Comparative Biochemistry and Physiology, Part A 158 (2011) 207–214

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A

j ourna l homepage: www.e lsev ie r.com/ locate /cbpa

Hypoxia tolerance and partitioning of bimodal respiration in the striped catfish(Pangasianodon hypophthalmus)

Sjannie Lefevre a,b,⁎, Do Thi Thanh Huong b, Tobias Wang a,b, Nguyen Thanh Phuong b, Mark Bayley a,b

a Biological Sciences, Zoophysiology Section, Aarhus University, Denmarkb College of Aquaculture and Fisheries, Can Tho University, Can Tho City, Vietnam

⁎ Corresponding author. BiologicalScience,AarhusUniveallé, Building 1131, 8000 Aarhus C, Aarhus, Denmark. Tel.: +

E-mail address: sjannie.lefevre@biology.au.dk (S. Lef

1095-6433/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.cbpa.2010.10.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 July 2010Received in revised form 26 October 2010Accepted 27 October 2010Available online 3 November 2010

Keywords:Air-breathingBimodal respirometryGill ventilation

Air-breathing fish are common in the tropics, and their importance in Asian aquaculture is increasing, but therespiratory physiology of some of the key species such as the striped catfish, Pangasianodon hypophthalmusSauvage 1878 is unstudied. P. hypophthalmus is an interesting species as it appears to possess bothwell-developed gills and a modified swim bladder that functions as an air-breathing organ indicating a highcapacity for both aquatic and aerial respiration. Using newly developed bimodal intermittent-closedrespirometry, the partitioning of oxygen consumption in normoxia and hypoxia was investigated inP. hypophthalmus. In addition the capacity for aquatic breathingwas studied throughmeasurements of oxygenconsumption when access to air was denied, both in normoxia and hypoxia, and the critical oxygen tension,Pcrit, was also determined during these experiments. Finally, gill ventilation and air-breathing frequency weremeasured in a separate experiment with pressure measurements from the buccal cavity. The data showedthat P. hypophthalmus is able to maintain standard metabolic rate (SMR) through aquatic breathing alone innormoxia, but that air-breathing is important during hypoxia. Gill ventilation was reduced duringair-breathing, which occurred at oxygen levels below 8 kPa, coinciding with the measured Pcrit of 7.7 kPa.The findings in this study indicate that the introduction of aeration into the aquaculture of P. hypophthalmuscould potentially reduce the need to air-breathe. The possibility of reducing air-breathing frequency may beenergetically beneficial for the fish, leaving more of the aerobic scope for growth and other activities, due tothe proposed energetic costs of surfacing behavior.

rsity,Zoophysiology,C.F.Møllers45 89 42 26 95.

evre).

l rights reserved.

© 2010 Elsevier Inc. All rights reserved.

1. Introduction

Air-breathing fish are abundant in tropical waters, where hypoxia iscommon and where high water temperatures decrease oxygensolubility and increase metabolism (Diaz, 2001; Graham and Wegner,2010). The striped catfish (Pangasianodon hypophthalmus, Sauvage1878) is a widespread and economically important teleost in southeastAsia that uses amodified swim-bladder for gas exchange (BrowmanandKramer, 1985; Danguy and Lenglet, 1988; Podkowa and Goniakowska-Witalinska, 1998). It has been classified as a continuous obligateair-breather (Browman and Kramer, 1985), but the seemingly well-developed gills (personal observation, see Fig. 1) indicate a highcapacity for aquatic oxygen uptake. The partitioning of oxygenconsumption has, however, not been quantified. P. hypophthalmus ismigratory in nature (So et al., 2006) and its stream-lined appearance,almost reminiscent of sharks points to an active lifestyle, where a highcapacity for aquatic breathing may be beneficial, because frequentsurfacing, while increasing the aerobic scope in hypoxic water, also

reduces the time available for other activities and increase the risk ofaerial predation (Kramer, 1983; 1987).

To study the partitioning of oxygen uptake and the effects of aquatichypoxia in P. hypophthalmus, we developed an intermittent-closedrespirometer for simultaneous measurements in both air and water.Therelatively short but frequentmeasurement intervals used in intermittent-closed respirometry make it possible to identify periods of spontaneousactivity thatmust be excludedwhendetermining standardmetabolic rate(SMR). Also, intermittent-closed respirometry minimizes problems ofwaste-product accumulation in the chamber and the necessity to correctfor washout time (Steffensen, 1989). The critical oxygen tension (Pcrit),definedas theoxygenpartial pressure of thewater (PO2w)where the SMRcan no longer be maintained was measured to provide a functionalcharacterization of the capacity for branchial gas exchange. In addition tothemetabolicmeasurements, changes in gill ventilation and air-breathingfrequencyweremeasured during exposure to stepwise hypoxia. Thiswasdone to investigatewhether changes ingill ventilationand the initiationofair-breathing were correlated with Pcrit. It was hypothesized that gillventilation would be reduced during air-breathing when PO2w wasdecreased. Furthermore, the survival and the ability tomaintain buoyancywere assessed in animals, both with and without access to air, in order toinvestigate the importanceofair-breathing for survival and inmaintaining

Fig. 1. (A)Whole gills from P. hypophthalmus. The individual was 18 cm long andweighed 80 g. None of the gill filaments are reduced, and the filaments appear to be the same lengthon all 4 gill arches. (B) Close-up of the tip of several gill filaments. This individual was 40 cm long and weighed 1 kg. Filaments and secondary lamellae have been pointed out.

208 S. Lefevre et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 207–214

buoyancy.We hypothesized that P. hypophthalmus has a high capacity forboth aquatic and aerial respiration and an associated low dependence onaerial respiration during aquatic normoxia.

