the first and second juvenile instars (crabs I and II) and adult males and females. Magnesium excretion improved during ontogeny, but haemolymph sulphate concentration was lowest in the zoeal stages. Neither haemolymph magnesium concentrations nor Ca2+:Mg2+ ratios paralleled activity levels of the life stages. Long-term (3 week) cold exposure of crab I to 1 °C caused a significant rise of haemolymph sulphate concentration and a decrease in magnesium and calcium concentrations compared to control temperature (9 °C). Spontaneous swimming activity of the zoeal stages was determined at 1, 4 and 9 °C in natural sea water (NSW, [Mg2+] = 51 mmol L 1) and in sea water enriched with magnesium (NSW + Mg2+, [Mg2+] = 97 mmol L 1). It declined significantly with temperature but only insignificantly with increased magnesium concentration. Spontaneous velocities were low, reflecting the demersal life style of the zoeae. Heart rate, scaphognathite beat rate and forced swimming activity (maxilliped beat rate, zoea I) or antennule beat rate (crab I) were investigated in response to acute temperature change (9, 6, 3, 1, − 1 °C) in NSW or NSW + Mg2+. High magnesium concentration reduced heart rates in both stages. The temperature–frequency curve of the maxilliped beat (maximum: 9.6 beats s 1 at 6.6 °C in NSW) of zoea I was depressed and shifted towards warmer temperatures by 2 °C in NSW + Mg2+, but antennule beat rate of crab I was not affected. Magnesium may therefore influence cold tolerance of highly active larvae, but it remains questionable whether the slow-moving lithodid crabs with demersal larvae would benefit from an enhanced magnesium excretion in nature.

Research Highlights

► The role of magnesium regulation in cold tolerance of Sub-Antarctic decapod crustaceans. ► Haemolymph inorganic ion composition changes during ontogeny of Paralomis granulosa. ► Cold exposure affects haemolymph ion composition of juvenile P. granulosa. ► Magnesium does not affect spontaneous swimming speed of zoeal stages. ► Cold tolerance of forced activity in zoea I is impaired by elevated magnesium concentration.

Introduction

Marine decapod crustacean diversity is low in Antarctic compared to Sub-Antarctic regions (Gorny, 1999). Over 130 benthic and pelagic decapod species occur in the Southern Ocean, but only 27 species are present south of the Polar Frontal Zone (PFZ). Brachyuran crabs are completely absent, whereas at least 9 species of the anomuran family Lithodidae have been found south of the PFZ (Gorny, 1999, García Raso et al., 2005, Thatje et al., 2005). Anomuran and brachyuran crabs still inhabited nearshore habitats of Antarctica in the late Eocene, but climatic cooling during the Miocene likely caused their extinction in this area (Feldmann and Zinsmeister, 1984a, Feldmann and Zinsmeister, 1984b, Gorny, 1999, Feldmann and Schweitzer, 2006, Aronson et al., 2007 and references therein). Today, brachyuran crabs occur in the warmer waters of the Sub-Antarctic (Gorny, 1999). The biogeography of lithodid crabs is probably constrained by low temperature as well, because this group has only been found in waters warmer than 0 °C (Hall and Thatje, 2009) with their southernmost habitat being the continental slope of the western Antarctic Peninsula in the Bellingshausen Sea (García Raso et al., 2005, Thatje et al., 2008). In contrast, caridean decapods tolerate temperatures as low as − 1.8 °C, and are frequently observed in shallower waters of the continental shelf of Antarctica (Gutt et al., 1991).

Besides temperature, the capability to regulate extracellular magnesium concentration below the concentration found in sea water (50 mmol L 1) has been proposed to be a factor to influence the biogeography of decapod crustaceans in the Southern Ocean (Sartoris et al., 1997, Frederich, 1999, Frederich et al., 2000a, Frederich et al., 2000b, Frederich et al., 2001). Sub-Antarctic brachyuran and lithodid crab species exhibit high (20–50 mmol L 1), Antarctic caridean decapods low (5–12 mmol L 1) haemolymph magnesium concentrations (Frederich, 1999, Frederich et al., 2000b, Wittmann et al., 2010). Increased magnesium concentration has a paralysing effect on vertebrates and invertebrates (Katz, 1936, Waterman, 1941, Pantin, 1948, Iseri and French, 1984, Lee et al., 1996). In crustaceans, this is caused by the action of the ion at least at two sites, which both are related to the block of calcium channels. Magnesium slows down neuromuscular transmission by reducing transmitter release of synapses (Parnas et al., 1994) and it reduces the contraction of muscle fibres as calcium influx is blocked at the postsynaptic membrane (Hagiwara and Takahashi, 1967, Ushio et al., 1993). Early studies on decapod nerve–muscle preparations showed that a 1.5- to 2-fold increase of external magnesium concentration above natural conditions weakened the response after stimulation, whereas a reduction of the magnesium concentration enhanced the facilitation of neuromuscular transmission and submaximal tensions of the locomotory musculature (Katz, 1936, Waterman, 1941, Boardman and Collier, 1946). Robertson, 1949, Robertson, 1953 was the first to note, that there may be an inverse relationship between the haemolymph magnesium concentration and the general activity level of a decapod crustacean species. Furthermore, he suggested that not just extracellular magnesium concentration, but also calcium concentration may play a role in determining the activity of decapods due to the antagonistic effects of these ions (Robertson, 1949, Robertson, 1953, Iseri and French, 1984). Because of this, Robertson, 1949, Robertson, 1953 compared Ca2+:Mg2+ ratios interspecifically: subjectively inactive species (e.g. of the genera Lithodes and Maja) exhibited low (0.19–0.31), more active species (e.g. Cancer, Palinurus) high (0.57–2.0) values. Several studies on decapods and amphipods supported this hypothesis quantifying activity by measuring heart rate, oxygen consumption and locomotory activity (Walters and Uglow, 1981; see review by Morritt and Spicer, 1993, Spicer et al., 1994, Sartoris et al., 1997, Watt et al., 1999).

