Journal of Thermal Biology 36 (2011) 128–137

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Journal of Thermal Biology

0306-45

doi:10.1

n Corr

E-m

URL

(S. Barr

journal homepage: www.elsevier.com/locate/jtherbio

Cancer pagurus (Linnaeus, 1758) physiological responses to simulated livetransport: Influence of temperature, air exposure and AQUI-Ss

Sara Barrento a,n, Antonio Marques a, Paulo Vaz-Pires b,c, Maria Leonor Nunes a

a Research Unit of Upgrading of Fishery and Aquaculture Products (U-VPPA), National Institute of Biological Resources (INRB I.P./L-IPIMAR), Avenida de Brasılia,

1449-006 Lisboa, Portugalb Institute of Biomedical Sciences Abel Salazar (ICBAS-UP), University of Porto, Largo Professor Abel Salazar 2, 4099-003 Porto, Portugalc Centre of Marine and Environmental Research, University of Porto (CIIMAR-UP), Rua dos Bragas 289, 4050-123 Porto, Portugal

a r t i c l e i n f o

Article history:

Received 8 October 2010

Accepted 21 December 2010Available online 29 December 2010

Keywords:

AQUI-Ss

Crustaceans

Edible crab

Stress

Vigour

Temperature

Air exposure

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016/j.jtherbio.2010.12.006

esponding author. Tel.: +351 21 3027025; fa

ail address: sarixbarrento@gmail.com (S. Barr

: http://ipimar-iniap.ipimar.pt/departamento

ento).

a b s t r a c t

Cancer pagurus is a commercially important crab, mostly appreciated in Southern European countries,

including Portugal, being usually live transported from UK and France. Once in Portugal, crabs are

redistributed across the country in small refrigerated vivier lorries in air or immersed conditions, while

some are sent exposed to air to the Azores archipelago with mortality reaching 40–60%. In order to

optimise transport conditions and survival, simulated live transport of immersed and air exposed crabs

sedated or not with an anaesthetic, AQUI-Ss, was tested at different temperatures. It was found that

crabs experienced stress during the experiment (with increased L-lactate, D-glucose and lowered pH),

but with different magnitudes according to temperature, treatment and transport duration, resulting in

100% mortalities at 16 1C in immersed conditions and when exposed to air with AQUI-Ss. Results

indicate that long duration transport in semi-dry conditions is viable at low temperatures (8 1C), while

immersed transport is viable at 12 1C. AQUI-Ss was not an efficient solution at low temperatures in

semi-dry conditions, but for short duration transport in immersed conditions at 16 1C it was the only

treatment without mortality.

& 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The European edible crab, Cancer pagurus, is one of the mostimportant commercial species in England, Wales and Ireland(Tully et al., 2006). However, this species is most appreciated insouthern European countries, such as France, Spain, Italy andPortugal where the demand for live edible crab is high, but theproduction is almost non-existent, e.g. Portugal (Barrento et al.,2008a). The journey is performed in vivier lorries from fishingcountries to Portugal, taking 2–3 days and live crabs are trans-ported in immersed conditions with transport mortalities of 1%(Barrento et al., 2008b). Once in Portugal crabs are either placed inplastic boxes exposed to air or immersed in tanks to be redis-tributed across the country in small refrigerated vivier trucks(12 1C), which further increases mortalities at the wholesalersand retailers (up to 40–60% respectively; Barrento et al. (2008a).Some crabs are sent by air freight to the Azores archipelago inStyrofoam sealed boxes (20 kg each; transport duration: 12–24 h)with coolers, but mortalities are also high (40%). There is, therefore,

ll rights reserved.

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ento).

s/inovacao-tecnologica.html

a high priority on improving national transport systems for ediblecrabs in order to diminish economical losses.

The transport of homarids, palinurid lobsters and crayfishexposed to air has proven to be efficient, as these animals manageto survive relatively long periods out of water (Jackson et al.,2001; Paterson and Spanoghe, 1997; Taylor and Whiteley, 1989;Whiteley et al., 1990; Whiteley and Taylor, 1988, 1990). This typeof transport can be an interesting approach in the live distributionof C. pagurus as it might elicit lower mortalities than thosereported by traders. However, to achieve better survival ratesseveral considerations must be taken into account: for instanceC. pagurus occupies subtidal habitats and, therefore, rarely experi-ences air exposure except during live marketing. Furthermore,environmental temperature are low (from 4 to 16 1C) comparedwith other species like Homarus americanus (from �1 to 24 1C;(Chartois et al., 1994; Lorenzon et al., 2007). Several studiesreported that gills collapse during emersion of subtidal species,resulting in respiratory acidosis, internal hypoxia and anaerobicmetabolism (Durand and Regnault, 1998). Therefore, the trans-port conditions (e.g. temperature, moisture, duration) must beoptimised to the specific needs of C. pagurus. Several studies havebeen conducted on the effect of emersion to physiologicalresponses of crustaceans, like lobsters (Fotedar et al., 2006;Jussila et al., 1997; Lorenzon et al., 2007; Marques-Rıos et al.,

Table 1Vigour scale of European edible crab, adapted from Barrento et al. (2008b).

Somatic response 0(dead)

1(moribund)

2(weak)

3(strong)

Eyestalk response No Yes Yes Yes

Claws and legs weightless Yes Yes No No

Slow claw and leg movements No No Yes No

Evident claw and leg movements No No No Yes

Strong claws with quick movements

(aggressive); claws and legs

strongly huddled to the abdomen

(defensive)

No No No Yes/No

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137 129

2007; Paterson and Spanoghe, 1997; Whiteley and Taylor, 1992)and crabs (Danford et al., 2001; Durand and Regnault, 1998;Otwell and Webb, 1977). However, as far as the European ediblecrab is concerned, only two studies were carried out so far byUglow et al. (1986) and Lorenzon et al. (2008). In the former, theeffect of air exposure and starvation were studied and it wasconcluded that both conditions were adverse to C. pagurus. Whilethe later study evaluated a consignment of crabs transported fromFrance to Italy during 36 h in semi-dry conditions and it wasconcluded that air transport of C. pagurus appears to have apositive impact on animal condition and welfare.

Anaesthetics are being tested in crustacean fisheries, followingtheir successful application in finfish aquaculture industry, notonly to avoid mortalities but also for welfare reasons (Seccombe,1999). Methods of paralyzing crabs can benefit commercialprocedures, but it is important to ensure that the quality of theproduct is not harmed by effects such as autotomy, which alsonegates the use of toxic chemicals (Gardner, 1997). AQUI-Ss is acommercial fish anaesthetic with isoeugenol as its active ingre-dient that is approved for food-safe use in New Zealand, Australia,Chile, Korea, Costa Rica and Honduras (Bowker et al., 2006). In theEuropean Union and USA, AQUI-Ss is undergoing the approvalprocess for use on seafood. This anaesthetic has the advantageof zero withdrawal period before animals can be slaughtered.Though the mode of action of isoeugenol is not yet fully under-stood, it has proven to be effective on freshwater prawns(Macrobrachium rosenbergii), but only at concentrations 5–10times higher (100–200 mg L�1) than those used on finfish (20 mgL�1; (Coyle et al., 2005; Meyer and Fish, 2008). AQUI-Ss efficacyhas never been tested so far in the edible crab.

