Oxygen binding properties of backswimmer (Notonectidae, Anisops) haemoglobin, determined in vivo

Journal of Insect Physiology 57 (2011) 1698–1706

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Oxygen binding properties of backswimmer (Notonectidae, Anisops) haemoglobin, determined in vivo Philip G.D. Matthews ⇑, Roger S. Seymour Environmental Biology, Darling Building, DP 418, University of Adelaide, Adelaide, SA 5005, Australia

a r t i c l e

i n f o

Article history: Received 5 July 2011 Received in revised form 12 September 2011 Accepted 12 September 2011 Available online 16 September 2011 Keywords: Haemoglobin Oxygen equilibrium curve Cooperativity Anisops

a b s t r a c t Aquatic backswimmers (Anisops spp.) collect oxygen from the atmosphere in order to breathe underwater, carrying it within a bubble of air on the ventral surface of their body and bound within haemoglobinfilled cells inside their abdomen. These oxygen stores are interconnected via the abdominal spiracles and the tracheal system. Fibre optic oxygen probes were used to measure PO2 changes within the air bubbles of submerged backswimmers (Anisops deanei) and these measurements were transformed into in vivo haemoglobin–oxygen equilibrium curves (OECs) using a biotonometric approach. The haemoglobin displayed a triphasic, highly sigmoid OEC with a P50 of 3.90 kPa. Comparisons made with a previous in vitro analysis of Anisops haemoglobin demonstrate that while the apparent cooperativity and oxygen affinity are considerably higher in vivo, both measurements share unusual Hb–O2 binding characteristics. The affinity and cooperativity of the backswimmers’ haemoglobin appears adaptive as it lengthens dives and promotes neutral buoyancy. While there are limitations associated with biotonometry, the in vivo OEC accurately represents the loading and unloading of biologically available oxygen within the backswimmers’ haemoglobin cells. Potential errors associated with determining the OEC are small, as evaluated with sensitivity analyses in numerical models. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Haemoglobin is the most common respiratory pigment found in nature. In the animal kingdom it is common to almost every vertebrate, but is patchily distributed among the invertebrates, where it is found among nematodes, annelids, molluscs, echinoderms and arthropods (Weber and Vinogradov, 2001). While it now appears that haemoglobin genes are fairly common among the Insecta (for a review see Burmester and Hankeln (2007)), only some Diptera and Hemiptera specialised for aquatic or semi-aquatic modes of life are known to possess haemoglobin in sufficient quantities to be visible to the naked eye. Aquatic blood-worms, the larvae of Chironomus spp., possess haemoglobin in their haemolymph where it facilitates diffusion of oxygen from their hypoxic burrows into their tissues (Walshe, 1951). The larvae of the horse bot fly (Gastrophilus intestinalis) are parasitic and spend part of their development anchored to the gastric mucosa inside a horse’s stomach. Within this semi-fluid environment they use large, haemoglobinfilled cells associated with their posterior spiracles to maximise oxygen storage during contact with ingested bubbles of air (Keilin, 1944). The oxygen bound by the haemoglobin then supplies the insect’s respiration during periods of anoxia. Aquatic backswimmers ⇑ Corresponding author. Tel.: +61 8 303 3694; fax: +61 8 303 4364. E-mail address: [email protected] (P.G.D. Matthews). 0022-1910/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.09.006

(Hemiptera, genera Anisops and Buenoa) are the only non-dipteran insects to possess large quantities of haemoglobin. As in Gastrophilus, the haemoglobin is produced in large, modified, fat-body cells associated with the abdominal spiracles (Bergtrom et al., 1976). The haemoglobin serves as an oxygen reserve during extended excursions underwater, but uniquely, also plays a critical role in buoyancy regulation (Matthews and Seymour, 2006, 2008). The oxygen binding properties of insect haemoglobins have been investigated in vitro in three insects: Chironomus, Gastrophilus, and Anisops. The haemolymph extracted from whole, homogenised Chironomus thummi larvae contains multiple haemoglobin types, all with extremely high oxygen affinities (Weber et al., 1985). Under biologically relevant conditions of 10 °C and pH 7.5, these haemoglobins become 50% saturated with oxygen (P50) at a PO2 of between 0.016 and 0.080 kPa. The haemoglobin subunits are monomeric or associate as dimers and thus do not exhibit the cooperative binding typical of tetrameric haemoglobins. The haemoglobin extracted from G. intestinalis has a slightly lower oxygen affinity than that of Chironomus (P50 = 0.65 kPa). The shape of its oxygen equilibrium curve (OEC) is hyperbolic, indicating no cooperativity between its haemoglobin dimers (Dewilde et al., 1998; Keilin and Wang, 1946). In contrast, the haemoglobin of the hemipteran backswimmer Anisops assimilis has a low oxygen affinity (P50 = 5 kPa) as well as a highly sigmoid OEC (Wells et al., 1981). A. assimilis haemoglobin is multimeric, associating as

