some considerations on calcium homeostasis in semi-terrestrial crabs

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OOFSome considerations on calcium homeostasis in
semi-terrestrial crabs

F.P. Zanottoa,*, F. Pinheiroa, L.A. Britoa, M.G. Wheatlyb

aUniversity of Sao Paulo, Department of Physiology, Sao Paulo, SP, BrazilbWright State University, Biological Sciences Department, Dayton, OH, USA

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RAbstract. Calcium (Ca), deposited as CaCO3 after molting events take place, indicates that Ca levels

in semi-terrestrial crabs should be regulated at the dietary level as well as through whole animal net

Ca flux. Two semi-terrestrial crabs found in salt marsh environments (Sesarma rectum and

Chasmagnathus granulata) were fed purified diets with variable Ca concentrations (0%, 2.22% and

6.66% Ca). Both animals displayed similar feeding strategies through consumption of higher

amounts of the diet containing more Ca (6.66% Ca). Sesarma, a predominantly herbivore crab, ate

more quantities of all the diets offered when compared to Chasmagnathus, a more carnivorous crab.

Whole animal net fluxes of Ca over 5 days in Sesarma show that fluxes vary from 2.5 to 1.5 mmol

kg�1 h�1, similar to values found earlier for a crayfish, Procambarus clarkii. However, the fluxes

over 5 days did not decrease significantly compared to fluxes for the first day postmolt. Overall,

semi-terrestrial crabs seem to maximize Ca influx through the gills and through dietary intake, a

mineral that is known to have great importance for biomineralization in crustaceans. D 2004

Published by Elsevier B.V.

Keywords: Calcium net flux; Dietary calcium; Calcium balance; Semi-terrestrial crabs

1. Introduction

Calcium (Ca) is of primordial importance for crustaceans because calcium carbonate

crystals (CaCO3) must be deposited in the new exoskeleton to harden it after the old

exoskeleton has been shed. Critical periods for mineral intake occur during molting

0531-5131/ D

doi:10.1016/j.

* Corresp

Consolacao 93

E-mail add

International Congress Series xx (2004) xxx–xxx

2004 Published by Elsevier B.V.

ics.2004.09.018

onding author. Present address: Universidade Presbiteriana Mackenzie, FCBEE, Rua da

0, 01302-907, Sao Paulo, SP, Brazil. Tel.: +55 11 3236 8145; fax: +55 11 3758 9803.

ress: [email protected] (F.P. Zanotto).

ICS-04635; No of Pages 7

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F.P. Zanotto et al. / International Congress Series xx (2004) xxx–xxx2

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related events, when crabs shed their old exoskeleton and calcify the new one during

postmolt. Whole body Ca uptake in freshwater crayfish, for example, occurs mainly

through the gills and changes from intermolt (zero net flux) to premolt (net efflux) and

postmolt (net influx at the rate of 2 mmol kg�1 h�1) [1,2]. Terrestrial crabs, on the other

hand, should have evolved mechanisms for calcium regulation through the gills but

additionally through dietary intake, and these regulatory mechanisms should be evident

during the whole life cycle of these animals. Therefore, dietary intake of calcium should

be especially important for terrestrial crabs, and it is already known that they display

different strategies to conserve ions such as storage in the hemolymph and in the

digestive system [3].

Specific work in the literature on crustacean nutrition using purified diets has been

focused mainly on shrimp, due to their commercial economic importance as human

food [4–8] (see Ref. [9]). Work on nutritional requirements of brachyuran crabs has

been neglected because of their slow growth rate, cannibalism and low meat/

exoskeleton ratio, characteristics which do not encourage their economic exploitation.

A few crabs from commercially important families such as Portunidae, Xanthidae and

Cancridae have been studied at semi-intensive conditions, fed, however, with natural

diets.

The focus of this study is to present an integrative view of calcium homeostasis in two

semi-terrestrial crabs, Sesarma rectum and Chasmagnathus granulata, by measuring

whole animal Ca net flux during postmolt as well as through the study of the effects of

dietary Ca changes in purified diets offered to the animals.