2. Methods and materials

2.1. Animals

Juvenile Pangasianodon hypophthalmus were obtained from a localfish farmer in Vietnam and transported to Can Tho University wherethey were kept at 27±1 ° C in a 500-L tank with continuous aeration(PO2 approximately 20 kPa). Water was changed every second day tokeep levels of NH3, NO2

−, and NO3− low (0, b 0.2, andb403 μmol L−1,

respectively). Fishwere fed commercialfloatingpellets (30%protein, 5%lipid, 2800 kcal kg−1) to satiation once a day, and uneaten food wasremoved after 1–2 h. Food was withheld for 2 days before measure-ments. All experiments were performed in accordance with nationalguidelines for the protection of animal welfare in Vietnam and theDanish guidelines for animal welfare in LBK726 of 9 September 1993.

2.2. Forced submergence

Forty-five fish (120–180 g) were randomly divided into 3 50-L tanksmaintained at 6.1, 12.3, or 20.4 kPa O2 (27 °C) using an oxygen controlsystem(MPA-48, InsiteIG, CA,USA).Air-breathingwasprevented for 24 hbyplacing anet 5 cmunder the surface, and thefishwere inspected every8 h. Forced submergence in normoxic water for 24 h caused no apparentdiscomfort and the effects of prolonged submergence on survival andbuoyancy in normoxic water were therefore investigated. Eighteen fish(120–180 g) were fasted for 3 days and submerged for 6 days innormoxic water as described above. The fish were inspected twice aday, and activity levels and posture were noted to assess buoyancy.Eighteen controlfishwerekept in a separate 500-L tankwith access to air.

2.3. Aquatic oxygen consumption, SMR, and Pcrit

2.3.1. Intermittent-closed respirometryOxygen consumptionwithout access to air (MO2)wasmeasuredusing

intermittent-closed respirometry for at least 20 h at 27.0±0.1 °C(Steffensen et al., 1984; 1989). The respirometer, which was submergedin water in a large tank (~30 L), consisted of a chamber containing thefish and a closed loop where water passed a galvanic oxygen electrodethat continuously measured the oxygen partial pressure of the water(PO2w) within the respirometer. In addition, an open loop could beactivated to flush the respirometer with fresh water from the tankcontaining the system. The opening and closure of the flush pump as

well as the logging of the PO2w measurements were automated by asoftware system (Respirometer 2.0) developed at the Zoophysiologysection at Aarhus University. Each individual MO2 measurement lasted15 min, where the respirometer was closed for 3 min, so the decline inPO2w could be used to calculate MO2, followed by 12 min of flush torenewoxygen levels. This setupprovided independentmeasuresofMO2

at 15-min intervals. MO2 for all points was calculated as:

MO2 =jΔPO2

Δt j⋅βO2⋅Vsys

Mb

where ΔPO2/Δt is the rate at which PO2w declined during the closedperiod, βO2 is the solubility of oxygen in water at 27 °C(1.617 μmol mm Hg−1 L−1), Vsys is the volume of water in the systemcorrected for the volume of the fish (density assumed to be 1 kg L−1),andMb is body mass. Values for MO2 were omitted when the R2 of thelinear regression for the decline in PO2w was less than 0.985. At thecompletion of the experiment, measurements were continued for 0.5–2 h after removing the fish from the respirometer to determinebackground MO2. Background MO2 was subtracted from the mea-suredMO2, to control for bacterial oxygen uptake (7±4 mg O2 kg−1 h−1). All parts of the respirometer system were carefully cleaned aftereach experiment and re-filled with chlorine free tap water, and thePO2w was allowed to reach equilibrium before proceeding with thenext fish. The O2 electrode was calibrated before each measurement.

2.3.2. Experimental protocolSince P. hypophthalmus survived forced submergence in normoxic

water for 6 days, we measured standard and routine metabolic rates(SMR and RMR, respectively) in 13 fish (136±24 g) denied access toair in normoxic water. SMR was determined as the average of the 5lowest MO2 determinations during the 24-h period. The 10% quantilewas also used as a measure of SMR for comparison. RMR wascalculated as the average MO2 during the entire measurement period.

In a further 7 fish (117±21 g), we measured SMR and RMR duringmild hypoxia (12–13 kPa) since preliminary studies showed that mildhypoxia reduced the variability in MO2, presumably as a result ofreduced spontaneous activity, and therefore allowed for amore reliabledetermination of SMR. This oxygen level was well above the criticaloxygen partial pressure (Pcrit) that was measured after 8 h of mildhypoxia (12–13 kPa) during preliminary trials. Pcrit was determined asthe PO2w, where MO2 was reduced below the SMR determined prior tohypoxia (Affonso and Rantin, 2005; Thuy et al., 2010). This methodwaschosen because fish were seldom at rest when the measurement of Pcritwas initiated, in which case the point at which SMR cannot be

209S. Lefevre et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 207–214

maintained was not revealed through the measurement of MO2 withdecreasing PO2 alone. The fish were then allowed to recover for 15 h at12–13 kPa before being exposed to an oxygen level close to the Pcrit ofthe individual fish for 6 h (6–8 kPa), followed by an additional 15-hrecovery. These studies were performed to investigate whether MO2

increased immediately after hypoxia as indication of an oxygen debtwhen confined to aquatic breathing in severe hypoxia.

2.4. Partitioning of oxygen consumption

2.4.1. Bimodal intermittent-closed respirometryIn a further 12 fish (46±5 g), aquatic oxygen uptake (MO2w) was

measured as described above, and the same intermittent-closedrespirometry principle applied to the air phase to measure aerialoxygen uptake (MO2a). A diagram of the bimodal intermittent-closedrespirometry system is shown in Fig. 2A. PO2 of the air phase (PO2a, seeFig. 2B and C) was measured using a mini optic O2 sensor (Fibox3,PreSens Precision Sensing GmbH, Regensburg, Germany) and datacollected by accompanying software (OxyView PST3, V6.02, PreSensPrecision Sensing GmbH, Regensburg, Germany). The system consistedof a plexiglass chamber submerged in water, with a small air-breathingchamber of plastic on top. The inlet and outlet of air were sealed withsolenoid valves (Type 6013, Bürkert Fluid Control Systems, Ingelfingen,Germany) connected to the computer controlled relays. An air pump,connected to the inlet, blew fresh air into the chamber when the valveswere opened, and this flushing of the air phase was only performedduring the intervals where water flushing was turned off (i.e., duringmeasurement ofMO2w). This prevented changes inwater level and thuschanges in the air space volume. The air chamber was flushed for 1 minevery hour, after which the outlet solenoid remained open for a further