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dimers, tetramers and hexamers, but is predominantly monomeric in the oxygenated state and hexameric when deoxygenated (Wells et al., 1981). It displays no significant Bohr effect. On a Hill plot, where the OEC is plotted as [S/(100  S)] vs. PO2 on log/log axes (where S is percent saturation), the steepest phase of A. assimilis haemoglobin occurs between 50% and 90% oxygen saturation, with the slopes above and below these saturations higher than 1. Clearly, this polyphasic OEC indicates that backswimmer haemoglobin does not conform to the two-state model of Monod et al. (1965). All in vitro studies on the binding properties of haemoglobin are necessarily carried out under conditions which differ, by greater or lesser degrees, from the physiological conditions within the animal. Since the ability of haemoglobin to associate with oxygen is sensitive to its concentration in solution, pH, temperature, and allosteric effectors (Imai, 1979, 1981), extracted haemoglobin is likely to behave differently from haemoglobin within the insect. This study uses a biotonometric method to examine the oxygenbinding properties of backswimmer haemoglobin in vivo, using the sigmoid decline in PO2 measured within the backswimmer’s air-stores to obtain haemoglobin OECs. The reliability of this method was then investigated by modelling air-store PO2 and oxygen release from the haemoglobin cells of a backswimmer under selected conditions. Knowledge of the in vivo OEC of backswimmers is necessary to understand the patterns of buoyancy regulation, in particular its role in producing a temporary condition of nearneutral buoyancy.

2. Materials and methods 2.1. Biotonometry The process of biotonometry was developed by Neville (1974) to determine haemoglobin OECs. While the conventional technique for determining an OEC uses spectrophotometry to measure haemoglobin–oxygen saturation in a chamber as it is flushed with oxygen or nitrogen, the biotonometric method relies upon an aerobic biological agent consuming oxygen from a sealed chamber containing a solution of oxygen-saturated haemoglobin, while the change in oxygen partial pressure (PO2) over time is recorded. In this study we use the backswimmer itself as a biological biotonometer: its air-store and associated haemoglobin cells comprise the sealed chamber while the insect performs the role of the aerobic biological agent. Biotonometry is based on the assumption that, in the absence of haemoglobin, an aerobic biological agent in a sealed chamber would cause the chamber’s PO2 to decline at a constant rate as it consumed the available oxygen. However, the presence of oxygenated haemoglobin within the chamber alters the rate at which PO2 declines by progressively unloading its oxygen in response to the falling PO2. At the beginning of a measurement the PO2 within the chamber is high and the haemoglobin is saturated with oxygen. Thus the biological agent will predominantly consume the free oxygen within the chamber, causing the PO2 in the chamber to decrease at a constant rate. The steadily declining PO2 eventually causes the haemoglobin to unload its oxygen into the chamber. This decreases the observed rate of PO2 change, as the biological agent is now respiring oxygen from the chamber supplemented with that released from the haemoglobin. Eventually the PO2 in the chamber reaches 0, at which point the haemoglobin is completely desaturated. The resulting sigmoid decrease in PO2 over time can be transformed into an OEC by equating the time axis extended parallel to the slope of the constant rate of oxygen consumption to declining haemoglobin–oxygen saturation (see Fig. 1, dashed lines). This analytical principle can be applied to

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Fig. 1. Example PO2 trace (solid line) recorded within the air-store of a submerged backswimmer. At the beginning of the dive the PO2 drops at a constant rate (A) due to the insect’s respiration until oxygen released from the haemoglobin (B) also begins to contribute to respiration. The maximum dive time in the absence of haemoglobin would therefore be at time (t LX ) while oxygen contributed by haemoglobin extends it to (t PX ). Point (t L0 ) indicates the time taken to consume all air-store oxygen in the absence of haemoglobin if respiration remained constant irrespective of PO2. The diagonal dashed lines parallel to the rate of oxygen consumption indicate the declining oxygen saturation of the haemoglobin. The backswimmer’s respiration ceases to be constant below the critical PO2 of 2 kPa (tPX ) and thus the haemoglobin must remain partially saturated, never reaching 0% (tP0 ).