2. Methods and results

2.1. Whole animal Ca flux during postmolt

To study the net influx of Ca 2+ after ecdysis took place, Ca 2+ net flux was measured in

S. rectum, a mangrove crab from Brazil, for 5 days after ecdysis. The animals were unfed

during the whole period. The net flux (Jca) was measured in Amol kg�1 h�1, using the

following equation:

Jca ¼ initialCa2þ concentration lmolð Þ

� finalCa2þ concentration lmolð Þvolume llð Þmass kgð Þelapsed time hð Þ

The artificial brackish water where the crabs were exposed contained the following

salts (in g L�1): NaCl 10.7; NaHCO3 0.1; NaBr (anhydrous) 0.03; Na2SO4 (anhydrous)

1.8; KCl 0.34; MgCl2d 6H2O 4.84 and CaCl2 (anhydrous) 0.51. Total Ca concentration in

the water was around 5 mM.

Ca2+ net flux in S. rectum, a Brazilian crab, was quantitatively very similar to flux

found in the crayfish Procambarus clarkii, a freshwater crustacean, at values around 2000

Amol kg�1 h�1 [1] (Fig. 1). The flux, however, did not decrease significantly over 5 days

after ecdysis (repeated-measures ANOVA, Pb0.05). This fact raises the question as to

what extent the uptake of Ca through the gills of semi-terrestrial crabs is an important

variable for Ca balance in these animals. Semi-terrestrial crabs usually undergo ecdysis in

OF

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Fig. 1. Ca 2+ net flux (Amol kg h�1) in P. clarkii and S. rectum for 5 days after ecdysis. Bars represent mean

values. The lower bars represent values of Ca2+ flux for intermolt animals.

t1.1t1.2

t1.3

t1.4t1.5t1.6t1.7t1.8t1.9t1.10t1.11t1.12t1.13t1.14t1.15t1.16

t1.17

t1.18

t1.19

F.P. Zanotto et al. / International Congress Series xx (2004) xxx–xxx 3

ED PROburrows, where ground water is available. Therefore, these animals still need to have

contact with water sources during the postmolt period. Another point to consider is that the

uptake of Ca in these crabs was qualitatively different from crayfish, showing a smaller

decrease as time after ecdysis went on. Perhaps these animals, unable to feed during the

experimental period, were relying on Ca uptake mainly through the gills. Moreover, semi-

terrestrial crabs are heavily calcified compared to their aquatic counterparts and could

partially explain why total Ca uptake over 5 days was apparently higher for the semi-

terrestrial crab studied here.

UNCORRECTTable 1

Diet composition offered to the crabs for 11 days

Ingredients Diet A Diet B Diet C

Casein 20.5 g 20.5 g 20.5 g

Peptone 5 g 5 g 5 g

Sucrose 5 g 5 g 5 g

Starch 11 g 11 g 11 g

Salt mixturea 3 g 3 g 3 g

Vitaminsb 0.1 g 0.1 g 0.1 g

Vitamin C 0.2 g 0.2 g 0.2 g

Betain 0.8 g 0.8 g 0.8 g

Corn oil 0.0019 L 0.0019 L 0.0019 L

Cod oil 0.0019 L 0.0019 L 0.0019 L

Cholesterol 0.5 g 0.5 g 0.5 g

CaCl2 0 g 1.11 g 3.33 g

Total 49.9 g 51.01 g 54.34 g

Diets A, B and C differed only in relation to Ca content. Diet A contained 0% Ca, Diet B 2.22% Ca and Diet C

6.66% Ca.a Calcium free salt mixture (ICN): KPO4: 52.81%; NaPO4: 10.31%; MgSO4d 7H20: 8.19%; NaCl: 23.13%;

iron citrate: 4.5%; KI: 0.13%; MgSO4d 1H20: 0.74%; ZnCl: 0.08%; CuSO4d 5H20: 0.05%; sodium selenite:

0.001% and chromium potassium sulfate: 0.06%.b AIN Vitamin Mixture 76 (ICN)—kg of mixture. Thiamine hydrochloride: 600 mg; riboflavin: 600 mg;

pyridoxine hydochloride: 700 mg; nicotinic acid: 3 g; d-calcium pantothenate: 1.6 g; folic acid: 200 mg; d-biotin:

20 mg; vitamin B12: 1 mg; vitamin A: 1.6 g (250,000 UI/g); ld-tocopherol acetate: 20 g (250 UI/g); vitamin D3:

250 mg (400,000 UI/g); vitamin K2: 5 mg and sucrose 972.9 g.

ROOF

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Fig. 2. Mean diet consumption (mg g�1) for S. rectum for every 2 days (total of 11 days), fed purified diets which

contained three different Ca concentrations (0%, 2.22% and 6.66% Ca). There was a higher consumption for diets

containing 6.66% Ca compared to the other diets (repeated-measures ANOVA, Pb0.01; N=10).


ECTED P2.2. Dietary effects of Ca during intermolt

Both Sesarma and Chasmagnathus were held without food for 48 h prior to

experimentation. Then, the diets were offered to the animals for the next 4 days, every

other day, before experiments started, to allow the animals to get used to the new purified

food. After the pre-treatment, diets were offered for 1 h in the dry part of the box

(considered day 1). The diets were removed and the animals were allowed to choose the

dry or wet part of the box. The wet part of the box contained artificial seawater [10] (Table

1). The diets were offered for the next 11 days, on days 1, 3, 5, 7, 9 and 11, again for 1 h,

and removed. The uneaten diets were dried for 48 h at 35 8C and weighed to calculate

amounts eaten.

UNCORR

Fig. 3. Mean diet consumption (mg g�1) for Chas. granulata for every 2 days (total of 11 days), fed purified diets

which contained three different Ca concentrations (0%, 2.22% and 6.66% Ca). There was a higher consumption

for diets containing 6.66% Ca compared to the other diets (repeated-measures ANOVA, Pb0.01; N=5 for diet

with 0% Ca and N=6 for the other diets).

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t2.1 Table 2

Total Ca consumption (mg) over 11 days for S. rectum and Chas. granulata fed three different diets (meanFS.E.;

N=10 for Sesarma and N=6 for Chasmagnathus)t2.2

Diet S. rectum Ca consumption (mg) Chas. granulata Ca consumption (mg)t2.3

0% Ca 0 0t2.42.22% Ca 11.39F2.15 12.78F6.28t2.56.66% Ca 48.29F5.52 28.29F1.19t2.6


UNCORRECTED PROOFS. rectum consumed more of the diet containing Ca at 6.66% compared to the other

diets (Fig. 2; repeated-measures ANOVA, Pb0.01). The overall consumption was

around 6 mg g�1 for the latter diet and around 3–4 mg g�1 for the other diets

containing Ca at 0% and 2.22%. There was an effect of day (repeated-measures

ANOVA, Pb0.01) showing that the overall consumption of diet varied over 11 days for

all diets.

Chasmagnathus fed the three diets showed the same pattern of consumption as seen in

Sesarma (Fig. 3). Chasmagnathus ate more of the diet containing Ca at 6.66% Ca, at

values around 3–6 mg g�1 (repeated-measures ANOVA, Pb0.01). There was also a strong

effect of days, the consumption over the days showing a large variation for all diets

(repeated-measures ANOVA, Pb0.001). Again, the consumption for diets containing Ca at

2.22 and 0% was lower and around 2–2.5 mg g�1 (Fig. 3). Ca consumption was higher for

diets containing 6.66% Ca, values around 4� higher compared to Sesarma fed diets

containing 2.22% Ca (Table 2) and 2.2� higher Ca ingestion was seen for Chasmagnathus

compared to animals fed 2.22% of Ca (Table 2).