Fig. 2. (A) The bimodal intermittent-closed respirometer. Inlet and outlet are solenoid valvesof a fish in normoxia. Pumps turning on/off are indicated. (C) Raw data from the air phase durespired air containing less oxygen. In hypoxia, the breathing frequency was increased and thwater. This decrease was included in the calculation of MO2a. This steady diffusion of oxyge

2 min to prevent changes in air pressure during closure of the inletsolenoid valve. During aquatic hypoxia, PO2w was lowered by bubblingthewater phase with N2 gas. The necessary flow of N2 gas was adjustedmanually until the required steady state PO2w was reached.

2.4.2. Experimental protocolAerial and aquaticMO2weremeasured in a groupoffish in normoxic

(19.1±0.5kPa) and hypoxic (6.5±0.9kPa) water, where the level ofhypoxia was below the Pcrit estimate. Both groups had access tonormoxic air, a water temperature of 29.6±0.3 °C and had fasted for24 h before experiments. The fish were placed in the respirometer at5–6 pm, and aerial and aquatic MO2 (MO2a and MO2w, respectively)were measured in 0.5-h intervals for approximately 22 h. BackgroundMO2 was subsequently determined for both phases. In water thebackgroundMO2was the sum of bacterial MO2 and diffusion of O2 fromthe air phase into the water. The contribution from diffusion betweenthe two phases was negligible in normoxia, but it was necessary toinclude it in calculations of MO2w and MO2a during hypoxia.

2.4.3. Data analysisMO2w was calculated for all points as described above, while MO2a

was determined from the decline in PO2 in the air phase (PO2a; dataexamples are shown in Fig. 2B and C). The data trace from the optic O2

sensor was analyzed by calculation of a slope for each interval (ΔPO2a)as in water phase respirometry. This ΔPO2a was then corrected for thedecline caused by diffusion into the water (~1.28 mg O2 h−1), whichonly occurred during hypoxia. MO2a was calculated from ΔPO2a using astandard curve made by injecting known volumes of N2 gas into the airchamber, removing the same volume of mixed air and N2, andcorrelating the volume of N2 gas with the corresponding ΔPO2a. The

controlled by a computer. (B) Raw data of PO2 from the air phase during measurementring measurement of a fish in hypoxia. The stepwise decreases in PO2a are exhalation ofere was a steady decrease in PO2a between breaths due to diffusion of oxygen from air ton from air to water was not evident in normoxia.

Time (h)

0 5

Su

rviv

al (

%)

0

20

40

60

80

100

10 15 20 25

Fig. 3. Survival percentage during the 24 h of forced submergence in 20 kPa (circles),12 kPa (triangles), and 6 kPa (squares). There was only one replicate (one tank) at eachoxygen level, hence no error bars. N=15 fish at each oxygen level.

210 S. Lefevre et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 207–214

volume of oxygen displaced was then 20.95% of the corresponding N2

volume. This system also allowed for a determination of breathingfrequency (fb). However, somefish had such a high breathing frequencyin hypoxia that single breaths were difficult to identify, causing anunderestimation of fb. SMRwas estimated as the average of the 5 lowestpoints of total MO2.

2.5. Measurement of gill ventilation

In a separate experiment, gill ventilation was measured at differentoxygen levels to investigate the ventilatory response to hypoxia.

Nine fish (81±10 g) were instrumented with a catheter in theopercula (PE90) for measurement of opercular pressure. Animals wereanesthetized (benzocaine0.1 g L−1) until cessationof the righting reflex,instrumented, and allowed to recover for minimum 15 h beforeinitiation of the experiment. The opercular pressure was measuredwith a pressure transducer, and data were collected at 50 Hz with aMP100 BIOPAC. The experiment was performed in a 30-L isolatedpolystyrene box, connected to an additional 40-L water reservoir, withfiltering, heating, and aeration. The connection to the reservoir wasclosedwhenwater PO2was lowered. The surfacewas covered, except fora small area (10×10cm) allowing the fish to surface and air-breathe. Arelatively large container was chosen for this experiment to minimizestress due to confinement.

2.5.1. Experimental protocolAfter connecting the opercular catheter to the pressure transducer,

opercular pressure was sampled for at least 1 h to allow the fish to settledown. The fish were then subjected to stepwise hypoxia, with 15 min ateachof 4 levels: 13.3, 8.0, 5.3, and2.7 kPa. Thewater PO2was loweredby aflow of N2 gas, and the time to reach the newwater PO2 was 10–20min.

2.5.2. Data analysisGill ventilation frequency and amplitude were measured for two 15-s

intervals in each treatment, and averaged. The intervals with activity(i.e., largeoscillations inamplitude)wereomitted.Aventilation indexwascalculated as:

VIxkPa =amplitude⋅frequencyð ÞxkPaamplitude⋅frequencyð Þ20kPa

⋅100%

Air-breaths made a distinct pattern of change in the opercularpressure, making it possible to count all the breaths during the entiremeasurement, and the air-breathing frequency for each oxygen levelcould therefore be calculated.

2.6. Statistics

Data were analyzed in the statistical analysis program JMP®7.0 (SASInstitute Incorporated, Cary, North Carolina, USA). A Shapiro-WilkW testwas used to test for normality. Normally distributed data were testedwith aone-wayANOVA, andaTukey'sHSDanalysiswasused to compareindividual means, while a non-parametric Kruskal–Wallis was used fornon-normally distributed data. A paired t-test was used to analyze theventilation data. Probabilities below 0.05 were considered significant.

