AQUATIC BIOLOGYAquat Biol

Vol. 15: 11–25, 2012doi: 10.3354/ab00396

Published online March 21

INTRODUCTION

Five volcanic islands rise up along the axis of theOkinawa Trough back-arc basin, which extends fromthe northeast of Taiwan to the Unzen volcano inKyushu. The southwesternmost island is KueishanDao (‘Turtle Mountain Island’; 24° 25’ N, 121° 57’ E).The hydrothermal vents are located at depths of 8 to

20 m and are characterized by low pH (1.8 to 4.6) andsulfur-rich discharges which reach temperatures of65 to 116°C. Moreover, the vents release variousgases, mainly carbon dioxide, nitrogen, oxygen, sul-fur dioxide, and hydrogen sulfide (Jeng et al. 2004).

This unique environment is inhabited by the hydro -thermal vent crab Xenograpsus testudinatus, whichis endemic to this area. It lives close to the hydrother-

© Inter-Research 2012 · www.int-res.com*Corresponding author. Email: reinhard.saborowski@awi.de

Metabolic energy demand and food utilizationof the hydrothermal vent crab Xenograpsus

testudinatus (Crustacea: Brachyura)

Marian Yong-An Hu1,4, Wilhelm Hagen1, Ming-Shiou Jeng2, Reinhard Saborowski3,*

1BreMarE − Bremen Marine Ecology Centre for Research and Education, University of Bremen (NW2), 28334 Bremen, Germany2Research Center for Biodiversity, Academia Sinica, Nankang, Taipei 115, Taiwan

3Alfred Wegener Institute for Polar and Marine Research, PO Box 120161, 27515 Bremerhaven, Germany

4Present address: The Sven Lovén Centre for Marine Sciences, University of Gothenburg, Kristineberg 566, 45034 Fiskebäckskil, Sweden

ABSTRACT: The hydrothermal vent crab Xenograpsus testudinatus (Crustacea: Brachyura) isendemic near Kueishan Island, Taiwan, where it lives in shallow waters close to the hydrothermalvents located in this area. X. testudinatus is adapted to a sulfur-rich and thus potentially toxicenvironment. It has established a specialized feeding strategy focusing on dead zooplanktonorganisms killed by the toxic discharges from the vents. During slack water, when there is little orno current, the crabs leave their crevices to feed on this ‘marine snow’. In the present study, weinvestigated the physiological aspects of nutritional adaptations of X. testudinatus. The crabsshowed high digestive capacities of major digestive enzymes and particularly high activities forproteolytic enzymes. This feature can be regarded as an adaptation to irregular food availability.Furthermore, enzymes were stable at elevated temperatures, in a wide pH range, and in the pres-ence of inorganic inhibitors like Cu2+, Fe2+, or Co2+. These enzyme properties can be consideredessential to functioning in a vent habitat over long exposure times. Moreover, X. testudinatus isable to store significant amounts of lipid (50 to 60% of dry mass in the midgut gland), which mayhelp to overcome periods of food scarcity. Fatty acid profiles revealed high amounts of saturatedand monounsaturated components (mainly 16:0, 16:1(n-7), 18:1(n-9), and 18:1(n-7)). These find-ings reflect physiological adaptations and energetic strategies that enable this crab to exist in thisextreme hydrothermal vent habitat.

KEY WORDS: Hydrothermal vents · Crustacea · Xenograpsus testudinatus · Digestive enzymes ·Fatty acids · Toxic environment · Heavy metals

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Aquat Biol 15: 11–25, 2012

mal vents and is one of the few known vent-endemicspecies at shallow depths of

Hu et al.: Feeding physiology of vent crab

order to minimize artefacts in lipid and enzymeanalysis due to sexual maturation and vitellogenesis.The withdrawal of the 200 specimens from their envi-ronment did not affect the population, which wasestimated to comprise some 3.6 million crabs (supple-ment in Jeng et al. 2004). In the laboratory, the crabswere maintained in 250 l tanks filled with naturalseawater (salinity: 33 to 35) and connected with anexternal pump (Alife, AE-1060) to a bottom gravel fil-ter system. The crabs were kept under a 12 h light:12 h dark regime at 24°C.

Crabs used for starvation experiments were main-tained in separate plastic jars (500 ml) to avoidaggression or cannibalism. The jars were puncturedto ensure water exchange. Eight to 9 of these jarswere placed in 60 l tanks equipped with external fil-ters (Alife, AE-1060). At the start of the experiments(Day 0), 20 specimens, which were caught the sameday, were anesthetized by cooling on ice and thenkilled by an incision in the frontal region of the cara-pace. The midgut gland from each specimen was ex -cised, weighed, immediately frozen in liquid nitrogen,and thereafter stored at −80°C. Another 80 speci-mens were maintained individually in the jars. Every10 d, 20 specimens were treated and sampled asdescribed for Day 0. At each sampling date, 10midgut glands were used for lipid analysis and theremaining 10 for enzyme assays. There was no crabmortality during the 30 d ex periment.

Foregut morphology

The stomachs of 8 specimens of Xenograpsus testu-dinatus were dissected on the same day the animalswere captured. Sections through all 3 planes were

made, and samples were treated ac -cording to a scanning electron micro -scopy (SEM) protocol (Lee et al.1996), including prefixation in 4%paraform aldehyde with 5% glutar -aldehyde (P4G5) for 10 h. Subse-quently, samples were transferredinto a 0.1 mol l−1 phosphate buffer(PB) and washed 3 times. In order tomaintain cellular structures, mem-brane fixation was performed with1% OsO4 in 0.1 mol l−1 PB for 30 minunder a hood. After fixation, sampleswere again washed in 0.1 mol l−1 PB.The samples were dehydrated in in -creasing concentrations of ethanol(50%, 70%, 80%, 95%, and 100%).