3. General discussion

Earlier work has shown that terrestrial crabs display behavioral regulation during food

intake [11,12]. Two species of herbivorous land crabs, Gecarcoidea natalis and

Cardisoma hirtipes displayed different feeding strategies when offered leaves found in

their natural environment [12] and as a result G. natalis had a higher intake of Ca in the

field when compared to Cardisoma, achieved through selection of yellow leaves in their

environment that already contained more Ca.

We found here that two phylogenetically related crabs with different feeding habits and

different degrees of terrestriality show similar patterns of regulation of ingested Ca.

Sesarma feeds on a mixed diet but the diet is composed mainly of mangrove leaves

(around 75% of the diet, see Ref. [13]) and Chasmagnathus feeds on a diet composed of

fewer plant matter, showing an opportunist behavior in relation to foods encountered [14].

The results showed that both crabs had the same behavioral response to food ingestion

when offered different levels of Ca in the diet, i.e., through more consumption of the high

Ca diet. The total consumption, however, was higher for Sesarma compared to

Chasmagnathus.

Work on a salt marsh crab, Armases cinereum, revealed that salt added as NaCl to the

diets stimulated feeding by Armases, in concentrations even higher than those found in

their natural environment [15]. Overall, it seems that salts in general, as well as Ca, are

upregulated for crabs living in salt marsh environments. This has been suggested before

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for terrestrial crabs that live in swamps far from the sea and where the water available is of

reduced salinity [16–18].

Presently, we cannot discriminate how our crabs detected and consumed more of the

high Ca diet. Interestingly, it has been found that hermit crab is able to behaviorally choose

shells in the environment [19] and detect calcium through chemoreceptors in the dactyls

which are sensitive to calcium [20]. This suggested bion sensitivityQ has also been noted in

terrestrial isopod antennae which showed sensitivity to calcium solutions in the range of

10–100 mM (cited in Ref. [20]). In our example, the crabs were able to detect and

consume more of the high Ca diet.

Ca net flux during postmolt has been studied before in crayfish [1,21] and there are

existing reports of first day postmolt fluxes in Carcinus maenas [22] and in Callinectes

sapidus [23]. Fluxes range from 3200 Amol kg�1 h�1 for the marine species Carcinus

and Callinectes to values as low as 1000 Amol kg�1 h�1 for the crayfish Cherax

destructor living in freshwater containing Ca at around 0.2 mmol L�1. Results

presented here suggest that although it is expected that terrestrial crabs do not rely

heavily on water sources for calcification, they do utilize available Ca in the water

under the experimental conditions presented here. The results also suggest that

calcification in these animals can last longer than expected compared to their aquatic

counterparts.

Acknowledgments

We would like to thank FAPESP (96/09756-9) and Universidade Presbiteriana

Mackenzie for the financial support and NSF grant IBN 0076035 to MGW.

References

[1] F.P. Zanotto, M.G. Wheatly, The effect of ambient pH on electrolyte regulation during the postmoult period

in freshwater crayfish Procambarus clarkii, J. Exp. Biol. 178 (1993) 1–19.

[2] F.P. Zanotto, M.G. Wheatly, Calcium balance in crustaceans: nutritional aspects of physiological regulation,

Comp. Biochem. Physiol. 133 (3 A) (2002) 645–660.

[3] P. Greenaway, Ion and water balance, in: W.W. Burggren, B.R. McMahon (Eds.), Biology of the Land

Crabs, Cambridge University Press, Cambridge, 1988, pp. 211–248.

[4] H.Y. Chen, C.F. Chang, Quantification of vitamin C requirements for juvenile shrimp (Penaeus monodon)

using polyphosphorylated l-ascorbic acid, J. Nutr. 124 (10) (1994) 2033–2038.

[5] S.Y. Shiau, C.W. Hsu, Dietary pantothenic acid requirement of juvenile grass shrimp, Penaeus monodon,

J. Nutr. 129 (3) (1999) 718–721.

[6] S.Y. Shiau, Y.H. Chin, Dietary biotin requirement for maximum growth of juvenile grass shrimp, Penaeus

monodon, J. Nutr. 128 (12) (1998) 2494–2497.

