J Comp Physiol B (2011) 181:941952 DOI 10.

1007/s00360-011-0573-3

ORIGINAL PAPER

Effects of environmental oxygen on development and respiration of Australian lungsh (Neoceratodus forsteri) embryos
Casey A. Mueller Jean M. P. Joss Roger S. Seymour

Received: 1 February 2011 / Revised: 20 March 2011 / Accepted: 22 March 2011 / Published online: 2 April 2011 Springer-Verlag 2011

Abstract The effects of oxygen partial pressure (PO2 ) on development and respiration were investigated in the eggs of the Australian lungsh, Neoceratodus forsteri. At 20C, embryonic survival and development was optimal at 15 and 20.9 kPa. Development was slowed at 5 and 10 kPa and embryos did not survive 2 kPa. At lower PO2 , the rate of oxygen consumption also decreased. Embryos responded to hypoxia by hatching at an earlier age and stage of development, and hatching wet and dry gut-free masses were reduced. The role of oxygen conductance (GO2 ) in gas exchange was also examined under selected environmental PO2 and temperatures. The breakdown of the vitelline membrane changed capsule geometry, allowed water to be absorbed into the perivitelline space and increased capsule GO2 . This occurred at embryonic stage 32 under all treatments and was largely independent of both PO2 and temperature (15, 20 and 25C), demonstrating that capsule GO2 cannot adaptively respond to altered environmental conditions. The membrane breakdown increased capsule diffusive GO2 and stabilised perivitelline PO2 , but reduced the convective GO2 of the perivitelline uid, as the large perivitelline volume and inadequate convective current resulted in a PO2 gradient within the egg prior to hatch.

Keywords Neoceratodus forsteri Embryos Development Respiration Hypoxia Capsule conductance

Introduction Vertebrate embryos are aerobic; they require oxygen from the environment for growth and development (Adolph 1979). Embryonic responses to low oxygen include decreased development and respiration rates (Alderdice et al. 1958; Garside 1959; Gruber and Wieser 1983; Hamor and Garside 1976; Mills and Barnhart 1999), retardation or promotion of hatching (Alderdice et al. 1958; Bradford and Seymour 1988; DiMichele and Powers 1984; Latham and Just 1989; Mills and Barnhart 1999; Petranka et al. 1982) and decreased hatching mass (Gruber and Wieser 1983; Hamor and Garside 1977). The effects of hypoxia are exacerbated by egg structures that restrict the exchange of oxygen from the environment to the embryo (Seymour and Bradford 1995). Amphibians and shes produce anamniote eggs in which the embryo is surrounded by perivitelline uid and an egg capsule. The capsules of amphibians consist of the vitelline membrane and jelly, and the perivitelline space appears under the vitelline membrane by absorption of water during development (Salthe 1963). The eggs of shes are described differently, with the vitelline membrane around the yolk, surrounded by the perivitelline space and a capsule of three to four layers (Groot and Alderdice 1985). Movement of oxygen through the capsule is via diffusion only and therefore relatively slow (Seymour 1994). It can be described by the Fick diffusion equation;   _ O2 GO2 PO M P 1 O 2 out 2 in

Communicated by I.D. Hume. C. A. Mueller (&) R. S. Seymour Ecology and Evolutionary Biology, University of Adelaide, Adelaide, SA 5005, Australia e-mail: casey.mueller@adelaide.edu.au J. M. P. Joss Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia

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_ O2 , nmol h-1) is related in which the rate of oxygen ux (M to the product of the capsule oxygen conductance (GO2 , nmol h-1 kPa-1) and oxygen partial pressure difference (PO2 , kPa) between the outside and inside of the capsule. The geometry of the capsule largely determines its GO2 , and in turn can inuence respiration and rate of development. Throughout development, amphibian egg geometry changes as water is absorbed through the capsule, creating the perivitelline space in which the embryo develops (Salthe 1965). This process is much faster in shes, with the perivitelline uid forming shortly after water immersion and the volume remaining constant throughout development in the limited species examined (Alderdice et al. 1984). Amphibian studies have demonstrated how changes in geometry can increase capsule GO2 and, therefore, oxygen ux (Seymour and Bradford 1987; Seymour et al. 1991; Seymour and Roberts 1995). Whether capsular GO2 can respond to environmental conditions, such as hypoxia that affects oxygen supply, or temperature that affects oxygen demand, apparently differs between amphibian species (Mills et al. 2001; Seymour et al. 1991). The importance of the egg capsule in shes has received some attention, with studies examining the GO2 (Rombough 1989) and oxygen diffusivity of the capsule (Daykin 1965; Hayes et al. 1951), while others have noted the increase in respiration upon hatching, highlighting the resistance of the capsule to gas exchange (Davenport 1983; Eldridge et al. 1977; Hayes et al. 1951). The Australian lungsh, Neoceratodus forsteri, has very large eggs with a thick egg capsule similar to amphibians. At approximately a third of the way through incubation, the vitelline membrane, which is located outside the perivitelline space, and some jelly break away from the inner layer of the capsule. A further layer of jelly dissolves, resulting in a thinner capsule, with membrane and jelly remnants lying at the bottom of the egg (Kemp 1982). This process is unlike that of amphibians and has never been investigated in terms of embryonic gas exchange. This study investigates the effects of environmental PO2 on development and respiration, and the importance of egg geometry changes in N. forsteri eggs. It also determines whether capsule conductance can respond adaptively to differences in oxygen supply (PO2 ) and metabolic demand (temperature) during incubation.

measured at pre-dawn and at 15:00 h, representing minimum and maximum measurements, using a portable dissolved oxygen meter (HQ10 LDOTM, Hach, Colorado, USA). Oxygen percent saturation readings were converted to partial pressure of oxygen (PO2 , kPa) using barometric pressure and saturated water vapour pressure at the relevant temperature. Measurements were taken at 15, 50 and 100 cm, at four locations in the pond from which eggs were collected. Effect of environmental PO2 on development, _ O2 and hatching M A total of 100 eggs of Neoceratodus forsteri were collected from the breeding pond at Macquarie University, Sydney, in October 2010, and were no more than 2 days old, judged by their stage of development. As eggs are laid individually, and there were a number of adults in the pond, the relationships between the embryos were unknown. The eggs were placed in two large snap-lock bags lled with autoclaved pond water and air and transported to the University of Adelaide. The eggs were at stages 1824 [closing of neural folds (Kemp 1982)] when they were randomly assigned to one of ve PO2 treatments: 2, 5, 10, 15 or 20.9 kPa (ambient air) at 20C. Eggs were placed individually in glass vials (25 mm diameter) on ten layers of Whatman No. 1 lter paper completely saturated with reverse osmosis water. This procedure exposed the eggs to the gas phase to avoid an external boundary layer, but prevented them from water loss. The vials were placed inside desiccators which were ushed with selected PO2 treatments created by mixing pure oxygen and nitrogen using a custom-made gas mixer that involved calibrated sapphire orices with precision gas pressure differences across them. A two-holed rubber stopper tted with plastic tubing allowed the gas mixture to enter the bottom of the desiccator and escape through the top. The PO2 leaving the desiccator was checked with a paramagnetic oxygen analyser (model 570, Taylor-Servomex, England), calibrated with N2 and air. When completely ushed, the desiccator was sealed. Eggs were exposed to ambient conditions for a few minutes when the desiccators were opened once a day _ O2 measurefor staging, and before and after GO2 and M ment. Treatments were then re-ushed and maintained at their target levels 0.5 kPa. Over a 24-h period, PO2 within the desiccator was estimated to decline by no more than 0.02 kPa, based on the number of eggs in the desic_ O2 measured in a cator and the highest rate of embryonic M previous study (Mueller et al. 2011). Embryos and hatchlings were staged according to Kemp (1982) and photographs from the Macquarie University lungsh laboratory Web site (http://www.bio.mq.edu.au/dept/centres/lungsh/ development/lungshGallerySQL.php?g=1).