3. Results

3.1. General behavior

In general, P. hypophthalmus showedhigh levels of swimmingactivityandwere rarely observed stationary in the water column. This species isknown to be sensitive to disturbance, with vigorous swimming andescape behavior after even the slightest stimulus. Surfacing and air-gulping were observed infrequently. Young fish (up to 150 g) had atendency to surface synchronously in large groups. During air-breathing,

the fish would swim towards the surface at an angle of approximately45° and, after surfacing where the breathing cycle seemed to be rapidlycompleted, turn quickly to submerge. Air bubbleswere occasionally seento be expelled through the gill slits during the descent from the surface.When confined in a respirometer without access to air, the fish weregenerally agitated for the first hour exhibiting pronounced gillventilation. Following this initial period, the fish continued to showregular bouts of spontaneous activity interspersedwith quiescence, evenafter many hours in the respirometer. The fish continued to be sensitiveto disturbance, for instance, when the flush pump had to be turned offduring measurement of Pcrit. It was therefore important that humandisturbancewaseliminatedduringmeasurement and that the laboratorywas completely undisturbed during this time. The fish seemed to be lesssensitive and calmer in a respirometer where access to air was allowed.

3.2. Forced submergence

When denied access to air in normoxic water, the fish initially madevigorous attempts to surface, but this behavior subsided within the first2 h. Swimming movements appeared slower after the first 2 days, andthe fish responded less to disturbance after 4–5 days. At that time, allsubmerged fish exhibited intensive gill ventilation. The fish were neverobserved sinking towards the bottom but generally positionedthemselves close to the net covering the surface and did thus notexhibit clear signs of negative buoyancy. All fish survived forcedsubmergence in normoxia, 90% survived in mild hypoxia (12 kPa),whereas 45% survived in severe hypoxia (6 kPa) (Fig. 3).

3.3. Aquatic MO2, SMR, and Pcrit

MO2 varied considerably when access to air was denied in normoxic(20 kPa) and slightly less when exposed tomildly hypoxic (12–13 kPa)water. The fish exhibited frequent bouts of spontaneous activityassociated with increased MO2, and as a consequence, the averageMO2 of differentfish at different timeswas considerable higher than theestimated SMR, both in 20 kPa and 12–13 kPa (Figs. 4 and 5B). RMR andSMR during normoxia (20 kPa), mild hypoxia (12–13 kPa), and severehypoxia (6 kPa) are represented in Table 1. The SMRestimated from the10% quantile was not used further, as it was highly variable, andprobably overestimated SMR due to the spontaneous activity. RMRwassignificantly reduced in 12–13 kPa (one-way ANOVA, pb0.05) and6 kPa (p b 0.0001), when compared to normoxia. RMR was alsosignificantly reduced in 6 kPawhen compared to 12–13 kPa (p b 0.001).SMR did not differ significantly between the different oxygen levels,although SMR tended to decrease when air-breathingwas prevented inhypoxicwater (pN0.05). Theoxygen levels during thehypoxia exposureare depicted in Fig. 5A. MO2 decreased gradually when oxygen levels

time (h)

0 50

50

100

150

200

250

0

5

10

15

20

VO2

PO2

MO

2 (m

g O

2 kg

-1 h

-1)

MO

2 (m

g O

2 kg

-1 h

-1)

0

50

100

150

200

250

SMR

PO

2 (k

Pa)

PO

2 (k

Pa)

4

6

8

10

12

14

16

18

20

mild hypoxia mild hypoxiaseverehypoxia

Pcrit

A

B

C

SMR

10 15 2520 30 35 40 45

Fig. 5. PO2 (A) andMO2 (B) duringmild hypoxiawithout access to air. PO2was decreasedafter 8 h to measure Pcrit, and decreased to near Pcrit after another 15 h. After 6 h, the fishwas allowed to recover for approximately 15 h in mild hypoxia. N=7. Data are mean±SEM. The grey bar represents SMR±SEM. (C) MO2 and PO2 data from an individual fish.

211S. Lefevre et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 207–214

declined for determination of Pcrit (Fig. 5B). This reductionwas probablycaused by a decline in spontaneous activity. Data from an individual fishare shown in Fig. 5C, and here it can be seen how MO2 increasesfrequently but returns to SMR. It is also evident thatMO2 is independenton PO2w during mild hypoxia. When the PO2w was reduced to close toPcrit (6–8 kPa depending on the fish) for 6 h, the MO2 decreased andremained stable on a level close to SMR but increased and becamevariable upon the return to 12–13 kPa O2 (Fig. 5B). There was thereforeno clear indication of an oxygen debt accumulated from anaerobicmetabolism. On average, Pcrit was 7.7±1.2 kPa, and all fish increasedmetabolism upon return to 12–13 kPa O2. In Fig. 6A, the mean MO2 forall fish is shown as a function of PO2. As can be seen in Fig. 6B, some fishwere calm and maintained a MO2 close to their SMR until below Pcrit.Fig. 6C shows another type of response where MO2 was elevated aboveSMR, causing a linear decrease in MO2 with PO2.

3.4. Partitioning of oxygen consumption

The oxygen consumption (MO2w, MO2a, total MO2, and SMR),partitioning, and air-breathing frequency for hypoxia and normoxiaare summarized in Table 2. Therewere no significant difference in eitherSMR (one-way ANOVA, pN0.05) or total MO2 (p N 0.5), between the twotreatments. Furthermore, the SMR determined with and without accessto air were not significantly different (one-way ANOVA, p N 0.05). TheMO2a, fb and %oxygen from air was significantly higher in hypoxia(Kruskal–Wallis, pb0.05) while the MO2w was significantly higher innormoxia (one-way ANOVA, pb0.001). The partitioning of oxygenconsumption during 22 h in normoxic (20 kPa) and hypoxic (6 kPa)water is shown in Fig. 7A andB, respectively. TotalMO2was initially highin both treatments (200–300 mgO2kg−1 h−1), but stabilized after 5–6 h(~ 150 mg O2 kg−1 h−1). In normoxia, MO2a was negligible throughoutthemeasurement (b7%, Table 2). In hypoxia, MO2a wasmore importantduring the first 15–16 h (8–9 am), after which the fish shifted towardsaquatic respiration within an hour. This shift occurred a few hours aftersunrise (6 am), and coincided with the arrival of staff at 8 am.