In this context, this study aimed to understand C. pagurus

physiological changes during different transport systems, namelyin immersed and semi-dry conditions, using AQUI-Ss as ananaesthetic at different transport temperatures. An increasedunderstanding of the physiological changes imposed on crabsduring transport may lead to improvements in commercialhandling methods with better survival rates, less waste and moreefficient and sustainable utilisation of this valuable resource.

2. Material and methods

2.1. Ethical statement

Though crustaceans are not protected in the scientific proce-dures act, all live animals utilised in the experiments have beentreated with proper care to minimise discomfort and distress.Also, the number of animals was kept to the minimum necessaryto obtain scientific results, considering that the gain in knowledgeand long term benefit to the subject species is high.

2.2. Collection and treatment of animals

A total of 95 intermoult crabs with nicked claws (procedure usedby fishermen to inactivate claws; average weight 667.3793.2 g;width 134.1729.6 mm and length 115.9729.1 mm) were sampledin this study. Crabs were obtained in summer from a local importer,where they were maintained in a well aerated seawater re-circulat-ing system at 12 1C without feeding for 6 days to recover fromcapture and transport prior to the experiment. Refrigerated trucktransport was simulated during 48 h in cooling chambers at threetemperatures (8, 12 and 16 1C). Crabs were put in plastic tankshaving a proportionally reduced size comparing to tanks used atdeposit facilities. These tanks measured 16.5 cm depth, 28.0 cmwidth and 38.0 cm length. Crabs were exposed to four treatments ateach temperature: (a) immersed in aerated seawater (WT; 1 kg

crabs L�1); (b) pre-anaesthetized and immersed in aerated seawaterwith dissolved AQUI-Ss (New Zealand, Ltd.; 40 mg L�1; WQS;1 kg crab L�1); (c) exposed to air and covered with seawater-soakedcloth (AIR); and (d) pre-anaesthetized, exposed to air and coveredwith dissolved AQUI-Ss (40 mg L�1) soaked cloth (AQS). Crabsexposed to air were placed in boxes with a drainage system toavoid direct contact with excretion fluids. Crabs’ pre-anaesthetisa-tion involved immersion in seawater at 12 1C with dissolved AQUI-Ss (300 mg L�1) for 10–15 min until absence of claw aggressivenessreflex and reaction to handling (AQUI-Ss NZ, 2009). Animals weremonitored by haemolymph bleeding and vigour determination at 0(immediately before being put in tanks), 3, 8, 24 and 48 h. Care wastaken to avoid extra stress attributable to sampling as haemolymphwas removed in less than 50 s. Vigour was visually estimated andscaled according to behavioural observations, such as eye-stalkresponse, strength of claws and walking legs (with the modifiedscale by Barrento et al. 2008b; Table 1). Animals used for simulatedtransport were chosen similarly to industrial common practices,i.e. selection of crabs without weakness signs (vigour 3).

Haemolymph was sampled (1 mL) from the fifth walking legwith 1.2 mL paediatric syringes (Sarstedt, S-monovet) and 20 Gx1(1/2)00) hypodermic needles (Sarstedt, S-monovet needle), andtransferred to microtubes where pH was measured. Blood para-meters of 10 undisturbed animals maintained for eight dayswithout feeding in the stocking tanks of the importer wereanalysed and considered as the control group. Several seawaterquality parameters (pH, temperature and dissolved oxygen) weremeasured in the beginning and end of the simulated live trans-port of immersed crabs with a Multi 350i compact precisionhandheld metre (WTW, Wissenschaftlich-Technische Werkstat-ten). Before measurements, calibrations were performed. Anaquarium test kit was used to measure ammonia (Nutrafin, Ref.A-7820) and nitrites (Nutrafin, Ref. A-7825).

2.2.1. D-glucose

Haemolymph was deproteinated according to the procedure ofPaterson and Spanoghe (1997). D-glucose concentration wasmeasured using a D-glucose assay kit (Sigma GAGO-20), accordingto the procedure described by Barrento et al. (2008b).

2.2.2. L-lactate

L-lactate concentration of deproteinated haemolymph wasmeasured using L-lactate reagent Trinity Biotech (no. 735-10),according to the procedure described by Barrento et al. (2008b).

2.2.3. Haemocyanine

Haemocyanine was determined spectrophotometrically (UnicamUV–Vis spectrometry Helios Alpha) in 10-mm quartz cuvettesusing 50 ml of thawed haemolymph diluted in 2 ml of distilledwater. Absorbance was measured at 335 nm, and haemocyanineconcentration (mg mL–1) was calculated from the molar extinction

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137130

coefficient Emg ml�1

1 cm ¼ 0:0283, as described by Harris and Andrews(2005).

2.2.4. pH

The pH of haemolymph samples were measured at 8, 12 or 16 1Cafter sampling. Samples were measured with a pH metre (HannaInstruments, HI 221) using a pre-calibrated micro-electrode (HannaInstruments, HI 1083 P) and pH buffers 4.01, 7.01, and 10.00 (WTW,Wissenschaftlich-Technische Werkstatten) as standards.

2.3. Statistics

All analyses were carried out in duplicate. Results wereexpressed as mean values7standard deviation. In order to satisfyANOVA assumptions data were transformed, followed by multiplecomparison tests (Tukey HSD) to identify differences betweengroups. If transformed data could not meet ANOVA assumptions,non-parametric analysis of variance (Kruskall–Wallis) was per-formed, followed by non-parametric multiple comparisons test(Dunn). Whenever two groups were compared, Student’s t-testwas applied instead of ANOVA, followed by the correspondentnon-parametric test (Mann–Whitney) if transformed data couldnot meet Student’s t assumptions. Transport duration, vigour andtemperature were subjected to correlation analyses, using thePearson r correlation coefficient. All statistical analyses weretested at the 0.05 level of probability with the software STATIS-TICATM 6.1 (Statsoft, Inc., Tulsa, OK74104, USA).

3. Results

3.1. Water quality

During transport in immersed conditions, seawater qualityrevealed a strong decrease in oxygen and pH, but an increase inammonia and nitrite levels (Table 2). After 48 h of simulatedtransport the lowest dissolved oxygen concentration was obtainedat 8 1C with AQUI-Ss and 16 1C without AQUI-Ss. In contrast,the highest oxygen and lowest nitrite values were obtained at 8 1Cin seawater without the anaesthetic, while in treatments withAQUI-Ss the highest oxygen content was obtained at 12 1C.Seawater became more acidic at 8 and 16 1C with AQUI-Ss.

3.2. Vigour

The effects of temperature and duration of simulated livetransport on C. pagurus vigour are shown in Fig. 1. In general,vigour was lower at higher temperatures and prolonged transportduration. C. pagurus was unable to withstand prolonged simu-lated live transport (48 h) at 16 1C in WT and AQS treatments. Atthis temperature WT treatment mortalities occurred after 8 h, andat 24 h most crabs had already died (75%), while AQS mortalities

Table 2Seawater pH, dissolved oxygen (mg L�1), ammonia and nitrites concentration

(mg L�1) before simulated live transport (0 h) and after 48 h.