The samples were dried in a critical point drier(Hitachi HCP-2 CPD), gold-coated (CressingtonSputter Coater 108), and examined in a SEM (FEIQuanta 200) within an electrical field of 25 kV.

Respiration measurements

Prior to the determination of routine metabolicrates, crabs were acclimated for 1 wk to the experi-mental conditions. Oxygen consumption rates werede termined in 5.8 l respiration chambers at atmo -spheric pressure. Five chambers were filled with filtered seawater (0.2 μm) and equipped with 1 crabeach. A sixth chamber without a crab served as acontrol. For acclimation, starved animals (24 h) werepre-incubated in the respiration chambers for 3 h,while the water was aerated to reach full oxygen saturation. Depending on the experimental tempera-ture (15, 20, 25 or 30°C), crabs were incubated for 5 to12 h in the dark. In order to keep the incubation tem-perature constant, test chambers were placed in awater bath equipped with a Dixell Prime tempera-ture control device that was connected to the circula-tion incubator (Dixell, Superflite). A decrease of oxy-gen concentrations be low 70% was avoided. Theappropriate incubation time for each temperaturewas empirically determined in preliminary experi-ments. Before and after incubation, water samples(3 × 50 ml plus overflow) were carefully siphoned offthe respiration chamber, and the amount of dissolvedoxygen was determined following the Winkler method(Hansen 1999). For each respiration measurement,5 new crabs were used.

In order to determine oxygen consumption of bacteria derived from the surfaces of the crabs, the

13

Fig. 1. Kueishan Island located off Taiwan’s east coast. Hydrothermal ventarea is delimited by stars. Source: Google Earth, photographed May 16, 2004,

and accessed October 20, 2011

Aquat Biol 15: 11–25, 201214

remaining seawater in each chamber was againtested for oxygen consumption, but without crabs.The water was aerated in order to saturate it withoxygen. The chambers were again incubated for 5 hand the oxygen concentrations were measured asdescribed in the previous paragraph. The amount ofoxygen consumed by bacteria was subtracted fromthe overall oxygen consumption of the crabs.

The relationship between oxygen consumption of thecrabs and the water temperature was demonstratedby calculating Q10 values according to van ’t Hoff’srule for metabolic processes:

(1)

where R1 and R2 represent the respiration rates attemperatures T1 and T2.

Lipid analysis

Frozen midgut glands were lyophilized for 48 hand the dry mass was determined. Lipid extractionwas done following Hagen (2000). The lyophilizedsamples were mechanically homogenized (Sarto-rius; Potter S) and extracted in dichloromethane(DCM)/ methanol (2:1 v/v). The amount of totalextractable lipid was determined gravimetrically ona micro balance (Sartorius precision balance R200D,precision: 10 μg) and presented as % of dry mass(%DM).

Lipid extracts adjusted to a final concentration of14 mg ml−1 in DCM were used to determine lipidclass composition using thin layer chromatographywith flame ionization detection (Iatron Laboratories,MK-5) according to Fraser et al. (1985).

FA analysis

Subsamples of lipid extracts (100 μg) were usedfor trans-esterification. After evaporation of the sol-vent under a continuous flow of nitrogen, 1 ml ofmethanolic sulfuric acid (3%) and 250 μl of hexanewere added to the sample and the mixture was incu-bated under nitrogen for 4 h at 80°C. Fifty μl of sam-ple were adjusted to a concentration of 0.02 mg ml−1

hexane and were then analyzed by gas chromatogra-phy (GC) according to Kattner & Fricke (1986). TheGC (HP 6890) was equipped with a cold-injectionsystem and a DB-FFAP column (30 m, inner diameterof 0.25 mm, 0.25 μm coating). Fatty acid methylesters (FAMEs) were quantified by a flame ionization

detector and identified by comparison with retentiontimes of an established fish oil standard (Marinol).

Preparation of crude enzyme extract

Samples (50 to 100 mg) of deep-frozen midgutglands of Xenograpsus testudinatus were extractedin 1 ml of demineralized water. The tissues werehomo genized with an ultrasonic cell disruptor (Bran-son Sonifier, B15) with 30% of maximum energy and3 bursts of 3 s each with a break of 7 s in between.The homogenates were centrifuged for 15 min at15 000 g and 4°C. The supernatants were then trans-ferred into new reaction cups and used for furtherenzyme analysis. All steps were performed on ice inorder to avoid thermal degradation or enzymatic pro-teolysis. For the characterization of proteases, weused only samples from freshly caught animals thatwere deep-frozen within 6 h after capture.

Protein concentrations of the crude extracts weredetermined according to Bradford (1976) with a commercial protein kit (Bio-Rad) and bovine serumalbumin as standard.