Most rates of locomotory activity as well as metabolic and developmental rates are slower in polar than in temperate species with similar ecological function. These processes thus display little or no compensation for temperature effects (Young et al., 2006, Barnes and Peck, 2008). At the neurophysiological level, low temperature, similar to magnesium, reduces the amount of transmitter released in crayfish axons, which is thought to be the result of declined calcium influx through calcium channels (Dunn and Mercier, 2003). Low temperature and high magnesium concentrations may therefore interact to diminish locomotory activity and affect the entire cardiovascular system of decapods. Referring to the concept of oxygen- and capacity-limited thermal tolerance in crustaceans (Frederich and Pörtner, 2000, Pörtner, 2002), anomuran and brachyuran crab species possessing high haemolymph magnesium concentrations may be constrained in their distribution to the subpolar temperature regime (Frederich et al., 2000a, Frederich et al., 2000b, Frederich et al., 2001). Vice versa, experimental reduction of haemolymph magnesium concentration in adult brachyuran crabs down to the level of caridean shrimp species led to an increase in oxygen consumption, walking and righting activity and cardiac output, extending the thermal tolerance window of these crabs towards lower temperatures and even below 0 °C (Frederich et al., 2000a, Frederich et al., 2000b).

Larval stages are thought to be more sensitive than the adults, therefore the thermal tolerance of larvae should be a crucial factor determining the biogeography of a species (Frederich, 1999, Anger, 2001, Anger et al., 2003, Thatje et al., 2003, Hall and Thatje, 2009, Storch et al., 2009). Hardly any data exist on thermal tolerance of adult and larval lithodid crabs from the Southern Ocean even though this group occurs farther south than the brachyurans. Furthermore, it is not clear whether extracellular ion regulation influences thermal tolerance and whether and to what extent the capacity for ion regulation changes during ontogeny in species of the family Lithodidae.

Temperature variations affect the regulation of ions, but the magnitude of the effect may depend on the season, exposure time and the ion species considered (Mantel and Farmer, 1983, Burton, 1986, Pequeux, 1995, Charmantier et al., 2009). Both passive (permeability of the epithelio-cuticular complex) and active components (ion transport) could be affected, but so far there is no consistent view on how temperature acts and which mechanisms account for the effects. In adult amphipods and caridean decapods, the haemolymph concentrations of magnesium rise once the animals are exposed to temperatures far below their acclimation temperature for up to one week (Campbell and Jones, 1989, Tentori and Lockwood, 1990, Spicer et al., 1994, Sartoris and Pörtner, 1997). We may therefore hypothesize that low temperature impairs extracellular regulation of this ion, which results in an increase in haemolymph magnesium concentration and subsequent anaesthesia of the animals.

Ontogenetic changes of osmoregulation in decapod crustaceans are well documented (Charmantier, 1998, Charmantier et al., 2009), but only few data exist on the development of the capacity to regulate single ion species (Brown and Terwilliger, 1992, Newton and Potts, 1993). It is known mostly from work on adults, that ion transport and excretion takes place through specialized epithelia in antennal glands, the gut including the hepatopancreas, and the branchial cavity including the gills (Mantel and Farmer, 1983, Gerencser et al., 2001, Freire et al., 2008). These epithelia are rich in the enzyme Na+/K+–ATPase, which generates at least part of the driving force for transepithelial ion transport (Lucu and Towle, 2003). Regulation of haemolymph sodium, chloride and calcium levels may take place through uptake and extrusion via the gill epithelium. Antennal glands are involved in magnesium and sulphate excretion via urine production as well as in the reabsorption of calcium and potassium (Freire et al., 2008). Sulphate excretion and calcium storage during the moulting cycle is facilitated by the hepatopancreas (Ahearn, 1996, Gerencser et al., 2001).