Treatment Temperature O2 pH Ammonia Nitrites

0 h 48 h 0 h 48 h 0 h 48 h 0 h 48 h

Water 8 1C 9.8 4.8 8.1 7.5 0.0 0.6 0.0 1.6

12 1C 9.5 2.8 8.0 7.5 0.0 0.6 0.0 3.3

16 1C 9.7 1.9 8.0 7.6 0.0 1.2 0.0 3.3

AQUI-Ss 8 1C 9.8 1.9 8.1 6.7 0.0 1.2 0.0 3.3

12 1C 9.7 3.2 8.1 7.6 0.0 0.6 0.0 3.3

16 1C 9.5 2.9 8.0 7.0 0.0 1.2 0.0 3.3

only started to occur after 24 h. At 16 1C, AIR had twice themortality than WQS at 24 h, despite both treatments ended with50% mortality at 48 h. The only treatment without any mortalityafter 48 h was WT at 12 1C. The treatment showing highermortalities and lower vigour at this temperature occurred inAIR, with 42.9% mortality and 57.1% moribund crabs after 48 h ofsimulated transport. At 8 1C, the lowest mortalities were observedin AIR and WQS (12.5%), while AQS crabs experienced the highestmortalities (37.5%) at this temperature.

3.3. Blood parameters

3.3.1. L-lactate

Haemolymph of crabs from the control group revealed lowlevels of L-lactate (0.2970.06 mM). The L-lactate concentration incrabs’ haemolymph at the three tested temperatures was alwaysabove the control levels (Table 3).

Significant differences in the L-lactate concentration wereobserved between temperatures and among treatments at the sametemperature (Table 3). WT crabs at 8 and 16 1C had more L-lactate inthe haemolymph than crabs at 12 1C (Table 3). These differenceswere detected after 8 h in crabs at 16 1C, but only after 24 h in crabsat 8 1C. Among WQS crabs, those at 12 1C started with the lowestL-lactate levels and kept low values compared to crabs at 16 1C. AIRcrabs at 8 1C showed the lowest L-lactate values between 8 and 48 hthan in the remaining temperatures. A similar trend was observed inthe AQS group, though with comparatively higher values.

Significant differences between treatments at 8 1C occurred from8 h onwards, with AIR treatment having lower L-lactate values, aswell as WQS at 48 h (Fig. 2). At 12 1C, significantly lower L-lactatevalues were detected from 8 h onwards in WT and WQS treatmentscompared to air exposed crabs independently of using anaesthetic(AIR and AQS). Crabs kept at 16 1C in WQS treatment had statisti-cally lower L-lactate levels between 8 and 24 h than AQS.

3.3.2. pH

Haemolymph pH of crabs from all treatments is presented inTable 3. All crabs had lower pH in the beginning of the experi-ment compared to the control group (7.6470.03). Regardless oftemperature, WQS crabs had pH levels similar to the controlgroup after 24 h. Particularly, crabs at 8 1C returned to pH controlvalues after 8 h and kept these values until the end of theexperiment. WT (12 1C) and AIR (8 1C) crabs also returned tocontrol levels after 24 h. WT crabs at 16 1C, had the most acidicpH of all groups in the beginning of the experiment (0 h) and didnot survive until the end of the experiment (Table 3). A similartrend occurred with AQS crabs at 16 1C.

Major differences were observed in the AIR treatment withcrabs at 8 1C having higher pH than crabs at 12 and 16 1C from 3 honwards.

Significant differences between groups at the same temperaturewere also detected (Fig. 2). In fact, at 8 1C WT and AQS crabs had themost acidic haemolymph from 3 h onwards. At 12 1C a clearseparation was observed between WT and AIR (since 8 h) and AQS(since 24 h), with WT crabs showing higher pH values. At 16 1C, WTcrabs and AQS crabs had the most acidic haemolymph values from 3to 8 h onwards, respectively, and these animals did not survive.

3.3.3. D-glucose

Haemolymph of all crabs was mostly hyperglycaemic throughoutthe experiments compared to the control group (0.1970.05 mM),except at 16 1C in WT (24 h) and at 12 1C in AQS (48 h; Table 4).Few significant differences were detected in glucose concentra-tion between temperatures and between treatments at the same

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137 131

temperature. Additionally, D-glucose variability during the experi-ments did not reveal any clear pattern.

3.3.4. Haemocyanine

In general, haemolymph haemocyanine showed great variabilityin all treatments (Table 4). Haemocyanine concentration of WT, AQSand AIR crabs was similar to the control group (28.275.0 mg L�1)at 8 1C, while generally higher haemocyanine values were obtainedat 12 and 16 1C (except in AIR at 12 1C and WQS at 16 1C). Nosignificant differences in haemocyanine concentration of crabs weredetected between treatments at the same temperatures.

3.4. Correlations

Correlations between each haemolymph parameter and trans-port temperature, duration and vigour of crabs are shown in

WT

100.088.9

77.8

44.422.2

33.322.2

11.122.2

11.1

33.3

22.2

11.1

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

100.0 100.0 100.0 100.0100.0

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

87.5

12.5

75.0

100.0

25.0

12.5

100.075.0

12.5

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

8 °C

12 °

16 °

n = 9

n = 7

n = 8

3 (strong) 2 (weak)

Fig. 1. Vigour of crabs during experimental conditions. Abbreviations: (WT) immersed c

semi-dry conditions; (AQS) air exposed crabs in semi-dry conditions pre-treated with

(2) weak crabs; (3) strong crabs.

Table 5. Considering that vigour in crabs treated with AQUI-Ss

can be misleading, correlations were not analysed in treatmentswith AQUI-Ss.

Significant positive correlations were detected between tem-perature and L-lactate (AIR and AQS crabs), as well as withD-glucose (WQS and AQS), whereas negative correlations wereobserved between pH (WQS and AIR) and haemocyanine (WQS).

In general, L-lactate concentration was positively correlated withtransport duration, except at 8 1C in AIR (negative correlation) andWQS (no correlation), as well as at 12 1C in WT (no correlation).Significant negative correlations with transport duration were regis-tered for pH at 12 and 16 1C in AQS, whereas positive correlationswere detected in WQS at all temperatures, at 12 1C in WT, and at 8 1Cin AIR. Significant correlations between transport duration andD-glucose were only observed at 8 1C in WQS (positive correlation)and AIR (negative correlation). As far as haemocyanine is concerned,the only positive correlation with transport duration was detected in

WQS

100.087.5

62.5

12.5

12.5

87.5

25.0

12.5

75.0

25.0

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

62.5

25.037.5

25.012.5

100.0

12.5 12.5

62.550.0 75.0

12.5

12.5

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

25.0

62.575.0

12.5

100.0 50.0

37.525.0

37.5

12.550.0

12.5

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

C

C

n = 8

n = 8

n = 8

1 (moribund) 0 (dead)

rabs in seawater; (WQS) immersed crabs with AQUI-Ss; (AIR) air exposed crabs in

AQUI-Ss; vigour-level index: (0) dead crabs (cumulative); (1) moribund crabs;