Api Zym enzyme assays

The Api Zym system (bioMérieux, REF 25200) wasused following Donachie et al. (1995) and Sabo rows -ki et al. (2006) to screen Xenograpsus testudinatusmidgut gland extracts for a set of 19 digestive en -zymes (see Table 3). Fifty μl of the midgut glandcrude extracts (50 mg ml−1) were added to each wellon the test trays. The trays were incubated at 30°Cfor 2 h. Then 1 drop of each of the reagents Zym Aand Zym B were added to the wells. The enzymeactivities were classified semi-quantitatively accord-ing to the intensities of the developed colors. Assayswere run in duplicate. However, this assay is not sen-sitive for crustacean chymotrypsin activity, due to theinappropriate substrate N-glutaryl-phenylalanine-2-naphthylamide.

Protease activity and stability

The activities of the serine proteases trypsin andchymotrypsin were determined colorimetrically withthe substrates N-benzoyl-L-arginine-4-nitroanilide-hydrochloride (BAPNA; Fluka 12915) for trypsin andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA;Sigma S7388) for chymotrypsin. In brief, 20 μl of

QRR

T T

102

1

10 2 1K

=−( )

Hu et al.: Feeding physiology of vent crab

crude extract were added to 960 μl of 0.1 mol l−1

Tris·HCl-buffer (pH 7) and pre-incubated for 3 min.The reaction was started by addition of 20 μl of sub-strate (50 mmol l−1 in DMSO). The increase of ab -sorbance at 405 nm was measured in a spectro -photometer at constant temperatures from 5 to 70°C.

The thermal stability of the enzymes was examinedin pooled extracts from 4 individuals. Sub-sampleswere pre-incubated for up to 5 h at 0 to 70°C. There-after, samples were cooled on ice and the residualproteolytic activity was subsequently measured asdescribed in the previous paragraph.

The effect of inorganic substances on the activitiesof proteolytic enzymes was examined with the fol-lowing metal ions and reagents: CuCl2, LiCl2, CoCl2,NaCl, FeCl2, AlCl3, MgSO4, HgNO3, and EDTA. Theconcentration of all salts and reagents in the reactionmixtures was 0.01 mol l−1.

The pH profiles of proteases were investigatedusing universal buffer following Ellis (1961). The pHstability was studied while crude extracts were incu-bated at pH 2, 4, 6, 8, 10, and 12 for 30 min at 30°C.Subsequently, samples were examined for proteaseactivity at pH 7.

Statistical analysis

Statistical analysis was performed with the com-puter program SigmaStat 3.1 (Systat). One-wayANOVA, including test of normality, equality of vari-ances, and all pair-wise multiple comparison proce-dures (Holm-Sidak method) were applied to compareproteolytic activities under varying experimental con -

ditions and during the starvation period. Student’st-test was performed to evaluate differences amonglipid profiles during starvation.

RESULTS

On June 3, 2007 we collected Xenograpsus testudi-natus and, simultaneously, had the unique opportu-nity to observe this species in its natural habitat. Thecrabs were sampled in 8 to 15 m depth. The habitatwas poor in flora and fauna and smelled of sulfurousplumes discharged by the vents. During SCUBA-diving we observed how a group of crabs fed on adead flying fish, which had probably been killed bythe toxic plumes. This observation shows that X. tes-tudinatus also scavenges on other available foodsources besides zooplankton.

Foregut morphology

The nomenclature of labeled components (Figs. 2 &3) and the terminology of the description of theforegut essentially follows Martin et al. (1998). Themedian tooth of Xenograpsus testudinatus can beseparated in 2 parts: a larger rectangular part witha large central dorsal projection and a smaller partthat is V-shaped. The median tooth is surrounded bysetae of different length, with 2 groups of strikingprojecting setae at the posterior edge of the tooth(Figs. 2 & 3a). The urocardiac ossicle is smooth andresembles a tongue carrying the median tooth on itsposterior end. The lateral teeth are well developed

15

Fig. 2. Xenograpsus testudinatus. Scanning electron microscopy (SEM) image of internal anatomy of the stomach. Dorsalview on the open stomach revealing the median tooth (MED), lateral tooth (LAT), accessory lateral tooth (ALT), urocardiac ossicle (UO), zygocardiac ossicle (ZO), and prepectineal ossicle (PO). The position of the cardiac stomach (CS; not shown)

is indicated by the arrow

Aquat Biol 15: 11–25, 2012

and carry one large tooth at their anterior end. Theposterior part consists of about 18 blade-shaped teeth(Figs. 2 & 3c), which become smaller in the posteriordirection. Along the ventral margin of the lateraltooth and the posterior end, several setae are pre-sent. These setae, like most of the other setae foundin the stomach of X. testudinatus, bear denticles(Fig. 3e,f). Accessory teeth are paired, attached toprepectineal ossicles, and located dorsally in theanterior part of the lateral teeth (Fig. 3b). Theseaccessory ossicles bear 11 medially directed spines,which carry denticles at their end. Only a few setaeare present on the anterior surface of the accessory

teeth. However, on the posterior side,these scaled setae form dense filters. Theentire foregut is densely equipped withmainly 2 types of setae. The first type con-sists of a central, slightly flattened bodycarrying 2 rows of laterally directedspines. These spines only occur at the tipof the setae. This type is found along thedorsal edge of the lateral teeth (Fig. 3e).The second type of setae is cylindrical,carrying small denticles that cover oneside of the round setae (Fig. 3f). The sec-ond type is widely distributed throughoutthe whole foregut.