PO2 changes measured within a backswimmer’s air-store, assuming that the insect’s respiration is constant and the air-store is sealed, to arrive at an OEC and establish the in situ oxygen binding properties of the haemoglobin. 2.2. Air-store PO2 measurement Australian backswimmers (Anisops deanei) carry a bubble of air within two lateral grooves on the ventral surface of their abdomen. This ‘air-store’ is held over the abdominal spiracles and, when the bug is submerged, is covered completely by a fringe of long hydrophobic hairs that arise from the outer margins of the abdomen’s ventral surface (see (Matthews and Seymour, 2008)). This study measured PO2 changes within the air-stores of backswimmers during simulated dives. Briefly, backswimmers (weight = 11.83 ± 1.85 mg, s.d., n = 9) were narcotised in pure CO2 for 3 min and then fixed on their back to a glass microscope slide with a small drop of cyanomethacrylate adhesive (Selleys Pty Ltd., Australia). After a 10 min recovery period, the insect was submerged in a 2 cm deep Petri dish containing air-equilibrated water at 20 °C. A fibre-optic oxygen probe with a 40 lm tip, held in a micromanipulator and connected to an oxygen meter (TX3, PreSens GmbH, Regensburg, Germany), was quickly placed within the air-store on the animal’s abdomen and PO2 recorded every 1 s for the duration of the dive. Only measurements made on inactive backswimmers were used for analysis. Backswimmers observed moving their large oar-like hind legs were excluded. 2.3. Analysis of air-store PO2 traces Measurements of air-store PO2 were transformed into oxygen equilibrium curves as follows: The constant rate of PO2 decline caused by the backswimmer’s respiration was found by fitting a straight line to the first stable 60–180 s of PO2 data, depending on the duration of the dive (Fig. 1, point A). As long as the PO2 trace dropped along this line it was assumed that the haemoglobin was 100% saturated. The release of oxygen at lower oxygen tensions caused the PO2 trace to diverge from the line of constant decrease in a sigmoid curve (Fig. 1, point B). In the absence of haemoglobin

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the PO2 would continue to drop to 0 kPa (Fig. 1, t L0 ). Assuming the backswimmer’s respiration remained constant, the oxygen would progressively disassociate from the haemoglobin until the PO2 trace reached 0 kPa, at which point it would be completely deoxygenated (Fig. 1, tP0 ). Under these circumstances the per cent oxygen saturation at any PO2 is calculated by:

S¼

  ðt P0  t L0 Þ  ðt Px  tLx Þ  100 ðt P0  tL0 Þ

ð1Þ

where S is the percentage of haemoglobin sites binding oxygen, t P0 is the time when the PO2 trace reaches 0 kPa (Fig. 1, t P0 ), tL0 is the time when the line of constant PO2 decrease crosses the abscissa (Fig. 1, t L0 ), t PX is the time at PO2 x on the trace (e.g., Fig. 1, t PX ) and t LX is the equivalent time at PO2 x on the line of constant decrease (e.g., Fig. 1, t LX ). However, as shown by experiments where the backswimmer’s haemoglobin was inactivated by carbon monoxide, the backswimmer’s oxygen consumption rate begins to decrease at very low oxygen tensions and the PO2 is not reduced to 0. Below a critical PO2 of approximately 2 kPa the resistance of the insect’s tracheal system limits the rate of oxygen uptake (Greenlee and Harrison, 2004). A critical PO2 of between 1.8 and 2.3 kPa has been determined experimentally for another similarlysized aquatic hemipteran, Agraptocorixa eurynome (Matthews and Seymour, 2010). Therefore, the OEC was calculated by disregarding the PO2 data below 2 kPa and substituting t P0 and t L0 with t P2 and t L2 (the times where the PO2 trace and line of constant PO2 decrease reach 2 kPa: see t PX and t LX Fig. 1). This calculation assumes that the haemoglobin is 0% saturated at 2 kPa. Since this is unlikely, a second OEC was calculated as being 10% saturated at 2 kPa (as determined from the in vitro oxygen-equilibrium curve of A. assimilis haemoglobin (Wells et al., 1981)), with % saturation calculated according to