Air

100.0 100.087.5

12.5

87.5 87.5

12.5 12.5

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

AQS

100.0

25.0

12.575.0

25.0

25.0

37.5

50.0

37.5

25.025.037.5

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

87.5

50.0

100.0

12.5

12.5

12.5

50.0

62.5

37.550.0

25.0

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

37.5

12.5

100.0

25.050.0

62.5

37.5

50.0

100.0

25.0

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

57.1

28.614.3

42.9

57.1

42.9

14.3

14.3

57.1

28.642.9

100.0

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

75.0 75.0

25.0

25.0

62.525.0

37.5

25.0

100.0

25.0 25.0

0%

20%

40%

60%

80%

100%

0 h 3 h 8 h 24 h 48 h

3 (strong) 2 (weak) 1 (moribund) 0 (dead)

12 °C

16 °C

n = 7 n = 8

n = 8 n = 8

n = 8 n = 8

Fig. 1. (Continued)

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137132

AQS at 12 1C, while the only negative correlation was registered forthe same treatment at 16 1C.

Correlations between vigour and blood parameters revealedthat L-lactate was negatively correlated at 16 1C in both treat-ments (WT and AIR), and at 12 1C in AIR. Regarding pH, WT at 8 1Chad a negative correlation with vigour, while AIR had a positivecorrelation at 12 1C. D-glucose concentration was negativelycorrelated with vigour in WT. Finally, haemocyanine was notcorrelated with vigour in any treatment.

4. Discussion

This experiment is an attempt to simulate transport systemsalready in use to distribute crabs in Portugal and to test possiblealternatives that might improve these systems. Therefore, it isimportant to bear in mind that the control group is not meant torepresent C. pagurus basal levels, as crabs have already been exposed

to several stressors (mainly capture and import transport). It is rathera control with unfed crabs not subjected to additional stressors.Similar results were reported by Lorenzon et al. (2008) and Ridgwayet al. (2006) for the control group of 15 crabs imported from France(transport duration 36 h) and rested at the premises of the Italianimporter company for 96 h feeding ad libitum (pH, 7.76; L-lactate,0.34 mM; D-glucose, 0.39 mM).

In the beginning of the experiments crabs already revealedstress signs, as L-lactate and D-glucose levels were above those ofthe control group and pH was more acidic. Such stress could beattributed to the handling procedures. It is also important to statethat crabs were affected in different ways, as apparently WQScrabs at 12 1C had lower L-lactate and D-glucose concentrationsthan the remaining crabs. The results indicate that crabs experi-enced physiological stress during simulated live transport, thoughwith different magnitudes depending on temperature, treatmentand transport duration. Previous studies have also reporteddifferent magnitudes of stress according to the above variables,

Table 3L-lactate (mM) and pH in C. pagurus haemolymph measured for each treatment at 8, 12 and 16 1C during 48 h. Values are presented as average followed by standard

deviation.

L-lactate T (1C) 0 h 3 h 8 h 24 h 48 h

WT 8 4.5471.50a*** 8.8873.43a*** 11.5772.83ab*** 12.6675.83a*** 15.7875.76a***

12 5.0672.16a*** 5.4270.91a*** 4.8071.50b*** 2.4870.26b*** 7.3075.12b***

16 5.5171.62a*** 8.8472.05a*** 14.6273.64a*** 21.1970.05a*** –

WQS 8 3.7070.50ab*** 9.8571.31ab*** 12.1071.65a***12.5875.03a %

a** 6.4974.07b***

12 2.9070.70b*** 5.2071.58b*** 6.3672.19b*** 8.3674.62a*** 6.5874.70b*

16 4.3870.81a*** 15.7473.11a*** 9.9773.51ab* 10.8275.18a*** 29.8875.76a***

AIR 8 4.4070.62a*** 8.6371.05a*** 7.9171.48b*** 2.9371.75b** 1.7570.28b***

12 5.0070.85a*** 8.3671.80a*** 13.1772.65a*** 19.7173.99a*** 35.95716.32a***

16 3.5370.95a*** 9.0772.05a*** 14.0473.15a*** 16.12711.31a** 41.9278.92a***

AQS 8 2.8371.22a*** 7.8873.53a*** 10.8073.60b*** 10.7873.57b*** 14.9673.35b***

12 4.1770.66a*** 8.4570.92a*** 15.1275.00ab*** 22.3474.98a*** 30.3176.62a***

16 4.5172.01a*** 10.0172.34a*** 16.4871.07a*** 24.0772.92a*** –

pH T1C 0 h 3 h 8 h 24 h 48 h

WT 8 7.2370.03a*** 7.2270.07b*** 7.1670.08b*** 7.2770.16b*** 7.3070.13a***

12 7.2970.04a*** 7.4170.07a*** 7.4270.12a*** 7.6570.09a 7.3670.06a***

16 7.1270.09b*** 7.1470.06b*** 7.1470.14b*** 6.9170.12b*** –

WQS 8 7.2870.08a*** 7.4970.10a** 7.5970.10a 7.6070.14a 7.6570.16a

12 7.2670.08a*** 7.2870.27a** 7.4370.25ab*** 7.5570.13a 7.4870.11ab**

16 7.2170.08a*** 7.3170.09a*** 7.2870.14b*** 7.6770.09a 7.2370.02b***

AIR 8 7.2570.05a*** 7.4970.09a*** 7.4470.11a*** 7.6370.08a 7.6370.08a

12 7.2170.12a*** 7.2670.20b*** 7.1570.12b*** 7.2270.04b*** 7.0370.09b***

16 7.2370.11a*** 7.3070.11b*** 7.3170.06ab*** 7.3570.24b** 6.6770.13c***

AQS 8 7.2870.07a*** 7.1870.20a*** 7.1570.17a*** 7.3270.17a*** 7.4770.23a***

12 7.3070.06a*** 7.3170.04a*** 7.2870.03a*** 7.3070.04a*** 7.1070.06b**

16 7.2370.06a*** 7.2470.08a*** 7.2070.05a*** 7.0370.12b*** –

For each treatment different letters in each column represent significant differences between temperatures (p values not shown); Statistical differences to control values

are represented as follows: ***po0.001; **po0.01; *po0.05. Abbreviations: (WT) immersed crabs in seawater; (WQS) immersed crabs with AQUI-Ss; (AIR) air exposed

crabs in semi-dry conditions; (AQS) air exposed crabs in semi-dry conditions pre-treated with AQUI-Ss.

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137 133

as well as trawling season, capture method and handling (Albalatet al., 2009; Harris and Andrews, 2005; Lund et al., 2009).

L-lactate and pH were the haemolymph parameters thatprovided clear indications of stress in response to simulatedtransport over a 48 h period. It is known that the developmentof anaerobic metabolism leads to the production of L-lactate andacidic pH (Jackson et al., 2001; Ridgway et al., 2006; Taylor andWhiteley, 1989; Whiteley and Taylor, 1990). Anaerobic metabo-lism can be elicited by a hypoxic environment and by an increasein metabolic rate of animals due to increase in temperature,which boosts the energy demands of the animal (Durand et al.,2000; Ridgway et al., 2006).