Oxygen consumption

The routine oxygen consumption (ROC)of Xeno grapsus testudinatus increasedwith temperature from 1.1 ± 0.2 μmolgFM−1 h−1 (where FM is fresh mass; errorvalues are SD throughout this paper) at15°C to 4.9 ± 1.5 μmol gFM−1 h−1 at 30°C.At 25°C it was 3.5 ± 1.0 μmol gFM−1 h−1

(Fig. 4). The Q10 value calculated over thewhole temperature range was 3.13. Q10values for the temperature ranges 20 to25°C and 25 to 30°C were approximately2. Be tween 15 and 20°C, the Q10 valuewas 5.43.

Respiration data allowed us to calculatethe metabolic energetic demand. Assum-ing that protein is a major food source ofXenograpsus testudinatus, we used a res-piratory quotient of 0.85 (Nelson et al.1977). At an ambient temperature of 25°C, average-sized adult X. testudinatus speci-mens of 10 gFM release 27.4 μmol CO2gFM−1 h−1. This is equal to 0.0789 g C d−1.

According to Salonen et al. (1976), the amount of en-ergy stored in 1 g organic carbon (zooplankton) cor-responds to 41.4 ± 0.9 kJ (9.9 kcal). Therefore, thedaily energy demand of a speci men with 10 g bodymass amounts to 0.525 kJ d−1, or 525 ± 71 J d−1 (=0.125 ± 0.017 kcal d−1).

Lipid content and composition

Mean total lipid contents of the midgut glands ofXeno grapsus testudinatus ranged from 53.5 ± 4.2 %DMin freshly sampled animals to 36.8 ± 16.3 %DM in ani-

16

Fig. 3. Xenograpsus testudinatus. Scanning electron microscopy (SEM)photographs of foregut ossicles and setae covering the internal surface ofthe foregut. (a) Lateral teeth (LAT) and median tooth (MED); note the se-tae covering the dorsal surface of the lateral tooth. (b) Close-up of acces-sory lateral teeth (ALT) from anterior view, showing the 11 medially di-rected spines. Detailed image of the blade-shaped lateral teeth from (c)lateral and (d) dorsal view. (e,f) Fine structure of the setae, showing 2

types of setae, differing in their denticle distribution

Hu et al.: Feeding physiology of vent crab

mals starved for 30 d (Table 1). Due to high variability,the differences were not statistically significant.

The majority of the lipids in the midgut gland wereTAG (neutral lipids) and polar lipids, which togetheraccounted for 95% of total lipids (%TL) in fed andin starved animals (Table 2). Free fatty acids as wellas sterols contributed only marginally to total lipids.After starvation for 30 d, the amount of TAG declinedfrom 92.0 ± 2.5 %TL to 76.3 ± 23.5 %TL. Simultane-ously, the proportion of polar lipids increased from4.0 ± 2.2 %TL to 17.4 ± 19.6 %TL. These results showa decrease of TAG during starvation, but due tohigher variation among starved animals, the differ-ences between fed and starved specimens were notstatistically significant.

FA composition

The FA compositions of fed and starved specimenswere dominated by saturated (SFA) and monounsatu-rated fatty acids (MUFA), while only a few polyunsat-urated fatty acids (PUFA) were present (Table 2). Themajor FA was the MUFA 18:1(n-7), which ac countedfor 22.8 ± 2.3% (field samples) and 24.7 ± 4.6% (starved30 d) of total FA. Other MUFAs were 16:1(n-7) (~10%of total FA) and 18:1(n-9) (~12% of total FA). The SFA16:0 amounted to ~17% and 18:0 to about 5% of totalFA. The major PUFAs, 22:5(n-3) and 22:6(n-3) to-gether, only contributed 11% to total FA. Duringthe 30 d of starvation, no statistically significantchanges in FA patterns were evident. Only 16:1(n-7)de creased significantly after 30 d of starvation.

Api Zym enzyme assays

The semi-quantitative enzyme screening of mid gutgland extracts of Xenograpsus testudinatus re vealeda wide range of enzyme activities (Table 3). Almost

17

Day 0 Starvation for 30 d

Total lipids (%DM) 53.5 ± 4.20 36.8 ± 16.3Lipid classes (%TL)Wax/sterol esters 2.4 ± 1.2 2.6 ± 1.3Triacylglycerols 92.0 ± 3.50 76.3 ± 23.5Free fatty acids 0.7 ± 0.9 1.0 ± 0.9Sterols 0.8 ± 0.2 2.9 ± 3.4Polar lipids 4.0 ± 2.2 17.4 ± 19.6

Table 1. Xenograpsus testudinatus. Lipid contents of midgutglands on Day 0 and after starvation for 30 d. Values aregiven in % of midgut gland dry mass (DM). Lipid classes arepresented as % of total lipids (TL) of 10 individual crabs

analyzed. Values are mean ± SD

Temperature (°C)10 15 20 25 30 35

Oxy

gen

cons

ump

tion

(µm

ol g

FM–1

h–1

)

0

1

2

3

4

5

6

7

Q10 = 5.43 (15–20°C)

Q10 = 2.02 (20–25°C)

Q10 = 1.94 (25–30°C)

Q10 = 3.13 (20–30°C)

Q10 = 1.96 (15–20°C)

Fig. 4. Xenograpsus testudinatus. Temperature-dependentoxygen consumption. Between 20 and 30°C, an exponentialregression was applied in accordance with van ’t Hoff’s rule.Oxygen consumption rates at 20 and 15°C are connected bya dashed line, indicating a deviation from the exponentialcourse. Q10 values are given for each temperature intervaltested and additionally for a theoretical value between 15and 20°C (dashed). Error bars indicate SD (n = 9 to 14).