S¼

  ðt P2  tL2 Þ  ðtPX  t LX Þ  100 ðt P2  tL2 Þ    ðt P2  t L2 Þ  ðt PX  t LX Þ  10 þ 1 ðtP2  t L2 Þ

ð2Þ

The OEC was produced by plotting S against PO2. The shapes of the in vivo OECs were analysed according to Hill (1910). 2.4. Model of in vivo OEC determination The procedure used for determining the in vivo behaviour of haemoglobin from measurements of air-store PO2 has several

Fig. 2. Schematic of the backswimmer model used for calculating PO2 changes in the air-store over time. Oxygen is consumed from the air-store at a rate equivalent to the backswimmer’s respiration (VO2). At the beginning of the simulation the airstore contains a quantity of oxygen which is supplemented with oxygen released from the haemoglobin cells and diffusing through the surface of the air-store from the surrounding water, which is affected by the oxygen conductance (G) of the haircovered air-store surface.

potential sources of error. Obtaining precise oxygen-equilibrium curves relies on fulfilment of the following assumptions: (a) the air-store and haemoglobin cells constitute a sealed vessel containing a finite amount of oxygen, (b) the rate of oxygen consumption by the backswimmer is constant during measurement, and (c) the haemoglobin is 100% saturated at the beginning of measurement and 0% saturated at the end. As these conditions were potentially invalid to some degree during measurement, a simple numerical model was created to understand how they would affect the position and shape of the derived OEC. The model assumes that the backswimmer respires oxygen from three sources: the air-store, the haemoglobin, and the water adjacent to the submerged air-store (Fig. 2). The starting conditions of the model assume that a 1.5 lL volume air-store (a typical volume as determined from buoyancy and direct measurement) contains 0.3069 lL of oxygen, assuming the air bubble was saturated with water vapour at 20 °C and contained 20.46% O2. As the haemoglobin contributes 47% of all oxygen consumed during a dive on average (Matthews and Seymour, 2008) the initial quantities of oxygen in the air-store and haemoglobin are considered to be equal. At 1 s intervals the model subtracts a constant volume of _ 2. O2 from the air-store, equivalent to the bug’s respiration rate VO _ 2 of 1.07  103 lL s1 was calculated as the mean oxygen A VO consumption rate of the submerged backswimmers at 20 °C. In

Fig. 3. Example of the output from the air-store model where V.O2 is constant at 1.07  103 lL s1 and air-store O2 conductance (G) is 2  105 lL s1 kPa1. The solid black line indicates the air-store’s PO2 during a dive, when oxygen is being released from haemoglobin (light dashed line) and diffusing in from the surrounding water (heavy dashed line). The solid grey line indicates the PO2 decrease during a dive, with oxygen diffusing from the water but without haemoglobin.

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100

A.

90

% Saturation

80 P75 = 4.17 kPa

70 60 50

P50 = 3.96 kPa

40 30

P25 = 3.55 kPa

20 10 0 100

B.

90

% Saturation

80 P75 = 4.14 kPa

70 60 50

P50 = 3.90 kPa

40 30

P25 = 3.30 kPa

20 10 0 0

2

4

6

8

10 12 PO2 (kPa)

14

16

18

20

Fig. 4. In vivo oxygen-equilibrium curve derived from the PO2 decrease in the backswimmers’ air-store. (A) Assumes that the haemoglobin was 0% saturated at 2 kPa. (B) Assumes that the haemoglobin was 10% saturated at 2 kPa. The solid line indicates a typical oxygen-equilibrium curve. The dashed line indicates the expected continuation of the oxygen-equilibrium curve. Circles indicate the mean P75, P50, P25. Error bars indicate ±95% CI (n = 9).

the absence of haemoglobin and oxygen diffusion from the surrounding water, the PO2 in the air-store is calculated at each 1 s interval by

PO2a ¼ Pb  P H2 O



Vi  Vr V T  ðVO2  V i Þ

 ð3Þ

where Pb is the barometric pressure (101.3 kPa), PH2 O is the saturated water vapour pressure (kPa), Vi is the volume of oxygen in the air-store at the ith interval, Vr is the volume of oxygen respired