The results of seawater quality after 48 h clearly indicate thata hypoxic environment was created in immerse systems, and pHdecreased probably due to L-lactate and CO2 excretion. Thedevelopment of anaerobic metabolism and the decrease in sea-water quality is an inevitable consequence of high animal densityand low dissolved oxygen. As expected at higher temperaturesthe consequences were more severe. Even though seawaterquality was poorer, an increase in temperature from 8 to 12 1Cin immersed crabs (WT and WQS) did not cause an increase in thestress response by crabs as measured by pH and lactate levels. Onthe contrary, WT crabs at 12 1C showed no mortalities and lessL-lactate concentration (7.3 mM) than crabs at 8 1C (lactate,15.8 mM; 22.2% mortality; Fig. 1). Also, the same temperatureincrease in WQS treatments did not reveal any clear effect onmortality, lactate concentration and pH.

The effect of raising temperature from 8 to 12 1C had a muchlarger detrimental effect on stress parameters of crabs exposed toair in both treatments, but the effect on vigour was greater in AIRcrabs (Tables 3 and 4; Fig. 1). L-lactate accumulation has beenextensively described as being associated with aerial exposure,

particularly in subtidal crustaceans, as emersion impairs gillfunctioning and the efficient release of excretion products, thusleading to further hypoxia or anoxia and anaerobic metabolism(Durand et al., 2000; Whiteley et al., 1990; Whiteley and Taylor,1990, 1992). Previous studies reported that L-lactate concentra-tion and pH are negatively influenced by air exposure andincreased temperature in several crustacean species, such asNephrops norvegicus (Lund et al., 2009; Ridgway et al., 2006;Spicer et al., 1990), Penaeus japonica (Chen and Chen, 1998), Jasus

edwarsii (Morris and Oliver, 1999) and Homarus americanus

(Lorenzon et al., 2007). On the other hand, low temperatureshave been referred as an anaesthesia inducer, which allowsanimals to be handled without apparent stress (Spanoghe andBourne, 1997). Therefore, survival rates were improved in crus-taceans exposed to air at low temperatures in H. americanus

(5–10 1C; McLeese, 1958), Callinectes sapidus (10 1C; Otwell andWebb, 1977), H. gammarus (5–10 1C; Whiteley and Taylor, 1992),Panulirus cygnus (17–20 1C; Spanoghe and Bourne, 1997),P. japonicus (6–12 1C; Chen and Chen, 1998) and N. norvegicus

(10 1C; Ridgway et al., 2006). In this study, haemolymph pH of AIRcrabs at the lowest temperatures (8 1C) was positively correlatedwith transport duration, while L-lactate was negatively correlated.A similar trend was reported by Bernasconi and Uglow (2008) forN. norvegicus during prolonged emersion (72 h) at 5 1C and byJackson et al. (2001) for the crayfish Austropotamobius pallipes

during prolonged emersion (24 h). Jackson et al. (2001) furtherexplains that this is due to lactate accumulation in the exoskele-ton of the crayfish in proportion to the haemolymph concentra-tion. In fact, the regression equation relating exoskeletal tohaemolymph concentrations indicated that the exoskeletal con-centration (mmol kg�1) averaged 48% of the haemolymph con-centration (mmol l�1; Jackson et al., 2001).

8 ºC

0.0

10.0

20.0

30.0

40.0

50.0

0 h 3 h 8 h 24 h 48 h C

Lact

ate

(mM

)6.46.66.87.07.27.47.67.8

0 h 3 h 8 h 24 h 48 h C

pH

12 ºC

0.0

10.0

20.0

30.0

40.0

50.0

0 h 3 h 8 h 24 h 48 h C

Lact

atet

(mM

)

6.46.66.87.07.27.47.67.8

0 h 3 h 8 h 24 h 48 h CpH

16 ºC

0.0

10.0

20.0

30.0

40.0

50.0

0 h 3 h 8 h 24 h 48 h C

Lact

ate

(mM

)

6.46.66.87.07.27.47.67.8

0 h 3 h 8 h 24 h 48 h C

pH

WT a b b c bWQS a a a ab a

Air a a a a aAQS a b b bc ab

p 0.347 0.000 0.000 0.001 0.006

WT a a ab a aWQS a a a a b

Air a a b b bAQS a a ab a a

p 0.058 0.260 0.028 0.003 0.000

WT a b ab ab -WQS a a b b a

Air a b ab ab aAQS a b a a -

p 0.171 0.000 0.026 0.041 0.083

WT a b b b -WQS a a ab a a

Air a a a ab bAQS a ab b b -

p 0.044 0.005 0.009 0.002 0.021

WT a a a a aWQS a a a ab ab

Air a a b b bAQS a a ab b b

p 0.204 0.218 0.006 0.000 0.001

WT ab b c b bWQS b a bc b b

Air a a ab a aAQS ab a a a a

p 0.011 0.001 0.001 0.000 0.001

Fig. 2. Changes in L-lactate concentration (mM) and pH values of C. pagurus during the experiments at 8, 12 and 16 1C in crabs exposed to air in semi-dry conditions (’;

AIR), immersed in seawater (K; WT), immersed in seawater with AQUI-Ss (J; WQS), and exposed to air in semi-dry conditions with AQUI-Ss (&; AQS). The p value is

shown for each column and different letters for each column represent significant changes between treatments. Control values shown as C (*).

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137134

The high survival rate monitored in air exposed crabs at 8 1C islikely due to the low L-lactate levels and high pH in haemolymph,which has also been shown to be positively correlated in othercrustaceans (Chen and Chen, 1998; Taylor and Whiteley, 1989;Whiteley and Taylor, 1990). Particularly, pH of crabs at 8 1C returnedto control values after 8 h, but not at 12 and 16 1C. This might be dueto a buffering capacity similar to that observed in H. gammarus

(Whiteley and Taylor, 1990), where the elevation of bicarbonatesupported 75% of haemolymph buffering capacity and compensatedfor an incipient acidosis after 14 h in air at 10 1C, but not at 20 1C.

Of all crabs that survived the 48 h experimental period, thebest treatments were AIR at 8 1C and WT at 12 1C, taking intoaccount L-lactate, pH and vigour measurements.

It is known that anaesthetics reduce or minimise fish stress bydecreasing metabolism (Iversen et al., 2003). In the current study,

it is likely that low temperature and AQUI-Ss decreased themetabolism far too much in AQS crabs, leading to deep anaesthesia,i.e. reduction of breathing and gas transfer, hypoxia and respiratoryacidosis due to the reduction of blood O2 tension and a concomitantrise in blood CO2. This is characteristic of deep anaesthesia, andprolonged maintenance of this stage may result in death (Ackermanet al., 2005). In fact, at 8 1C the highest mortality rate was observedin the AQS treatment (37.5%); this rate was greater than at 12 1C forWT (0%), WQS (12.5%) and AQS (25%), but similar to the AIRtreatment at 12 1C (42.9%). Therefore, the tested concentration ofAQUI-Ss was not an efficient solution for live transport of crabs at8 1C in semi-dry conditions, but can be an option at 12 1C. Never-theless, further studies are still required to test different AQUI-Ss

concentrations that might optimise the use of this anaesthetic undersuch particular conditions.