3.5. Gill ventilation

Gill ventilation amplitude, frequency, ventilation index, and air-breathing frequency are depicted in Fig. 8. The overall ventilation,

MO

2 (m

g O

2 kg

-1 h

-1)

MO

2 (m

g O

2 kg

-1 h

-1)

0

50

100

150

200

250

SMR

time (h)

0 50

50

100

150

200

250

300

SMR

A

B

10 15 20

Fig. 4. (A) MO2 during exposure to normoxic water. N=13. Data are mean±SEM.The horizontal bar represents SMR±SEM of the 13 fish. (B) Data from an individual fish.

measured as the ventilation index, was significantly reduced below 8 kPa(Fig. 8C, pb0.01). This reduction was primarily caused by a significantdecrease in ventilation amplitude (Fig. 8A, p b 0.01), while the ventilationfrequency was significantly reduced at 2.7 kPa (Fig. 8B, p b 0.05). The air-breathing frequency increased significantly at 8kPa (Fig. 8D,pb 0.01), andincreased even further at 5.3 and 2.7 kPa (p b 0.005).

4. Discussion

Using a novel design to automate intermittent-closed respirometryfor simultaneous measurements of oxygen uptake from water and air,we have shown that P. hypophthalmus is a facultative air-breather, witha high capacity for both aerial and aquatic respiration. As such,P. hypophthalmus differs from many other air-breathing species,where the gills are reduced to minimize branchial oxygen loss tohypoxic water when the oxygenated blood from the swimbladderpasses through the gills to reach the systemic circulation (Olson, 1994;Graham, 1997; Graham and Wegner, 2010). In this study, we were

Table 1Standard metabolic rate and routine metabolic rate in fish exposed to three differentoxygen levels.

Treatment N SMR SMR (10% quantile) RMR

Severe hypoxia (6–8 kPa) 7.a 90±2 – 104±3a

Mild hypoxia (12–13 kPa) 7.a 97±3 106±7 147±7b

Normoxia (20 kPa) 13 107±6 122±11 171±6c

a Seven individuals were exposed to both mild and severe hypoxia. Data are mean±SEM. Different letters indicate significant differences.

0

30

60

90

120

150

0

30

60

90

120

150

RMR

SMR

A

B

2 4 6 80

50

100

150

200C

10 12 14

MO

2 (m

g O

2 kg

-1 h

-1)

MO

2 (m

g O

2 kg

-1 h

-1)

MO

2 (m

g O

2 kg

-1 h

-1)

PO2 (kPa)

Pcrit

Pcrit

Fig. 6. (A) TheaverageMO2 of 7fishduring themeasurement ofPcrit (filled circles) and theaverage SMR (open squares). (B) Data from an individual fish. This fish was not showingspontaneous activitywhen the Pcritmeasurementwas initiated. (C)Data fromanotherfish.This individual had an increased metabolism when the Pcrit measurement was initiated.

0

100

200

300

400Water

Air

Total

time (h)

0 5 200

100

200

300

400

Water

Air

Total

A

B

normoxia

hypoxia

SMR

MO

2 (m

g O

2 kg

-1 h

-1)

MO

2 (m

g O

2 kg

-1 h

-1)

1510

Fig. 7.MO2a (open circles), MO2w (closed circles), and total MO2 (triangles) during 22 hin normoxic (A) and hypoxic (B) water. The grey bar represents the SMR±SEM. At 15 h(~8:00 h), people entered the room to check on experiments, causing a shift inpartitioning from air to water. There was a higher variation in partitioning betweendifferent fish during hypoxia than in normoxia. N=6. Data are mean±SEM.

212 S. Lefevre et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 207–214

unable to detect a branchial oxygen loss in P. hypophthalmus withbimodal intermittent-closed respirometry even when water PO2 wasallowed to drop to as low as 2 kPa (Lefevre et al., unpublished), whichcould be due to a reduction in both gill ventilation amplitude andfrequency when water PO2 was below Pcrit. A similar reduction in gillventilation in hypoxic water has also been observed in other facultativeair-breathing fish (Graham et al., 1978;Mattias et al., 1998, Geiger et al.,2000). In addition, theopercula of P. hypophthalmushaveamembranousflap of skin along the edge that seems to diminish the contact betweengills and water when the opercula are closed. Further measurements offor example post-branchial water PO2 or pre- and post-branchial bloodPO2 are needed to fully reveal whether a branchial oxygen loss iscompletely prevented by these ventilatory and morphological adapta-tions. Whether air-breathing and the swimbladder in P. hypophthalmus,in addition to its respiratory function, have importance for buoyancyregulation is difficult to discern from the present study, as therewere noclear indications of negative buoyancy. It would, however, be likely thatthe swimbladder serves a dual function considering the active lifestyleof the species, as has also been found in other air-breathers such asAmiacalva (Hedrick and Jones, 1993; 1999).

Table 2Average aerial, aquatic, and total oxygen consumption (mg O2 kg−1 h−1), breathingfrequencies (breath h−1), oxygen partitioning (%), and standardmetabolic rate (SMR) of fishexposed to hypoxia and normoxia.

Group n MO2a MO2w Total MO2 % O2 from air fb SMR

Normoxia 6 11±4b 150±13b 161±11 7±3b 1±0.4b 118±11Hypoxia 6 89±13a 62±18a 151±13 60±9a 11±1.6a 99±6

Data are mean±SEM. Different letters indicates significant differences between groups.