(1.07  103 lL), VT is the total initial air-store volume of 1.5 lL, and VO2 is the initial volume of oxygen in the air-store (0.3069 lL). Carbon dioxide is assumed to be constant, because it is so highly buffered by the body fluids and the surrounding water. To simplify the model, nitrogen volume is also considered to be constant. The decreasing air-store PO2 establishes a partial pressure gradient that drives oxygen diffusion from the surrounding water at a rate according to

_ 2aq ¼ GðPO2aq  PO2a Þ VO

ð4Þ

100 nH = 5.46 2 R =1 nH = 2.49

Phase III

10 S 100-S

nH = 15.17 2 R = 0.98

nH = 5.21

Phase II

1 nH = 1.29 Phase I

nH = 2.61 2 R = 0.95

0.1 1

10 PO2 (kPa)

100

Fig. 5. Hill plot of A. deanei the OEC determined in vivo, assuming 10% saturation at 2 kPa (solid line). Filled circles indicate mean PO2. Error bars indicate ±95% CI (n = 9). Dashed line indicates Hill plot of A. assimilis OEC measured in vitro at a 500 mg Hb mL1 and 25 °C. Data replotted from Wells et al. (1981).

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25

A.

PO2 (kPa)

20

15

10

5

0 0

5

10

15

20

25

6

8

10

Time (min) 100

B.

90 80

% saturation

70 60 50 40 30 20 10 0 0

2

4 PO2 (kPa)

Fig. 6. Model simulation showing that oxygen uptake into the air-store from the water slows the reduction of the air-store’s PO2 during a dive (A) and makes the derived in vivo oxygen equilibrium curves less sigmoid (B). The dashed line in (B) shows the arbitrary shape of the sigmoid function used to represent the behaviour of haemoglobin in the model. The solid sigmoid lines are derived from models in which conductance was set at 0, 1, 2, 3 and 4  105 lL s1 kPa1, lightest to darkest lines, respectively.

5

7 6

4.9

4.8

4 nH

P50 (kPa)

5

3

4.7

2 4.6 1 4.5

0 0

1

2

3

4

-5 -1 -1 Air-store O2 conductance ( 10 L s kPa )

Fig. 7. Model results showing that increasing oxygen diffusion into the air-store (G) produces an apparent increase in oxygen affinity (P50, filled circles) while reducing the slope of the OEC (nH open circles).

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25

A.

PO2 (kPa)

20

15

10

5

0 0

2

4

6

8

10

6

8

10

Time (min) 100

B.

90

% saturation

80 70 60 50 40 30 20 10 0 0

2

4

PO2 (kPa) Fig. 8. Model simulation of the air-store’s PO2 during a dive showing that increases in respiration rate during a dive causes the oxygen in the air-store to drop rapidly (A) shifting the derived in vivo oxygen equilibrium curves to the right (B). The dashed line in (B) indicates the arbitrary shape of the sigmoid function used to represent haemoglobin in the model. All simulations began with a VO2 of 1.07  103 lL s1 and (G) was 0. Solid lines show models where the rate of respiration increased linearly during the simulation at rates of 0%, 0.1%, 0.2%, 0.4% and 0.8% s1, lightest to darkest.

_ 2aq is the rate of oxygen diffusion (lL O2 s1), G is the conwhere VO ductance of the air-store (lL O2 s1 kPa1) and PO2aq is the partial pressure of O2 in the surrounding water. PO2 also affects haemoglobin–oxygen saturation (S), modelled as a sigmoid function:

S¼



 1  100 1 þ eð  1:07  ðPO2a  5ÞÞ

ð5Þ

The model became unstable if the haemoglobin–oxygen saturation responded instantly to air-store PO2. Calculating saturation using the average PO2 of the previous eight intervals introduced a lag which allowed the haemoglobin to respond smoothly to declining air-store oxygen tensions. The sigmoid curve produced according to Eq. 5 has a P50 of 5 kPa and an nH of 5.2 between 20% and 80% oxygen saturation. These values were chosen to emulate the OEC produced by Wells et al. (1981) from A. assimilis haemoglobin. While this function does not exactly match the multiple phases of A. assimilis OEC, it is sufficient for examining how the shape and position of this standard curve would be altered if it were derived from the PO2 drop within an air-store and altered by the addition of oxygen from the surrounding water. The volume of oxygen released from the haemoglobin’s oxygen store at each 1 s interval (VO2Hb) is calculated by the drop in haemoglobin–oxygen saturation between at each interval, thus