FM: fresh mass

Fatty acid Duration of starvation (d)0 10 20 30

14:0 1.1 ± 0.3 2.0 ± 0.7 2.3 ± 0.4 0.7 ± 0.316:0 16.5 ± 5.8 18.8 ± 3.3 20.4 ± 0.8 18.0 ± 3.2016:1(n-7) 10.8 ± 1.2 11.1 ± 1.8 11.4 ± 1.9 8.3 ± 1.716:1(n-5) 0.5 ± 0.1 0.7 ± 0.2 0.6 ± 0.1 0.5 ± 0.116:2(n-4) 0.6 ± 0.2 0.5 ± 0.2 0.5 ± 0.2 0.6 ± 0.117:0 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.6 ± 0.218:0 5.2 ± 0.6 5.1 ± 1.0 5.0 ± 0.6 5.8 ± 0.718:1(n-9) 11.3 ± 1.7 13.0 ± 3.0 11.4 ± 2.3 11.8 ± 0.9018:1(n-7) 22.8 ± 2.2 18.9 ± 6.4 21.7 ± 3.4 24.6 ± 4.7018:2(n-6) 1.1 ± 0.3 1.4 ± 0.7 1.0 ± 0.4 1.1 ± 0.418:3(n-3) 0.6 ± 0.1 0.7 ± 0.2 0.6 ± 0.2 0.5 ± 0.220:1(n-9) 0.9 ± 0.2 0.9 ± 0.4 0.7 ± 0.5 1.0 ± 0.220:1(n-7) 2.1 ± 0.4 1.9 ± 0.8 2.1 ± 0.4 2.7 ± 0.920:4(n-6) 2.0 ± 0.5 2.4 ± 1.7 1.9 ± 0.8 2.6 ± 1.120:5(n-3) 4.9 ± 1.1 4.7 ± 1.8 4.2 ± 1.0 4.7 ± 1.522:1(n-11) 0.5 ± 0.1 0.6 ± 0.4 0.5 ± 0.2 0.5 ± 0.222:5(n-3) 0.8 ± 0.2 0.7 ± 0.3 0.6 ± 0.2 0.6 ± 0.322:6(n-3) 7.7 ± 1.7 6.4 ± 2.6 5.9 ± 1.7 6.3 ± 2.2Unknown 4.1 ± 2.5 4.7 ± 3.4 3.3 ± 0.8 3.6 ± 1.4∑SFA 23.4 ± 6.8 26.4 ± 5.0 28.2 ± 1.9 25.1 ± 4.40∑MUFA 48.9 ± 6.0 47.2 ± 13.0 48.5 ± 8.9 49.4 ± 8.70∑PUFA 17.8 ± 4.2 16.6 ± 7.5 14.8 ± 4.6 16.3 ± 5.70∑(n-7) 35.7 ± 3.8 32.0 ± 8.9 35.3 ± 5.7 35.6 ± 7.30

Table 2. Xenograpsus testudinatus. Fatty acid (FA) composi-tion (mass% of total FA) of total lipid extracts of midgutglands. SFA: saturated FA, MUFA: monounsaturated FA,PUFA: polyunsaturated FA, and (n-7): FAs from the (n-7) se-ries. Fatty alcohols (

Aquat Biol 15: 11–25, 2012

all enzymes tested showed positive reactions, exceptα-chymotrypsin and lipase (C-14). The highest activ-ity levels, on a scale of 0 to 5 (see Table 3), wereshown by leucine arylamidase, trypsin, α-gluco -sidase, and alkaline phosphatase. Phosphate hydro-lases, including alkaline phosphatase, acid phospha -tase, and naphthol-AS-BI-phosphohydrolase, showedhigh activities as well. All glucosidases tested showedintermediate ac tivities.

Effect of starvation on lipolytic and proteolyticenzyme activities

Starvation for 30 d caused a significant decrease inproteolytic as well as lipolytic activities (Fig. 5). Atthe beginning of the starvation period, the reductionin total proteolytic activity was low but became sig-nificant after 20 and 30 d (60 to 70% residual activ-ity). The course of lipase/ esterase activity duringstarvation was similar but the decrease in activitywas much more distinct. After 10 d of star vation, theresidual lipolytic activity de creased to

Hu et al.: Feeding physiology of vent crab

trypsin showed similar profiles. After 30 min of incu-bation at pH 2, the residual activities were

Aquat Biol 15: 11–25, 2012

on dead zooplankton during short periods of slackwater (Jeng et al. 2004). The species has de velopedbehavioral, anatomical, and physiological adapta-tions to cope with the specific conditions around shal-low-water hydrothermal vents.

Foregut morphology

Comparison of the foregut morphology of Xeno -grapsus testudinatus with other brachyuran crabs(Meiss & Norman 1977), including the deep-seahydrothermal vent crab Bythograea thermydron(Martin et al. 1998), confirmed that the general orien-tation of the median tooth, the lateral teeth, and theaccessory lateral teeth is similar to that in otherbrachyurans. The calcified median tooth and lateralteeth are mobile in order to grind food into fine parti-cles. The setae of the foregut are thought to beinvolved in mixing and filtering the digestive fluidsinside the stomach and directing the flow of thechyme to the midgut gland, where the absorption ofnutrients takes place (Ceccaldi 1989, Salindeho &Johnston 2003). However, in contrast to the foregut ofdeep-sea vent crabs described by Martin et al.(1998), the foregut of X. testudinatus is densely covered with scales bearing setae in the micro meterrange. This high density of setae can be consideredadvantageous for the specialized feeding of X. testu-dinatus crabs in terms of optimized processing of

ingested food and effective transport ofthe chyme. The morphology of the gastricmill indicates that X. testudinatus is omni -vorous (Jeng et al. 2004). Opportunisticfeeding, including scavenging, can be con -sidered the most efficient feeding modefor this species.