4.1. Bimodal intermittent-closed respirometry

To our knowledge, this is the first study to use automatedintermittent-closed respirometry simultaneously in air and water topartition gas exchange of a bimodal breather. As reasoned by Steffensen(1989), automated intermittent-closed respirometry benefits from thesensitivity of traditional closed respirometry, but the short durations ofthe closed periods alleviate waste product accumulation and preventlong-term oxygen depletion, while also avoiding the need to correct forwashout time. A problem arisingwhenmeasuring simultaneously in airand water is the potential diffusion of oxygen from air to water(Graham, 1997). Themagnitude of this diffusion depends on the area ofthe interface between the two media, the partial pressure differencebetween water and air, and the extent of the boundary layer at theinterface. Furthermore, the level ofmixing due tomovements of the fishmight influence the magnitude of diffusion. Although backgroundmeasurements in the present study were performed without thepresence of a fish, they were similar to the diffusion measured ininter-breath intervals when a fish was present (see Fig. 2C), indicatingthat the additional mixing caused by the fish was negligible. Diffusionwas undetectable when the water was normoxic, but some diffusionoccurred when the water was hypoxic (~1.28 mg O2 h−1).

4.2. SMR, Pcrit, and spontaneous activity

Determinations of SMR require that stress is reduced to a minimum,which can be achieved by leaving the animal undisturbed in theexperimental setup overnight, while measuring MO2. Nevertheless,spontaneous bouts of activity attended by increased metabolism persistin most species, even when undisturbed (e.g., Zimmermann andKunzmann, 2001; Steffensen, 2002; Hölker, 2003; Thuy et al., 2010).These periods are easily identifiedwith intermittent-closed respirometry,as long as the closed periods are kept relatively short and frequent. In thepresent experiments, P. hypophthalmus displayed frequent bouts ofactivity when prevented from surfacing. This made the determination ofSMR difficult, since conventional analyses, such as the fitting of twonormal distributions (Steffensen et al., 1994) and calculation of the 10thquantile (Chabot and Claireaux, 2008), tends to overestimate SMR in fishwith high spontaneous activity, which was also the case with

Am

plit

ud

e (c

m H

2O)

0

1

2

3

4

air-

bre

ath

ing

(h

-1)

0

50

100

150

200

0

50

100

150

200

20,013,38,05,32,7

ven

tila

tio

n in

dex

(%

)

0

10

20

30

40

50

a

a

b

c

c

a

aa

b

c

aaaa

b

aa

a

b

c

gill

ven

tila

tio

n (

min

-1)

PO2 (mmHg)

A

B

C

D

Fig. 8. Gill ventilation amplitude (A), gill ventilation frequency (B), ventilation index (C),and air-breathing frequency (D) in gradual steps of hypoxia. N=9. Data are mean±SEM.Different letters indicate significant differences.

213S. Lefevre et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 207–214

P. hypophthalmus (see Table 1).When air-breathingwas allowed, the fishshowed the more common decline and stabilisation of MO2

(see Steffensen et al., 1984; 1994). Despite the increased spontaneousactivity when air-breathing was prevented, the determinations of SMRwith andwithout access to air are surprisingly similar (see Table 1 and 2),indicating thatwaterphasemeasurementswithout access to air, at least innormoxic water, allow an assessment of the efficiency of oxygen uptakeover the well-developed gills, and a determination of SMR. In normoxicwater, evenwith access to air, it is apparent that the gills have the capacityto sustain anoxygenuptake at least three timeshigher thanSMR(Fig. 7A).When denied access to air, RMR decreased with the severity of hypoxia,which probably stems from reduced spontaneous activity, highlightingthe reduction in aerobic scope occurring with decreased oxygenavailability if air-breathing is not possible (Claireaux and Lagardere,1999; Jordan and Steffensen, 2007; Wang et al., 2009). When deniedaccess to air, somefishhada classic response to graduallydecliningPO2: atfirst, they behaved as oxyregulators, maintaining ametabolic rate close toSMR, but at a certain point, they became oxyconformers, and MO2

decreased gradually with PO2 (see Fig. 6B). Other fish had a response thatat first glance looked as an oxyconformer, since MO2 decreasedimmediatelywhenPO2decreased (Fig. 6C). Thiswas, however, an artefactcaused by the fish being spontaneously active at the time the Pcritmeasurement was started, thus having an MO2 above the SMR. The fishcould then decreaseMO2 by reducing its activity. Since these fish are verysensitive, itwas inevitable to disturb themwhen theflushpumphad to be

turned off to decrease PO2. The Pcrit of 7.7 kPa (55 mm Hg) when P.hypophthalmuswas denied access to air is similar to other facultative air-breathing fish for which Pcrit has been measured (Graham et al., 1978;Mattias et al., 1998; Oliveira et al., 2004; Affonso and Rantin, 2005; Nelsonet al., 2007). In some air-breathing fish, Pcrit coincides with the thresholdfor air-breathing (Affonso and Rantin, 2005; Perna and Fernandes, 1996),while other species initiate air-breathing at oxygen levels above Pcrit(Mattias et al., 1998; Oliveira et al., 2004). Here we found that air-breathing was initiated at 6–8 kPa, which is close to the measured Pcrit.Because of the potential costs of surfacing in terms of transport andpredation (Kramer, 1983; 1987; Kramer et al., 1983), it may be beneficialfor a fish to initiate air-breathing only at the point where SMR can nolonger be maintained through aquatic respiration alone (i.e., at Pcrit).

4.3. Modification of air-breathing behavior

Consistentwithother air-breathingfish (Ojhaet al., 1979;GrahamandBaird, 1982; 1984; Mattias et al., 1998), aerial oxygen consumption of P.hypophthalmus dominated in hypoxicwater. Disturbance by the presenceof people in the laboratory, however, shifted the partitioning of oxygenuptake towardsaquatic respirationasnoted inFig. 7Bwhenstaff arrived inthe morning. A similar response to disturbance has been reported forother air-breathing fish and can be regarded as a trade-off mechanismbetween the risk of predation and the gain of oxygen from air-breathing(Kramer, 1983; 1987; Smith and Kramer, 1986; Wolf and Kramer, 1987;Chapman and Mckenzie, 2009). The observation that air-breathingbehavior can be modified and even suppressed by external factors andthemere perception of ‘predation threats’ indicates that higher level brainprocesses may be involved in the control of air-breathing, possiblyoverruling the signals from external and internal chemoreceptorsnormally controlling the hypoxia-induced reflex to air-breathe. Furtherstudies are needed to confirm this hypothesis.