[7] S.Y. Shiau, J.S. Liu, Quantifying the vitamin K requirement of juvenile marine shrimp (Penaeus monodon)

with menadione, J. Nutr. 124 (2) (1994) 277–282.

[8] H.Y. Chen, F.C. Wu, S.Y. Tang, Thiamin requirement of juvenile shrimp (Penaeus monodon), J. Nutr. 121

(12) (1991) 1984–1989.

[9] G. Cuzon, J. Guillaume, C. Cahu, Composition, preparation and utilization of feeds for Crustacea,

Aquaculture 124 (1994) 253–267.

[10] C.F.A. Pantin, Notes on Microscopical Technique for Zoologists, Cambridge University Press, Cambridge,

1948.

[11] P. Greenaway, Calcium and magnesium balance during molting in land crabs, J. Crustac. Biol. 13 (2) (1993)

191–197.

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177

178179

180

181

182183

184

185

186187

188

189

190

191


ED PROOF

[12] P. Greenaway, S. Raghaven, Digestive strategies in two species of leaf-eating land crabs (Brachyura:Ge-

carcinidae) in a rain forest, Physiol. Zool. 71 (1) (1998) 36–44.

[13] T.D. Steinke, A. Rajh, A.J. Holland, The feeding-behavior of the red mangrove crab Sesarma meinerti-De

Man, 1887 (Crustacea, Decapoda, Grapsidae) and its effect on the degradation of mangrove leaf-litter, S.

Afr. J. Mar. Sci.-Suid-Afrikaanse Tydskrif Vir Seewetenskap 13 (1993) 151–160.

[14] L.C. Kucharski, R.S.M. Da Silva, Effect of diet composition on the carbohydrate and lipid metabolism in an

estuarine crab, Chasmagnathus granulata (Dana, 1851), Comp. Biochem. Physiol. 99 (A) (1991) 215–218.

[15] S.C. Pennings, et al., Feeding preferences of a generalist salt–marsh crab: relative importance of multiple

plant traits, Ecology 79 (6) (1998) 1968–1979.

[16] D.L. Wolcott, T.G. Wolcott, Food quality and cannibalism in the red land crab, Gecarcinus lateralis,

Physiol. Zool. 57 (3) (1984) 318–324.

[17] T.G. Wolcott, D.L. Wolcott, Availability of salts is not a limiting factor for the land crab Gecarcinus lateralis

(Freminville), J. Exp. Mar. Biol. Ecol. 120 (1988) 199–219.

[18] T.G. Wolcott, D.L. Wolcott, Ion conservation by reprocessing of urine in the land crab Gecarcinus lateralis

(Freminville), Physiol. Zool. 64 (1) (1991) 344–361.

[19] K.A. Mesce, Calcium-bearing objects elicit shell selection behavior in a hermit crab, Science 215 (4535)

(1982) 993–995.

[20] K.A. Mesce, Morphological and physiological identification of chelar sensory structures in the hermit-crab

Pagurus hirsutiusculus (Decapoda), J. Crustac. Biol. 13 (1) (1993) 95–110.

[21] S. Zare, P. Greenaway, Ion transport and the effects of moulting in the freshwater crayfish Cherax destructor

(Decapoda:Parastacidae), Aust. J. Zool. 45 (1997) 539–551.

[22] P. Greenaway, Uptake of calcium at the postmoult stage by the marine crabs Callinectes sapidus and

Carcinus maenas, Comp. Biochem. Physiol. 75 (2 A) (1983) 181–184.

[23] J.N. Cameron, Post-moult calcification in the blue crab (Callinectes sapidus): relationship between apparent

net H+ excretion, calcium and bicarbonate, J. Exp. Biol. 119 (1985) 275–285.

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some considerations on calcium homeostasis in semi-terrestrial crabs

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