  Si1  Si VO2Hb ¼  VO2HbT 100

ð6Þ

where Si1 is the percent oxygen saturation of the interval preceding the ith, Si is the oxygen saturation calculated for the current ith interval and VO2HbT is the total volume of oxygen stored by the haemoglobin at saturation. Both the surrounding water and haemoglobin are contributing oxygen to the air-store as the backswimmer’s respiration is consuming it, so the net rate of oxygen change within the air-store during each interval is

VO2Net ¼ ðVO2aq þ VO2Hb Þ  V r

ð7Þ

But because both VO2Hb and VO2aq are influenced by the airstore’s PO2, which is in turn determined by the backswimmer’s respiration (Vr), then

V r  VO2aq þ VO2Hb

ð8Þ

This causes the air-store’s PO2 to constantly decline over time (Fig. 3). To examine how oxygen diffusion from the water affects the shape and P50 of an in vivo OEC, the model was run with the airstore’s conductance for oxygen set at 0, 1, 2, 3 or 4  105 lL s1 kPa1. The effect of a steadily increasing rate of oxygen consumption during the dive was also tested by increasing the volume of oxygen respired each interval. This was done by cumulatively increasing the amount of oxygen consumed at each 1 s interval by a percentage of the initial Vr (0.1%, 0.2%, 0.4% or 0.8% initial Vr). The models were then run until the PO2 in the air-store reached 0. These simulated PO2 traces (Fig. 3) were then used to produce

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5.4

7 6

5.3

5.2

4

nH

P50 (kPa)

5

3

5.1

2 5

1 4.9

0 0

0.1

0.2

0.3

Increase in respiration rate(% s)

0.4

-1

Fig. 9. Model results showing that increasing respiration during a dive causes a decrease in apparent oxygen affinity (filled circles) and an increase in the slope (nH open circles) of a derived OEC.

OECs using the same biotonometric method as described previously. 3. Results

the duration of the dive period (Fig. 8a) and shifting the OEC to the right (Fig. 8b). This results in a steeper mid-slope and an artificially increased nH (Fig. 9). An increasing rate of oxygen consumption causes the PO2 trace to drop in a curve, invalidating the assumption that the initial decrease in PO2 is linear.

3.1. In vivo OEC and Hill plot 4. Discussion Linear regressions fitted to the PO2 traces recorded at the beginning of the dive revealed a constant linear decline in air-store PO2 (R2 = 0.9934 ± 0.01 s.d.), consistent with the backswimmers maintaining a stable VO2. Once the PO2 in the air-store reached 6 kPa, it no longer dropped linearly but began to decline in a curvilinear fashion. This transition from a linear drop in air-store PO2 can be explained by the release of oxygen from the backswimmer’s haemoglobin. The transformed experimental PO2 traces produced highly sigmoid OECs, with the haemoglobin’s oxygen affinity dropping rapidly below 5 kPa. Assuming 0% saturation at 2 kPa gave a P50 of 3.96 ± 0.15 (95% CI) kPa (Fig. 4a). Correcting for the partial saturation of the haemoglobin at 2 kPa gave 3.90 ± 0.15 kPa (Fig. 4b). The oxygen equilibrium curve assuming 10% saturation was then analysed as a Hill plot (Fig. 5). The plot indicated three distinct phases, the first being the shallowest (nH = 2.61) followed by an exceptionally steep phase above 35% O2 saturation (nH = 15.17) before again dropping off above 90% (nH = 5.46). 3.2. Model If the air-store is considered to be sealed (G = 0) and the backswimmer’s respiration is constant, the air-store’s PO2 drops rapidly as a sigmoid curve (Fig. 6a) and the derived OEC follows the shape of the sigmoid function (Fig. 6b). However, if the airstore is permeable to oxygen diffusion (G > 0) then the air-store’s PO2 drops at a slower rate, increasing the time taken for all the oxygen stores to be depleted (Fig. 6a). The addition of external oxygen changes the haemoglobin’s response to declining oxygen partial pressure, as it is impossible to distinguish between the oxygen released from the haemoglobin and that diffusing into the airstore. When these PO2 traces are transformed into OECs, they show artificially increased haemoglobin–oxygen saturations at lower PO2 as well as lower P50 (Figs. 6b and 7). As the PO2 appears to drop more slowly with increasing G, when examined as a Hill plot the nH values, plotted from the slope between 20% and 80% saturation, become increasingly shallow (Fig. 7). A linear increase in the rate the insect consumes oxygen from the air-store has the opposite effect to increasing G, decreasing