4.4. Relevance to the aquaculture of tropical air-breathing fish

While hypoxia is a common natural phenomenon, it also occursregularly in tropical aquaculture (Egna and Boyd, 1997; Lefevre et al.,2010). It is therefore not surprising that many of the fish cultured intropical aquaculture are air-breathers. The respiratory physiology ofsome cultured air-breathers, such as Channa sp., has been studiedextensively (e.g., Hughes and Munshi, 1973; Ishimatsu and Itazawa,1981; Itazawa and Ishimatsu, 1981; Olson et al., 1994), while others,such as P. hypophthalmus, have hardly been studied. This is paradoxicalconsidering that theworld's production of Channa is less than one-thirdof the production of Pangasianodon (FAO, 2007). We show thatP. hypophthalmus does not need to breathe air under well-aeratedconditions but initiates air-breathing at 6–8 kPaO2. The oxygen levels ofless than 5 kPa experienced by fish in typical Vietnamese catfish ponds(Lefevre et al., 2010) indicate that most farmed fish must devote largeamounts of time and energy on air-breathing. As a result, anintroduction of aeration of aquaculture ponds is likely to benefit growthand thereby production.

5. Conclusions

Using a new bimodal intermittent-closed respirometry, we demon-strate that P. hypophthalmus has a high capacity for aquatic respiration,being able to survive without air-breathing in normoxic water, and thatit can be characterized as a facultative air-breather. Aerial respiration isnegligible in normoxia, and these data therefore indicate that surfacingin this species may be reduced in well-aerated conditions. Thepossibility of reducing air-breathing frequency would probably beenergetically beneficial for thefish, leavingmore of the aerobic scope forgrowth and other activities, due to the proposed energetic costs ofsurfacing behavior.

214 S. Lefevre et al. / Comparative Biochemistry and Physiology, Part A 158 (2011) 207–214

Acknowledgments

This study was performed at Can Tho University (Vietnam) as part ofthe PhysCAM-project supported by Danida (Danida Fellowship Centre,Denmark).

References

Affonso, E.G., Rantin, F.T., 2005. Respiratory responses of the air-breathing fishHoplosternum littorale to hypoxia and hydrogen sulfide. Comp. Biochem. Physiol. C.141, 275–280.

Browman, M.W., Kramer, D.L., 1985. Pangasius sutchi (Pangasiidae), an air-breathingcatfish that uses the swimbladder as an accessory respiratory organ. Copeia 1985,994–998.

Chabot, D., Claireaux, G., 2008. Quantification of SMR and SDA in aquatic animals usingquantiles and non-linear quantile regression. Comp. Biochem. Physiol. A. 150, S99.

Chapman, L.J., Mckenzie, D.J., 2009. Behavioral responses and ecological consequences.Fish Physiol. 27, 25–77.

Claireaux, G., Lagardere, J.P., 1999. Influence of temperature, oxygen and salinity on themetabolism of the European sea bass. J. Sea Res. 42, 157–168.

Danguy, A., Lenglet, G., 1988. Anatomical–microscopic study of the gas bladder inPangasius sutchi Fowler (Teleostei, Siluriformes). Annal. Soc. R. Zool Belg. 118,199–209.

Diaz, R.J., 2001. Overview of hypoxia around the world. J. Environ. Qual. 30, 275–281.Egna, H.S., Boyd, C.E., 1997. Dynamics of Pond Aquaculture. CRC Press.FAO, 2007. Global Aquaculture Production. http://www.fao.org/fishery/statistics/

global-aquaculture-production/en.Geiger, S.P., Torres, J.J., Crabtree, R.E., 2000. Air breathing and gill ventilation frequencies in

juvenile tarpon, Megalops atlanticus: responses to changes in dissolved oxygen,temperature, hydrogen sulfide, and pH. Environ. Biol. Fish. 59, 181–190.

Graham, J.B., 1997. Air-breathing fishes. Academic Press, San Diego.Graham, J.B., Baird, T.A., 1982. The transition to air breathing in fishes.1. Environmental-

effects on the facultative air breathing of Ancistrus chagresi and Hypostomusplecostomus (Loricariidae). J. Exp. Biol. 96, 53–67.

Graham, J.B., Baird, T.A., 1984. The transition to air breathing in fishes. 3. Effects of bodysize and aquatic hypoxia on the aerial gas-exchange of the swamp eel Synbranchusmarmoratus. J. Exp. Biol. 108, 357–375.

Graham, J.B., Kramer, D.L., Pineda, E., 1978. Comparative respiration of an air-breathingand a non-air-breathing characoid fish and the evolution of aerial respiration incharacins. Physiol. Zool. 51, 279–288.

Graham, J.B., Wegner, N.C., 2010. Breathing air in water and in air: the air-breathingfishes. In: Nilsson, G.E. (Ed.), Respiratory Physiology of Vertebrates: Life With andWithout Oxygen. Cambridge University Press, pp. 174–221.

Hedrick, M.S., Jones, D.R., 1993. The effects of altered aquatic and aerial respiratory gasconcentrations on air-breathing patterns in a primitive fish (Amia calva). J. Exp. Biol.181, 81–94.

Hedrick, M.S., Jones, D.R., 1999. Control of gill ventilation and air-breathing in thebowfin Amia calva. J. Exp. Biol. 202, 87–94.

Hölker, F., 2003. Themetabolic rate of roach in relation tobody size and temperature. J. FishBiol. 62, 565–579.