In several decapod species, the development of ion regulatory sites during life history, by use of Na+/K+–ATPase as a marker, has been shown to correlate with different osmoregulatory capacities of larval, juvenile and adult stages (Felder et al., 1986, Lignot and Charmantier, 2001, Cieluch et al., 2004, Cieluch et al., 2005, Khodabandeh et al., 2005, Khodabandeh et al., 2006). Osmoregulatory capacity does not necessarily improve linearly with stage, but matches environmental conditions, which may be very different for each stage if life history is spent in a variety of habitats (marine, estuarine, limnic or semi-terrestrial, Cieluch et al., 2004, Anger et al., 2008). Similarly, it can be hypothesized that the lifestyle of a given stage is linked to the regulation of single ion species. Planktonic zoeal stages may benefit from the specific regulation of magnesium and calcium as these ions might influence swimming activity, and from the regulation of sulphate, which influences buoyancy (Newton and Potts, 1993). In agreement with Robertson's rationale, highly active zoeal stages should exhibit low and the settling, predominantly benthic megalopa elevated haemolymph magnesium concentrations. Juveniles should have lower magnesium concentrations than the adults because they can be expected to be more agile than the adults as a result of their higher metabolic rates and associated food requirements in relation to body weight. A prerequisite for this is, however, that the larvae already possess functional tissues for the regulation of these ions. Measurements of haemolymph ion concentrations in the lobster Homarus gammarus (Newton and Potts, 1993) and Cancer magister (Brown and Terwilliger, 1992) suggest that the capacity for magnesium and sulphate excretion increases during the life history as the antennal glands differentiate progressively (Khodabandeh et al., 2006).

The key objectives of this study were to identify ontogenetic and temperature-dependent changes in the capacity for extracellular ion regulation in the Sub-Antarctic lithodid crab Paralomis granulosa. Furthermore, we quantified larval activity in relation to the haemolymph magnesium concentration and investigated whether it is possible to shift the thermal limits of larvae and juveniles by experimentally altering the extracellular magnesium concentration. It was not feasible to raise the larvae in artificial sea water, therefore only the effects of an increase in magnesium concentration were tested. These investigations intended to elucidate the question whether the capacity for magnesium regulation influences thermal tolerance of the larvae and juveniles and thereby the biogeography of lithodid crabs. As a model organism we chose the Sub-Antarctic lithodid crab P. granulosa. This species occurs in the Magellanic province, which extends from the north of Chiloé Island in the Eastern Pacific (42°S) to the southern tip of South America, including the Falkland islands, and up to the South-western Atlantic to about 35°S (but here into deeper waters, see Boschi, 1979, Boschi, 2000). Around the tip of South America and at the Falkland Islands the animals encounter water temperatures in summer of 9–11 °C and in winter of 2–4 °C (Hoggarth, 1993, Lovrich and Vinuesa, 1993, Arntz et al., 1999, Arkhipkin et al., 2004). Larval development is fully lecithotrophic and highly abbreviated consisting of only two demersal zoeal stages and a megalopa stage (Campodonico and Guzman, 1981). The development of P. granulosa is completed in a relatively wide thermal range of 3–15 °C with highest survival rates at 6 and 9 °C; the species has therefore been classified as cold-eurythermal (animals from the Beagle Channel, Anger et al., 2003).

We present the first study of haemolymph sodium, magnesium, calcium, chloride and sulphate levels throughout ontogeny in an anomuran crab in relation to activity and thermal tolerance in its early stages. We discuss the results in the context of the hypothesis that extracellular magnesium regulation co-determines the biogeography of decapod crustaceans in the Southern Ocean.

Section snippets

Animals

Male and ovigerous female P. granulosa were obtained from local fishermen in Punta Arenas, Chile in April 2008. The animals were transported to the Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany (AWI) on board RV Polarstern (ANT-XXIV/4) and thereafter kept in a recirculated aquarium system at 4 °C, 32.5 PSU and an artificial 12:12 h light:dark cycle. Two females were transferred to the biological laboratory on Helgoland, Germany (BAH) in June and placed in individual

Ontogeny of haemolymph ion composition

Significant hyporegulation of haemolymph magnesium compared to the sea water magnesium fraction (4.7 ± 0.1% of the sum, 51 ± 1 mmol L 1, Table 1) was observed in the megalopa stage (4.1 ± 0.2% of the sum, Fig. 1). The megalopa exhibited a similar magnesium fraction as the first juvenile stage and adult male specimens (4.0 ± 0.2% of the sum, 40 ± 2 mmol L 1). The second juvenile instar and the ovigerous females displayed the lowest haemolymph magnesium fractions of 3.7 ± 0.1% of the sum and 3.6 ± 0.2% of the sum

Ontogeny of haemolymph ion regulation

Haemolymph magnesium fractions decreased during larval development of P. granulosa. This implies that the active magnesium hyporegulation improved successively. The high haemolymph magnesium fractions in the zoeal stages may indicate that the antennal gland was not yet entirely functional during early ontogeny (Khodabandeh et al., 2006). The megalopa stage already exhibited the same capacity for magnesium extrusion as the first juvenile stage and adult males. This suggests that the antennal

Acknowledgements

We greatly appreciate the help of the crew of RV Polarstern and Fredy Véliz Moraleda during the transport of live animals. We would like to thank Uwe Nettelmann and Dana Pargmann for their assistance with culturing larvae and juveniles and Charlyn Völker and Marc Bullwinkel for their help with the analysis of the videos. The experiments comply with the current laws of the country in which they were performed. This study was supported by Deutsche Forschungsgemeinschaft grants no. SA 1713/1–1 and

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