Energy demand

The increase in respiration of Xeno -grapsus testudinatus with rising tempera-tures is common for poikilothermic organ-isms living under atmospheric pressure(Ali et al. 2000, Taylor & Peck 2004, Kunz-mann et al. 2007). The magnitude ofincrease followed van ’t Hoff’s rule in thetemperature range between 20 and 30°C,with Q10 values around 2. The deviation ofQ10 values below 20°C may have resultedfrom adaptation to higher temperaturesand physiological in tolerance of low tem-

peratures. Metabolic rates of organisms from polar ortemperate regions, like northern krill Meganyc-tiphanes norvegica, follow van ’t Hoff’s rule in thelower temperature range (Saborowski et al. 2000,2002, Chausson et al. 2004). In tropical species, van ’tHoff’s rule applies to the higher temperature range(Démeusy 1957, Kunzmann et al. 2007). Surfacewater temperatures in the area of Kueishan Islandare usually higher than 20°C (M. S. Jeng pers. obs.),and the temperature at the bottom varies between 20and 27°C (Chen et al. 2005a). Thus, X. testudinatusdoes not experience water temperatures of ≤15°C inthat setting, but in this temperature range in the pre-sent study, the crabs showed an excessive reductionin respiration rates.

At 25°C, an individual Xenograpsus testudinatusof 10 g body mass converts an energy equivalent of525 J d−1 or 125 cal d−1 for maintaining routine meta-bolic activity. Assuming that 1 g of copepods (ash-free dry mass) contains between ~22 and 31 kJ, adaily zooplankton (copepods) uptake of 85 to 120mgFM (assuming a mean water content of 80%) isnecessary to maintain basic energy demands (Salo-nen et al. 1976). Taylor & Peck (2004) determineddaily energy demands of the sand shrimp Crangonseptemspinosa. Large shrimps (1.5 gFM) metabolize300 to 400 J d−1 at an ambient temperature of 20°C.On a ‘per gram’ basis, X. testudinatus converts 52.5 Jand the shrimp 200 to 267 J. This difference is cer-tainly due to the allometric relationship between

20

Ref. CuCl2 LiCl2 CoCl2 AlCl2 NaCl FeCl2 NaSO4 MgSO4 HgNO3 EDTA

Res

idua

l act

ivity

(%)

0

20

40

60

80

100

120

140

160

180

CHYMTRY

**

*** *

* **

* *

Fig. 9. Xenograpsus testudinatus. Effects of inorganic inhibitors on trypsin(TRY) and chymotrypsin (CHYM) activity. Residual activity is presented as% of uninhibited activities (mean ± SD, n = 3). *Significant difference from

reference (Ref.) group (Holm-Sidak, p < 0.05)

Hu et al.: Feeding physiology of vent crab

body mass and energy demand. But the lower valuefor X. testudinatus may also be explained by reducedactivity and a higher amount of calcified shell com-pared to the shrimp (Weymouth et al. 1944). The shellcontributes significantly to the mass of the crab but isotherwise not metabolically active.

Lipid stores

In decapods, the midgut gland is the major lipid-processing and lipid-storage organ (O’Connor &Gilbert 1968, Lawrence 1976, Wen et al. 2006). Theability to accumulate energy stores is an essentialphysiological prerequisite for survival in environ-ments with deprived or variable food availability.High lipid contents have been reported for sub-polarand polar species, which have to cope with long seasonal starvation periods and tightly coordinatedreproduction periods (Albers et al. 1996, Falk-Petersen et al. 2000, Kreibich et al. 2010). However,crabs from temperate or tropical regions may alsoshow elevated lipid contents. The robber crab Birguslatro can accumulate lipid contents of up to 75% drymass (Chakravarti & Eisler 1961), which enablesit to survive for >1 yr without food (Storch et al. 1982,Greenaway 2003). Xenograpsus testudinatus’ mid -gut gland contained >50% lipids (%DM). Neutrallipids were predominantly stored as TAG. The abilityof X. testudinatus to store high amounts of lipids canbe regarded as beneficial in the animal’s habitat.The only input of particulate organic matter (POM)consists of plankton (and nekton) that were killed bytoxic discharges and sunk to the ground. This foodsource is not continuously available and the supplystrongly depends on tidal and local currents. Further-more, Jeng et al. (2004) observed that X. testudinatusswarm out and leave their crevices only during slackwater. Apparently, the crabs roam and search forfood in certain conditions, but they do not seem to ac -tively hunt for food, even when the supply is limited.Accordingly, X. testudinatus must be capable of sur-viving food-deprived periods by using their lipid stores.