Hughes, G.M., Munshi, J.S.D., 1973. Nature of the air-breathing organs of the Indiansfishes Channa, Amphipnous, Clarias and Saccobranchus as shown by electronmicroscopy. J. Zool. Lond. 170, 245–270.

Ishimatsu, A., Itazawa, Y., 1981. Ventilation of the air-breathing organ in the snake-headChanna argus. Jap. J. Ichthyol. 28, 276–282.

Itazawa, Y., Ishimatsu, A., 1981. Gas-exchange in an air-breathing fish, the snakeheadChanna argus, in normoxic and hypoxic water and in air. B. Jap. Soc. Sci. Fish 47,829–834.

Jordan, A.D., Steffensen, J.F., 2007. Effects of ration size and hypoxia on specific dynamicaction in the cod. Physiol. Biochem. Zool. 80, 178–185.

Kramer, D.L., 1983. The evolutionary ecology of respiratory mode in fishes—an analysisbased on the costs of breathing. Environ. Biol. Fish. 9, 145–158.

Kramer, D.L., 1987. Dissolved-oxygen and fish behavior. Environ. Biol. Fish. 18, 81–92.Kramer, D.L., Manley, D., Bourgeois, R., 1983. The effect of respiratory mode and oxygen

concentration on the risk of aerial predation in fishes. Can. J. Zool. 61, 653–665.Lefevre, S., Huong, D.T.T., Wang, T., Phuong, N.T., Bayley, M. 2010. A telemetry study of

swimming depth and oxygen in a Pangasius pond in the Mekong delta. Submitted toAquaculture.

Mattias, A.T., Rantin, F.T., Fernandes, M.N., 1998. Gill respiratory parameters duringprogressive hypoxia in the facultative air-breathing fish, Hypostomus regani(Loricariidae). Comp. Biochem. Physiol. A. 120, 311–315.

Nelson, J.A., Rios, F.S., Sanches, J.R., Fernandes, M.N., Rantin, F.T., 2007. Environmentalinfluences on the respiratory physiology and gut chemistry of a facultative air-breathing, tropical herbivorous fish Hypostomus regani (Ihering, 1905). In:Fernandes, M.N., Rantin, F.T., Glass, M.L., Kapoor, B.G. (Eds.), Fish Respiration andEnvironment. Science Publishers, USA, pp. 191–217.

Ojha, J., Mishra, N., Prasadsaha, M., Munshi, J.S.D., 1979. Bimodal oxygen-uptake injuveniles and adults amphibiousfish, Channa (Ophiocephalus)marulius. Hydrobiol. 63,153–159.

Oliveira, R.D., Lopes, J.M., Sanches, J.R., Kalinin, A.L., Glass, M.L., Rantin, F.T., 2004.Cardiorespiratory responses of the facultative air-breathing fish jeju, Hoplerythrinusunitaeniatus (Teleostei, Erythrinidae) exposed to graded ambient hypoxia. Comp.Biochem. Physiol. A. 139, 479–485.

Olson, K.R., 1994. Circulatory anatomy in bimodally breathing fish. Am. Zool. 34, 280–288.Olson, K.R., Roy, P.K., Ghosh, T.K., Munshi, J.S.D., 1994. Microcirculation of gills and

accessory respiratory organs from the air-breathing snakehead fish, Channapunctata, C. gachua, and C. marulis. Anat. Rec. 283, 92–107.

Perna, S.A., Fernandes, M.N., 1996. Gill morphometry of the facultative air-breathingloricariid fish, Hypostomus plecostomus (Walbaum) with special emphasis onaquatic respiration. Fish Physiol. Biochem. 15, 213–220.

Podkowa, D., Goniakowska-Witalinska, L., 1998. The structure of the airbladder of thecatfish Pangasius hypophthalmus Roberts and Vidthayanon 1991. Folia Biol. Krakow46, 189–196 previously P. sutchi Fowler 1937.

Smith, R.S., Kramer, D.L., 1986. The effect of apparent predation risk on the respiratorybehavior of the Florida gar (Lepisosteus platyrhinchus). Can. J. Zool. 64, 2133–2136.

So, N., Van Houdt, J.K.J., Volckaert, F.A.M., 2006. Genetic diversity and population historyof the migratory catfishes Pangasianodon hypophthalmus and Pangasius bocourti inthe Cambodian Mekong River. Fish. Sci. 72, 469–476.

Steffensen, J.F., 1989. Some errors in respirometry of aquatic breathers—how to avoidand correct for them. Fish Physiol. Biochem. 6, 49–59.

Steffensen, J.F., 2002. Metabolic cold adaptation of polar fish based on measurements ofaerobic oxygen consumption: fact or artefact? Artefact! Comp Biochem. Physiol. A.132, 789–795.

Steffensen, J.F., Johansen, K., Bushnell, P.G., 1984. An automated swimming respirom-eter. Comp. Biochem. Physiol. A. 79, 437–440.

Steffensen, J.F., Schurmann, H., Bushnell, P.G., 1994. Oxygen consumption in 4 species ofteleosts from Greenland—no evidence of metabolic cold adaptation. Pol. Biol. 14,49–54.

Thuy, N.H., Tien, L.A., Tuyet, P.N., Huong, D.T.T., Cong, N.V., Bayley, M.,Wang, T., Lefevre, S.,2010. Critical PO2 increases during digestion in the Eurasian perch (Perca fluviatilis).J. Fish Biol. 76, 1025–1031.

Wang, T., Lefevre, S., Huong, D.T.T., Cong, N.V., Bayley, M., 2009. The effects of hypoxiaon growth and digestion. Fish Physiol. 27, 361–396.

Wolf, N.G., Kramer, D.L., 1987. Use of cover and the need to breathe—the effects ofhypoxia on vulnerability of dwarf gouramis to predatory snakeheads. Oecologia 73,127–132.

Zimmermann, C., Kunzmann, A., 2001. Baseline respiration and spontaneous activity ofsluggish marine tropical fish of the family Scorpaenidae. Mar. Ecol. Prog. Ser. 219,229–239.