Table 4D-glucose (mM) and haemocyanine (mg mL�1) in C. pagurus haemolymph measured for each treatment at 8, 12 and 16 1C during 48 h. Values are presented as average

followed by standard deviation.

D-glucose (mM) T1C 0 h 3 h 8 h 24 h 48 h

WT 8 0.8770.59a** 0.9570.64a** 1.7070.45a*** 0.7770.64a*** 1.1070.87a*

12 0.6570.24a*** 0.6470.31a** 1.0270.41b*** 0.6370.16a*** 0.3570.09a**

16 1.8170.82a*** 0.7670.15a*** 1.4870.53ab*** 0.3970.33a –

WQS 8 0.1070.06b*** 0.6970.56a* 1.0770.56a*** 1.1270.52a*** 0.8870.73a*

12 0.0670.03b*** 0.9970.44a*** 0.8770.40a*** 1.3070.50a*** 0.4370.31a*

16 2.0570.58a*** 1.2370.32a*** 1.2070.17a*** 1.4670.37a*** 1.2370.35a***

AIR 8 1.2470.45a*** 1.0070.47a*** 1.0870.43a*** 0.4770.26a* 0.8270.74a*

12 0.8870.55a** 0.8470.67a* 0.9470.52a*** 0.5970.41a* 0.9770.19a***

16 1.5970.90a*** 0.7070.49a* 1.1570.53a*** 1.2070.84a** 1.1170.62a**

AQS 8 0.8970.34b*** 1.3370.54a*** 1.4870.74a*** 1.3970.63a*** 1.2370.44a***

12 1.1870.66a** 1.3570.57a*** 1.4670.48a*** 1.1370.56a*** 0.6270.60a

16 1.9770.58a*** 1.3970.57a*** 1.7170.28a*** 1.6570.42a*** –

Haemocyanine T1C 0 h 3 h 8 h 24 h 48 h

WT 8 37.5715.2a 37.6713.3a 38.0714.4ab 38.0717.2 %a 48.8721.0a

12 4576.8a*** 43.674.4a*** 48.075.6a*** 46.777.7a*** 48.876.6a***

16 36.979.8a* 40.6712.8a* 23.979.3b 48.2715.3a** –

WQS 8 41.4712.4a* 54.9713.3a*** 49.1711.0a*** 51.6714.4a*** 55.4713.5a***

12 37.079.3a* 42.079.5a** 41.079.1ab*** 43.078.8ab** 43.478.7a**

16 37.8715.4a 42.478.8a** 25.1710.3b 28.674.0b 46.576.6a*

AIR 8 30.9710.5a 39.7710.7a* 37.1711.8a 34.9711.3a 31.3718.1a

12 36.0710.2a 35.7711.0a 36.679.8a 36.2712.5a 35.0722.0a

16 39.7714.0a* 45.7716.9a* 22.3713.9a 28.5712.9a 51.8711.0a*

AQS 8 28.4718.5a 31.0716.0a 29.2715.5ab 31.6716.3a 39.1718.6a

12 40.2712.1a* 40.5710.2a** 39.878.7a** 47.979.3a*** 51.0710.7a***

16 43.4711.7a** 46.0714.6a** 19.777.4b* 36.2710.2a –

For each treatment different letters in each column represent significant differences between temperatures (p values shown); Statistical differences to control values are

represented as follows: ***po0.001; **po0.01; *po0.05. Abbreviations: (WT) immersed crabs in seawater; (WQS) immersed crabs with AQUI-Ss; (AIR) air exposed crabs

in semi-dry conditions; (AQS) air exposed crabs in semi-dry conditions pre-treated with AQUI-Ss.

Table 5Pearson’s correlation coefficient of haemolymph parameters of each treatment in relation to temperature, transport duration and vigour.

Parameter Treatments T 1C Transport duration Vigour

8 1C 12 1C 16 1C 8 1C 12 1C 16 1C

L-lactate WT r¼�0.05ns r¼0.68*** r¼�0.22ns r¼0.88*** r¼�0.66ns r¼0.26ns r¼�0.57**

WQS r¼0.15ns r¼0.21ns r¼0.41* r¼0.58** – – –

AIR r¼0.43*** r¼�0.59** r¼0.96*** r¼0.79*** r¼0.32ns r¼�0.67*** r¼�0.43*

AQS r¼0.24* r¼0.81*** r¼0.94*** r¼0.96*** – – –

pH WT r¼�0.15ns r¼0.13ns r¼0.43* r¼�0.04ns r¼�0.48** r¼0.13ns r¼0.06ns

WQS r¼�0.37*** r¼0.68* r¼0.49** r¼0.44* – – –

AIR r¼�0.46* r¼0.81* r¼�0.33ns r¼�0.06ns r¼0.30ns r¼0.50** r¼0.04ns

AQS r¼�0.22ns r¼0.20ns r¼�0.50** r¼�0.54** – – –

D-glucose WT r¼0.08ns r¼0.00ns r¼�0.32ns r¼�0.32ns r¼�0.24ns r¼�0.38* r¼0.26ns

WQS r¼0.40*** r¼0.54** r¼0.32ns r¼�0.34ns – – –

AIR r¼0.11ns r¼�0.47** r¼�0.01ns r¼�0.13ns r¼�0.18ns r¼�0.22ns r¼0.09ns

AQS r¼0.23* r¼0.32ns r¼�0.29ns r¼�0.09ns – – –

Hcy WT r¼�0.01ns r¼0.10ns r¼0.23ns r¼�0.18ns r¼�0.05ns r¼0.29ns r¼0.31ns

WQS r¼�0.45*** r¼0.29ns r¼0.22ns r¼�0.11ns – – –

AIR r¼0.04ns r¼0.09ns r¼0.16ns r¼�0.08ns r¼0.11ns r¼�0.24ns r¼0.21ns

AQS r¼0.13ns r¼0.23ns r¼0.42* r¼�0.37* – – –

Symbols: ***po0.001, **po0.01, *po0.05, ns, not significant. Abbreviations: Hcy, haemocyanine; (WT) immersed crabs in seawater; (WQS) immersed crabs with AQUI-Ss;

(AIR) air exposed crabs; (AQS) air exposed crabs pre-treated with AQUI-Ss. The correlation between haemolymph parameters and vigour was not analysed for crabs

treated with AQUI-Ss.

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137 135

Crabs stored at 16 1C were not able to adapt to such hightemperature and, consequently, stress responses developed after3 h either in immersed or exposed to air conditions. The stressresponses were cumulative to a point that some crabs were forcedbeyond their tolerance limits and died (WT and AQS treatments).

Nevertheless, AQUI-Ss had a positive effect on C. pagurus survivalrate at higher temperatures, and in blood L-lactate and pH valuesduring simulated transport in AQS (12 1C) and WQS (16 1C) treat-ments. Probably, the anaesthetic is counteracting the negativeeffects of high temperatures by delaying metabolic and energy

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137136

demands. Other authors suggested that temperature increase islikely to increase the efficacy in anaesthetics (Olsen et al., 1995;Stehly and Gingerich, 1999). However, at such high temperatures(16 1C) transport duration is detrimental, as confirmed by thepositive correlation with L-lactate.