4.1. In vivo OEC and Hill plot The in vivo OEC produced in this study is, effectively, a whole animal OEC, as it is the product of all aspects of the backswimmers’ respiratory physiology, not just the interaction between haemoglobin and oxygen. The previously determined in vitro OEC of A. assimilis haemoglobin revealed a high degree of triphasy and a high level of cooperativity (nH = 6) (Wells et al., 1981). A high degree of triphasy is also a feature of the whole animal OEC (Fig. 5). However, when analysed on a Hill plot, this in vivo OEC displays an exceptionally high midslope (nH = 15.17). A conventional interpretation would suggest that this slope results from the cooperative interaction of at least 16 haemoglobin subunits (Wells and Dales, 1976). This far exceeds the cooperativity of any known haeme proteins – including the giant multimeric Lumbricus earthworm haemoglobin (n50 = 9.5 (Krebs et al., 1996; Weber and Vinogradov, 2001)). Rather than assume that this result accurately reflects the haemoglobin’s true OEC (as this would demand the existence of exceptionally large, tight-knit aggregates of haemoglobin subunits), the most likely explanation for such spuriously high Hill coefficients is a failure to achieve 100% oxygen saturation (Lapennas et al., 1981). This is an important issue, both for this study and the previous measurements of Anisops OECs, due to the relatively low oxygen affinity of this haemoglobin and the use of air to achieve complete saturation. Indeed, as the backswimmer’s haemoglobin cells are in equilibrium with the PO2 of their tracheal system and not the atmosphere in this study, it is unlikely that they were completely saturated at the beginning of measurement. Other errors directly associated with the biotonometric technique, including the leakage of oxygen into the air-store and changes in the insects’ rate of oxygen consumption during measurement, could also potentially account for the high nH values observed in this study. Mathematically modelling this process shows that a high oxygen conductance between the air-store and surrounding water actually reduces the apparent Hill coefficient, and so cannot explain the high nH observed (Fig. 7). An increase

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in the rate of respiration during measurement increases the slope of the OEC, as well as increasing the P50 (Figs. 8 and 9), but modelling shows that the impact of this error is small. For example, a 2.4fold increase in respiration rate by the end of a simulated dive causes the P50 to increase by 0.2 kPa when compared to a simulation where the respiration rate was constant, while the n50 increased by 1.2. Additionally, our measurements show that the insects’ oxygen consumption rate is more likely to decrease towards the end of a forced dive due to the low PO2 of the air-store. The similarities between the whole animal OEC and the in vitro haemoglobin OEC of Wells et al. (1981) suggest that the unusual polyphasic oxygen binding of Anisops haemoglobin in vivo is a real phenomenon (Fig. 5), but without using a more direct method than biotonometry the accuracy of this result remains to be conclusively demonstrated. 4.2. Functional constraints of backswimmer haemoglobin The oxygen-binding properties of backswimmer haemoglobin are restricted by the need for it to function both as a buoyancy regulator and respiratory oxygen source. The haemoglobin is located in the abdomen, but the most aerobically active tissues are in the thorax (i.e., the muscles associated with the oar-like hind legs). Evidence suggests that oxygen mainly diffuses from the abdominal haemoglobin cells to the thorax through the external film of air connecting the thoracic spiracles with the air-store and not necessarily through the longitudinal trunks of the tracheal system (Bare, 1929; Miller, 1966; Wells et al., 1981). Therefore, unlike the extracellular haemoglobins of Chironomus larvae, which have highly left-shifted oxygen-equilibrium curves (P50  0.17 kPa (Weber et al., 1985)), the haemoglobin carried by backswimmers must release its oxygen at a high enough PO2 to diffuse down a partial pressure gradient from the abdomen, through the air-store, and in through the thoracic spiracles. The backswimmer’s buoyancy is controlled by the volume of its air-store. During a dive the insect’s respiration consumes the airstore’s oxygen fraction, reducing its volume. For neutral buoyancy to occur, the haemoglobin must stabilise the air-store’s PO2, and therefore volume, in the face of continuing oxygen uptake by the insect. However, for a stable PO2 to produce a period of nearneutral buoyancy, it must occur when the air-store volume balances the backswimmers tendency to sink. This places two constraints on the initial volume of air collected from the surface. Firstly, as the air-store’s oxygen level and volume can only decrease during a dive, the initial volume must be larger than required for neutral buoyancy. Secondly, as the haemoglobin only unloads its oxygen at a low PO2, the volume of the air-store must reach neutral buoyancy before it becomes anoxic. In other words, if the collected volume of air is too large, then the volume of nitrogen collected would be so great that even an oxygen-depleted air-store would continue to confer positive buoyancy; if it is any smaller, the bug would become negatively buoyant before the haemoglobin stabilises the air-store’s PO2 and volume. The fractional decrease in air-store volume which occurs before haemoglobin stabilises the PO2 (FS) is calculated as:

FS ¼

FO2amb ðPb  PH2 O Þ  P50 Pb

ð9Þ

where FO2amb is the fraction of oxygen in dry atmospheric air (0.2095), Pb is atmospheric pressure, and PH2 O is the partial pressure of water vapour at saturation (2.34 kPa at 20 °C), and P50 the PO2 at which the haemoglobin is 50% saturated. Therefore, if the initial volume of air collected at the surface (VI) is larger or smaller than the volume required at neutral buoyancy (VN) such that

V I –V N þ ðV N  F S Þ

ð10Þ

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then the haemoglobin cannot stabilise the bubble’s volume at neutral buoyancy. The oxygen-binding properties of the haemoglobin dictate how the backswimmer uses its oxygen stores. The insect swims actively during the initial phase of the dive to overcome the positive buoyancy of its refilled air-store. This activity increases the backswimmer’s rate of oxygen consumption, causing the air-store’s volume to shrink quickly at the beginning of the dive due to the removal of a large fraction of its oxygen. The reduced volume of the bubble then closely offsets the tendency of the insect to sink. The haemoglobin’s P50 of 3.9 kPa ensures that it releases its bound oxygen only once the volume and PO2 of the air-store have both decreased significantly, thereby conserving the bound oxygen almost entirely for the neutrally buoyant phase, and so lengthening the dive. If the haemoglobin had a lower oxygen affinity, oxygen would be unloaded from the haemoglobin at a higher PO2 following a smaller decrease in bubble volume. Under these circumstances neutral buoyancy could only be achieved with a smaller bubble of air and the dive would be sustained primarily by oxygen released from the haemoglobin. As soon as the haemoglobin’s oxygen store was exhausted the bug would become negatively buoyant and would need to surface and replenish its air-store while it still contained oxygen. The shape of A. deanei’s oxygen equilibrium curve is also critical in determining the stability of the neutrally buoyant phase. The in vivo oxygen equilibrium curve is extremely steep – a PO2 change of less than 1 kPa (between 4.14 and 3.30 kPa) is sufficient to unload half of haemoglobin’s oxygen (Fig. 4). This high sensitivity to declining PO2 ensures that, after a drop in initial air-store volume of 16.4% to the P75, half of the oxygen carried by the haemoglobin is released as the volume declines by a further 1%. In contrast, the in vitro oxygen-equilibrium curve from A. assimilis haemoglobin (at a concentration of 500 mg mL1) indicates that the steepest phase, beginning at a PO2 of 8.5 kPa, only unloads 40% of the oxygen bound by haemoglobin and requires a 3 kPa drop to do so. This would stabilise the volume of the air-store between 12% and 15% below the initial volume, resulting in a shorter, and comparatively imperfect, near-neutral buoyancy phase.

5. Conclusions The in vivo OEC for Anisops haemoglobin determined using the biotonometric method provides a valuable insight into how the haemoglobin behaves within the insect’s abdomen. While this information is relevant to the animal’s physiology, reflecting changes in the biologically available oxygen stored within the haemoglobin cells during a dive, this technique only manages to capture a limited portion of the haemoglobin’s true OEC, being unable to guarantee complete oxygenation and de-oxygenation at the beginning and end of measurement. A comparison with the only available in vitro OEC determined for Anisops haemoglobin shows that the OEC produced using the biotonometric method is qualitatively similar, as both curves exhibit unusual oxygen binding characteristics. However, the limitations associated with oxygenating and deoxygenating the haemoglobin within the insect means that this technique cannot be used to understand oxygen-haemoglobin binding from 0% to 100% saturation.

Acknowledgements This research was supported by the Australian Research Council and the University of Adelaide. We thank the two anonymous reviewers for their constructive comments and advice.

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