Starvation experiments confirmed the ability ofXeno grapsus testudinatus to survive starvation peri-ods of at least 30 d. The daily energy demands of a10 g individual X. testudinatus amounted to 525 ±71 J. In turn, the average lipid content in the midgutgland accounted for 75 mg. According to the physicalenergetic equivalents for lipids (39.7 kJ g−1) (Salonenet al. 1976), the lipid reserves in the midgut glandwould theoretically be exhausted after 5 to 6 d. Thefact that X. testudinatus survived at least 30 d with-

out food indicates that they may catabolize additionalenergy stores such as proteins or carbohydrates. Inaddition, X. testudinatus may switch to energy-savingmetabolic pathways and reduction of metabolic rates.Unfortunately, it was not possible in the frame of thepresent work to continue respiration measurementsafter 30 d of starvation, because the availability ofcrabs was limited. An additional transfer and expo-sure of crabs from the 30 d starvation experiment todifferent temperatures could have caused negativeeffects on the quality of tissue samples that we usedfor lipid and enzyme analysis.

FA composition

The FA composition of the midgut of Xenograpsustestudinatus was dominated by MUFAs of the (n-7)and (n-9) series and the SFA 16:0, but ω3 PUFAs like20:5(n-3) (eicosapentaenoic acid or EPA) and 22:6(n-3) (docosahexaenoic acid or DHA) were also present.SFA can be synthesized by most species. The MUFA18:1 (n-9) is the major FA in most marine animals and,thus, it may serve as a marker for carnivory (Dals-gaard et al. 2003). Accordingly, high amounts of18:1(n-9) in the midgut glands of X. testudinatus sug-gest a mainly carnivorous nutrition (Jeng et al. 2004).Moreover, this predominantly carnivorous feedingmode in X. testudinatus is supported by low amountsof longer-chain PUFAs such as 20:5 (n-3), which aretypical markers of diatoms (Kattner & Hagen 1995).Nevertheless, the essential PUFAs of the (n-3) serieswere present in the midgut gland of X. testudinatus.These FAs are predominantly synthesized by pri-mary producers because they possess the enzymesΔ12 and Δ15 desaturase to form (n-3) PUFAs. Theseessential FAs can directly derive from phytoplanktonthat co-agglomerates with zooplankton in the marinesnow. They may also come from herbivorous or omni -vorous zooplankton in which these FAs accumulated.

Many deep-sea vent organisms depend on bacter-ial primary production. Consequently, all trophic levels are more or less interspersed with bacterial FAtrophic markers (Pond et al. 1998, Dalsgaard et al.2003, Phleger et al. 2005a, 2005b). FAs belonging tothe MUFA (n-7) series are predominantly producedby phytoplankton and bacteria, but not by animals(Dalsgaard et al. 2003). Since deep-sea thermal ventenvironments contain negligible amounts of phyto-plankton, 18:1(n-7) or 16:1(n-7) FAs can be directlytraced back to bacterial pro duction. Hydrothermalvent species like the tube worm Riftia pachyptila,Munidopsis subsquamosa, Bathymodiolus sp., and

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Bythograea thermydron contain significant amountsof these bacterial markers (Phleger et al. 2005a,2005b). These may also be present in shallow-waterhydrothermal habitats. Kharlamenko et al. (1995)showed that the symbiont-containing clam Axinop-sida orbiculata exhibits high amounts of 18:1(n-7),16:1(n-7), and 16:0 FAs. However, the origin of 18:1(n-7) (25% of total FA) and 16:1(n-7) FAs (10% oftotal FA) in Xenograpsus testudinatus midgut glandscannot be clearly traced back to bac teria, as the ventsare located within the euphotic zone. It remains to beinvestigated whether the high concentrations of (n-7)MUFAs, which were a striking feature of the FA profile of X. testudinatus, are of bacterial origin orderived from other sources.

Digestive potential

The midgut glands of Xenograpsus testudinatusshowed a wide range of highly active digestiveenzymes comprising various glucanases, esterases,lipases, and peptidases. Since X. testudinatus feedson dead zooplankton, we paid special attention to theimportant proteolytic enzymes with tryptic and chy-motryptic activities (Saborowski et al. 2004, Muhlia-Almazán et al. 2008). The activities of both enzymeswere higher than in many other crustacean speciesmeasured under the same conditions. The trypticactivities in the midgut gland of freshly caught X. tes-tudinatus reached the same levels as in Antarctickrill Euphausia superba (Fig. 10), which is knownfor very high enzyme activities (Anheller et al. 1989,

Turkiewicz et al. 1991). The high digestive capacityof krill has been explained as an adaptation of feed-ing on patchy and unpredictable phytoplankton(Morris et al. 1983, Saborowski & Buchholz 1999).The animals have to ingest and to utilize their foodas fast and efficiently as possible. Similar to Antarc-tic krill, X. testudinatus feeds on an irregular foodsource, which only appears during slack water. Dur-ing this short period, the crabs swarm out of theircrevices and start to feed rapidly, making the most ofthe scarce food.

The midgut gland extracts showed high chitinolyticactivities of N-acetyl-β-glucosaminidase (chitobiase).Crustaceans require chitinolytic enzymes for molting(Oosterhuis et al. 2000) but they also synthesize chiti-nolytic enzymes in the digestive organs (Saborowskiet al. 1993, Peters et al. 1999). High activities ofdigestive chytinolytic enzymes may indicate that thevent crabs are capable of digesting and utilizingchitin from crustacean shells as a source of both car-bon and nitrogen (Saborowski & Buchholz 1999).