Vigour was a good visual estimate of physiological stress inC. pagurus, especially in treatments at higher temperatures andexposed to air, where crabs experience more severe stress andwider variations in physiological haemolymph variables.

Overall, D-glucose and haemocyanine did not change muchover the 48 h transport period. It is possible that the initial airexposure was sufficient to increase D-glucose levels to such anextent that no further increase was elicited. In this study, areduction of D-glucose was only detected in WT crabs at 16 1Cafter 24 h and at 12 1C in AQS treatment after 48 h. This reductionin D-glucose to control levels might have been due to the elevatedmetabolic rates that lead to a depletion of tissue glycogenconcentrations and a more premature cessation of glucoserelease. Once exhausted of glycogen, tissues are unable to re-supply glucose to haemolymph and maintain carbohydratehomoeostasis (Ridgway et al., 2006). On the other hand, haemo-cyanine is the most important protein in crustacean’s haemo-lymph (75–100% of total blood proteins), is responsible foroxygen transport and is also a food reserve (Uglow et al., 1986).The haemocyanine concentration in haemolymph of crabsrevealed great variation between individuals (from 7.2 to 72.5 mgmL�1). Haemocyanine in C. pagurus haemolymph has been pre-viously monitored by Uglow et al. (1986), who also reportedvariable values ranging from 20.42 to 63.62 mg mL�1. Consider-ing that this protein is an important food reserve, the greatvariability among crabs is probably due to different nutritionalconditions, which may further influence their ability to cope withstressful conditions during transport and subsequent recoverywithout feeding (Uglow et al., 1986).

5. Conclusions

From a commercial perspective it is important to strive, toensure that it is possible to transport live C. pagurus with lessmortality than the systems currently employed by the crabindustry. This can be achieved by keeping the animals at lowtemperatures (8 1C or lower), in humid conditions and ensuringthat they are not pilled. Whiteley and Taylor (1988) also high-lighted the importance to maintain crabs and lobsters at lowtemperatures along the trade chain and to reduce handling stressduring handling. It is also essential to bear in mind that transportduration strongly affects the crabs’ quality and mortality; there-fore, transport journeys should be as short as possible. Moreover,grading animals prior to transport is fundamental since the initialcondition strongly influences survival. Whilst it is also feasible tohave zero mortality during 48 h transport of immersed crabs at12 1C, it requires complex logistics and is expensive compared tosemi-dry transport, which is more advantageous from a commer-cial perspective. Therefore, it is highly recommended to developin situ tests in industrial operating conditions.

In this study the advantage of using AQUI-Ss was mainlyobserved for short transport duration at 16 1C. Yet, more studiesare still required to test different concentrations in order tooptimise the use of anaesthetics at different temperatures.

The key issue is the handling procedure before and aftertransport and the temperatures employed in semi-dry transport.It is important to bear in mind that even though mortalities canbe lowered during transport, the subsequent procedures cannotbe neglected and further attention must be paid to avoid tem-perature changes and stressfull handling procedures.

Acknowledgments

The first and second authors acknowledge the PortugueseFoundation for Science and Technology (FCT) for supportingrespectively a Ph.D. scholarship and a Research contract (Refs.SFRH/BD/24234/2005 and Ciencia 2008). The European Commis-sion supported this research through the Collective ResearchProject ‘‘CrustaSea: development of best practise, grading andtransportation technology in the crustacean fishery sector’’ (Ref.COLL-CT-2006-030421). The authors also acknowledge the con-tributions of reviewers.

References

Ackerman, P.A., Morgan, J.D., Iwama, G.K., 2005. Anesthetics. In: (CCAC), C.C.o.A.C.(Ed.), Guidelines on the care and use of fish in research, teaching and testing,pp. 87.

Albalat, A., Gornik, S.G., Atkinson, R.J.A., Coombs, G.H., Neil, D.M., 2009. Effect ofcapture method on the physiology and nucleotide breakdown products in theNorway lobster (Nephrops norvegicus). Marine Biology Research, 441–450.

Barrento, S., Marques, A., Pedro, S., Vaz-Pires, P., Nunes, M.L., 2008a. The trade oflive crustaceans in Portugal: space for technological improvements. ICESJournal of Marine Science 65, 551–559.

Barrento, S., Marques, A., Vaz-Pires, P., Nunes, M.L., 2008b. Live shipment ofimmersed crabs Cancer pagurus from England to Portugal and recovery instocking tanks: stress parameter characterization. ICES Journal of MarineScience 67, 435–443.

Bernasconi, C.J., Uglow, R.F., 2008. Effects of emersion-induced hypoxia on somehaemolymph constituents of Nephrops norvegicus. Diseases of Aquatic Organ-isms 82, 135–143.

Bowker, J.D., Carty, D., Bowman, M.P., 2006. The efficacy of AQUI-Ss as ananaesthetic for use on juvenile and adult largemouth bass Micropterussalmonides, Drug Research Report US, p. 10.

Chartois, H., Latrouite, D., Le Carre, P., 1994. Stocking and transportation of livecrustaceans. Report of the directorate of live resources, IFREMER, Institut Franc-ais de Recherche pur L’Exploitation de la Mer, Brest, p. 65.

Chen, J., Chen, J., 1998. Acid–base balance, ammonia and lactate levels in thehaemolymph of Penaeus japonicus during aerial exposure. Comparative Bio-chemistry and Physiology—Part A: Molecular & Integrative Physiology 121,257–262.

Coyle, S.D., Dasgupta, S., Tidwell, J.H., Beavers, T., Bright, L.A., Yasharian, D.K., 2005.Comparative efficacy of anesthetics for the freshwater prawn Macrobrachiurnrosenbergii. Journal of the World Aquaculture Society 36, 282–290.

Danford, A.R., Uglow, R.F., Rosas, C., 2001. Physiological responses of blue crabs(Callinectes sp.) to procedures used in the soft crab fishery in La Laguna deTerminos, Mexico. University of Alaska Sea Grant, AK-SG-01-03, Seattle.

Durand, F., Devillers, N., Lallier, F.H., Regnault, M., 2000. Nitrogen excretion andchanges in haemolymph components during emersion of the subtidal spidercrab Maja squinado (L.). Comparative Biochemistry and Physiology, Part A:Molecular & Integrative Physiology 127, 259–271.

Durand, F., Regnault, M., 1998. Nitrogen metabolism of two portunid crabs,Carcinus maenas and Necora puber during prolonged air exposure and sub-sequent recovery: a comparative study. Journal of Experimental Biology 201,2512–2528.

Fotedar, S., Evans, L., Jones, B., 2006. Effect of holding duration on the immunesystem of western rock lobster, Panulirus cygnus. Comparative Biochemistryand Physiology—Part A: Molecular & Integrative Physiology 143, 479–487.

Gardner, C., 1997. Options for humanely immobilizing and killing crabs. Journal ofShellfish Research 16, 219–224.

Harris, R.R., Andrews, M.B., 2005. Physiological changes in the Norway lobsterNephrops norvegicus (L.) escaping and discarded from commercial trawls onthe West Coast of Scotland. 2. Disturbances in haemolymph respiratory gases,tissue metabolites and swimming performance after capture and duringrecovery. Journal of Experimental Marine Biology and Ecology 320, 195–210.

Iversen, M., Finstad, B., McKinley, R.S., Eliassen, R.A., 2003. The efficacy ofmetomidate, clove oil, Aqui-STM and Benzoaks as anaesthetics in Atlanticsalmon (Salmo salar, L.) smolts, and their potential stress-reducing capacity.Aquaculture 221, 549–566.

Jackson, D., Wang, T., Koldkjaer, P., Taylor, E., 2001. Lactate sequestration in thecarapace of the crayfish Austropotamobius pallipes during exposure in air.Journal of Experimental Biology 204, 941–946.

Jussila, J., Jago, J., Tsvetnenko, E., Dunstan, B., Evans, L.H., 1997. Total anddifferential haemocyte counts in western rock lobsters (Panulirus cygnusGeorge) under post-harvest stress. Marine and Freshwater Research 48,863–867.

Lorenzon, S., Giulianini, P.G., Libralato, S., Martinis, M., Ferrero, E.A., 2008. Stresseffect of two different transport systems on the physiological profiles of thecrab Cancer pagurus. Aquaculture 278, 156–163.

Lorenzon, S., Giulianini, P.G., Martinis, M., Ferrero, E.A., 2007. Stress effect ofdifferent temperatures and air exposure during transport on physiological

S. Barrento et al. / Journal of Thermal Biology 36 (2011) 128–137 137

profiles in the American lobster Homarus americanus. Comparative Biochem-istry and Physiology—Part A: Molecular & Integrative Physiology 147, 94–102.

Lund, H.S., Wang, T., Chang, E.S., Pedersen, L.F., Taylor, E.W., Pedersen, P.B.,McKenzie, D.J., 2009. Recovery by the Norway lobster Nephrops norvegicus(L.) from the physiological stresses of trawling: influence of season and live-storage position. Journal of Experimental Marine Biology and Ecology 373,124–132.

Marques-Rıos, E., Gomez-Jimenez, S., Ocano-Higuera, V.M., Castillo-Yanez, F.J.,Pacheco-Aguilar, R., 2007. Quality parameter of spiny lobster Panulirus inter-ruptus tails as affected by short term emersion at two different temperatures.Ciencia Marina 33, 73–82.

McLeese, D.W., 1958. Air Shipment of Lobsters. Trade News. Department ofFisheries Canada, pp. 5–6.

Meyer, R.E., Fish, R.E., 2008. Pharmacology of injectable anaesthetics. Sedativesand Tranquilizers Academic Press.

Morris, S., Oliver, S., 1999. Respiratory gas transport, haemocyanin function andacid–base balance in Jasus edwardsii during emersion and chilling: simulationstudies of commercial shipping methods. Comparative Biochemistry andPhysiology—Part A: Molecular & Integrative Physiology 122, 309–321.

Olsen, Y.A., Einarsdottir, I.E., Nilssen, K.J., 1995. Metomidate anaesthesia in Atlanticsalmon, Salmo salar, prevents plasma cortisol increase during stress. Aqua-culture 134, 155–168.

Otwell, W.S., Webb, N.B., 1977. Investigation of containerization for transportationof live blue crabs, Callinectes sapidus. Cheesepeak Science 18, 340–346.

Paterson, B.D., Spanoghe, P.T., 1997. Stress indicators in marine decapod crusta-ceans, with particular reference to the grading of western rock lobster(Panulirus cygnus) during commercial handling. Marine and FreshwaterResearch 48, 829–834.

Ridgway, I.D., Taylor, A.C., Atkinson, R.J.A., Stentiford, G.D., Chang, E.S., Chang, S.A.,Neil, D.M., 2006. Morbidmortality in Norway lobsters, Nephrops norvegicus.Physiological, Immunological and Pathological effects of air exposure andJournal of Experimental Marine Biology and Ecology 328, 251–264.

Seccombe, J., 1999. Live fish handling strategies from boat to retail establishment.In: Paust, B.C., Rice., A.A. (Eds.), Proceedings of the Second International

Conference and Exhibition. University of Alaska Sea Grant, AK-SG-01-03.USA, Seattle, WA, pp. 57–62.

Spanoghe, P.T., Bourne, P.K., 1997. Relative influence of environmental factors andprocessing techniques on Panulirus cygnus morbidity and mortality duringsimulated live shipments. Marine and Freshwater Research 48, 839–844.

Spicer, J.I., A.D., H., Taylor, A.C., Strang, R.H.C., 1990. Effect of aerial exposure onconcentrations of selected metabolites in the blood of the Norwegian lobsterNephrops norvegicus (Crustacea: Nephropidae). Marine Biology 105, 129–135.

Stehly, G.R., Gingerich, W.H., 1999. Evaluation of Aqui-STM (efficacy and minimumtoxic concentration) as an anaesthetic/sedative for public aquaculture in theUnited States. Aquaculture Research 30, 365–372.

Taylor, E.W., Whiteley, N.M., 1989. Oxygen transport and acid–base balance in thehaemolymph of the lobster, Homarus gammarus, during aerial exposure andresubmersion. Journal of Experimental Biology 144, 417–436.

Tully, O., Robinson, M., O’Keeffe, E., Cosgrove, R., Doyle, O., Lehane, B., 2006.The Brown Crab (Cancer pagurus L.) Fishery: Analysis of the Resource in2004–2005. Fisheries resource series, Bord Iascaigh Mhara (Irish Sea FisheriesBoard), Dun Laoghaire, p. 48.

Uglow, R.F., Hosie, D.A., Johnson, I.T., Macmullen, P.H., 1986. Live handling andtransport of crustacean shellfish: an investigation of mortalities. In: Ministryof Agriculture, F.a.F.(MAFF), UK, R&D Commission (Eds.), Seafish TechnologySR280, pp. 24.

Whiteley, N.M., Al-Wassia, A.H., Taylor, E.W., 1990. The effect of temperature,aerial exposure and disturbance on oxygen consumption in the lobster,Homarus gammarus (L.). Marine Behaviour and Physiology 17, 213–222.

Whiteley, N.M., Taylor, E.W., 1988. Handling transport and storage of live crabs andlobsters. In: Authority, S.F.I. (Ed.), Sea Fish Industry Authority—Open LearningModule. Sea Fish Industry Authority. Her Majesty’s Stationary Office, pp. 115.

Whiteley, N.M., Taylor, E.W., 1990. The acid–base consequences of aerial exposurein the lobster, Homarus gammarus (L.) at 10 and 20 1C. Journal of ThermalBiology 15, 47–56.

Whiteley, N.M., Taylor, E.W., 1992. Oxygen and acid–base disturbances in thehaemolymph of the lobster Homarus gammarus during commercial transportand storage. Journal of Crustacean Biology 12, 19–30.