Due to high ambient water temperatures, highmetal ion concentrations, and extreme pH variationsin hydrothermal vent habitats (McMullin et al. 2000),adaptations at the protein/enzyme level appearprob able. The trypsin-like proteinase of this speciesshowed remarkable thermal stability and remainedunimpaired after exposures to 60°C for 5 h. Com-pared to other crustaceans (Dittrich 1992, Garcia-Carreño & Haard 1993, Johnston & Freeman 2005),Xenograpsus testudinatus proteinases show a sig -nificantly elevated thermal tolerance. Dittrich (1992)studied trypsin-like proteases of crustaceans fromtropical, temperate, and subarctic regions. The low-est thermal stabilities were present in polar specieslike the copepod Calanoides acutus (loss of >50%,2 h at 40°C) or the shrimp Chorismus antarcticus (lossof >90%, 2 h at 40°C). In contrast, tropical specieslike the hermit crab Clibanarius striolatus and Ocy-pode ryderi retained maximum activities after 2 h at40°C or 50°C. At 60°C, residual activities in bothtropical species totally vanished after 30 and 90 min,respectively (Dittrich 1992). In X. testudinatus, thepresence of trypsin-like enzymes, which function atelevated temperatures of 60 to 65°C, and proteinases,which function at up to 55°C for 5 h without loss ofactivity, may be re garded as adaptations to a habitatwith temporarily elevated temperatures.

The pH optima of trypsin and chymotrypsin havebeen investigated in several species (Garcia-Carreño& Haard 1993, Hernandéz-Cortés et al. 1997, Navar-rete del Toro et al. 2006), revealing slightly alkalineworking optima. Dionysius et al. (1993) reported a

22

0

2

4

6

8

10

12

14

Crangoncrangon

Cancer pagurus

Pagurus bernhardus

Euphausia superba

Xenograpsus testudinatus

a

b b

c

a

Act

ivity

(U g

FM–1

)

Fig. 10. Comparison of tryptic activity at 30°C in midgutglands of X. testudinatus (this study) and several othermarine crustaceans (modified after Teschke & Saborowski2005). Different letters indicate significant differences be-tween groups (Holm-Sidak, p < 0.05). tMean + SD, n = 3 to 6

Hu et al.: Feeding physiology of vent crab

pH optimum of 8 for a trypsin-like protease isolatedfrom the sand crab Portunus pelagicus. Similar re -sults with highest activities at pH 7.5 to 8 were ob -served for trypsins from Paralithodes camchatica(Rudenskaya et al. 2000). The present study demon-strates that both trypsin and chymotrypsin of Xeno -grapsus testudinatus show a wide range of stabilityfrom slightly acidic to alkaline (pH 6 to 10) condi-tions. Since the hydrothermal vent site of KueishanIsland is characterized by highly acidic discharges,enzymatic tolerance over a wide pH range can beconsidered an essential feature of this crab. Thehasty and nonselective feeding of X. testudinatusduring the short slack-water period (Jeng et al. 2004)prohibits a proper selection of food particles. As aconsequence, digestive enzymes might be shortlyexposed to ingested food particles or water withextreme pH. In order to tolerate these pH variations,a wide pH-stability range can be regarded as animportant feature of the crab’s digestive enzymes.

The water around the hydrothermal vents containshigh concentrations of metal ions such as Mg2+, Ca2+,Fe2+, Cu2+, Al3+, and Mn2+ (Chen et al. 2005a). Someof these ions show inhibitory effects on enzymes.Thus, the resistance of digestive en zymes againstthese inhibitors is an important issue (Dreyfus &Iglew ski 1986, Edgcomb et al. 2004, Cardigos et al.2005). Our results confirm earlier findings that de -monstrated that Fe2+ and Cu2+ inhibit enzyme activi-ties (Sakharov & Prieto 2000). The concentrationsreported for hydrothermal vent habitats were usuallyin the micro- to nanomolar range. Ac cordingly, theconcentrations applied in the present study (10 mmoll−1) can be considered as extremely high for aquaticorganisms (Olaifa et al. 2004, Cardigos et al. 2005).Nevertheless, the activities of trypsin and chy-motrypsin were not completely inhibited. This indi-cates a high tolerance against metal ions, which is anadvantage for the functioning of these enzymes inthe vicinity of hydrothermal vents.

CONCLUSIONS

The shallow-water hydrothermal vent crab Xeno -grapsus testudinatus exhibits physiological adap -tations to successfully inhabit a highly challengingenvironment. These physiological properties aremainly reflected in digestive features and energystorage. A digestive tract designed for omnivorousfeeding combined with a set of highly active and sta-ble enzymes enable X. testudinatus to efficiently uti-lize dead zooplankton, which is only available during

slack water and absence of currents or strong winds.These features can be regarded as essential for X.testudinatus to survive long periods close to thehydrothermal vent system. Substantial lipid reservesin the midgut gland ensure survival during pro-longed periods of food scarcity. Future research isneeded to study the physiological properties offemale and juvenile crabs as well. Also, questionsabout the distribution and migration of larvae andthe ontogenesis of their physiological characteristicsshould be addressed.

Acknowledgements. We are grateful to P. P. Hwang at theInstitute of Cellular and Organismic Biology, AcademiaSinica for providing lab facilities, and we greatly acknowl-edge the assistance of Y. C. Tseng during preparation ofsamples for the SEM analysis. For assistance and guidanceduring sample collection at Kueishan Island, we particularlythank D. J. Kuo.

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Editorial responsibility: Christine Paetzold, Oldendorf/Luhe, Germany

Submitted: August 17, 2011; Accepted: November 25, 2011Proofs received from author(s): February 28, 2012

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