Methods Pond measurements Oxygen level and temperature of the Neoceratodus forsteri breeding pond at Macquarie University, Sydney, were

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Briey, stages 26 cleavage; stages 711 blastulation; stages 1216 gastrulation; stages 1730 neurulation; stage 30 tail bud, gill clefts; stage 35 mouth, ns; stage 41 lateral line system (Kemp 1982). Oxygen uptake was determined throughout development from the decrease in PO2 in closed water-lled chambers, made from 5-mL glass syringes, ushed with water of the treatment PO2 . Each syringe had a 5-mm hole in the side into which an oxygen electrode (model 730, Diamond General, MI, USA) was inserted and compression sealed with silastic tubing. The syringe barrels were suspended horizontally across plastic water baths and the spouts closed by three-way valves. The baths were perfused from a thermocirculator (Thermomix 1442D, B.Braun, Melsungen, Germany) set at 20C. Currents from the electrodes were measured with an oxygen analyser (ReadOx-4H, Sable Systems, NV, USA). The electrodes were calibrated each day with a fresh zero solution of 20 mg sodium sulphite in 1 mL of 0.01 M sodium tetraborate and with air-equilibrated water (Tucker 1967). Individual eggs were placed in each syringe and air bubbles expelled through the electrode mount when the electrode was removed. The electrode was then inserted into the silastic tube and the chamber ushed with water at the treatment PO2 2 kPa, obtained by bubbling N2 in reverse osmosis water. The chamber size was set by the syringe plunger and varied from 1.2 to 1.6 mL, depending on embryo stage and egg size. After a 1-h period of stabilisation, readings of PO2 were taken every 5 min. Measurements were taken for approximately 2 h, in which time PO2 dropped by a maximum of 5 kPa under ambient levels _ O2 was calculated from chamber volume, (20.9 kPa). M decrease in PO2 and the oxygen capacitance of water at the relevant temperature according to Dejours (1981). Air_ O2 equilibrated water was measured after each run and M corrected for electrode drift, which was assumed to be _ O2 of capsules removed from the eggs was mealinear. M _ O2 measurements to sured separately and subtracted from M account for any oxygen uptake by the electrode, microorganisms in the water and the capsules themselves. In addition to the effects of PO2 on development and respiration, another experiment was undertaken to assess the hatching response of embryos to hypoxia. In November 2009, 20 eggs, collected from the Macquarie University breeding pond, were placed in the same oxygen treatments described above at approximately the 15-day-old age (stages 3637). The age and stage of hatchlings were recorded for eggs placed in the treatments at 15 days old (2009) and at 2 days old (2010). Hatchlings from both years were killed and placed in Tylers xative to assist removal of yolk before being dissected into body and residual yolk, weighed, dried over silica gel and reweighed.

Effect of environmental PO2 on capsule GO2 Eggs randomly selected from treatments were placed in reverse osmosis water in a plastic, water-jacketed chamber (35 mL) thermostated to 20C using a water bath (Julabo HC, Seelbach, Germany) and held by slight suction on the at end of a vertical tube (2 mm) through the chamber oor. Egg capsule geometry was measured by viewing the egg through the side of the chamber with a horizontal dissecting microscope tted with an ocular micrometer and a bre-optic microscope light. Internal and external radii were measured at the top, bottom and sides of the egg to take into account uneven egg geometry. The vertical and horizontal outer diameters were measured and then halved to nd the midpoint of the egg, from which the top, bottom, left and right inner radii were measured. Measurements were taken while the eggs were submerged in water to prevent light refraction in the jelly. Capsule diffusive GO2 (nmol h-1 kPa-1) was calculated from the following equation using egg geometry and Kroghs diffusion coefcient (a measure of oxygen permeability) of jelly (KO2 : nmol mm-1 h-1 kPa-1): 4 p ri ro KO2 Go2 2 ro ri GO2 depends on the effective surface area of a sphere, ESA = 4priro, where the radius is the geometric mean of the inner (ri, mm) and outer radii (ro, mm) of the jelly layers, and the thickness of the jelly L = ro - ri mm (Seymour and Bradford 1987). A Kroghs coefcient of the jelly that is 76% of water was assumed, a value determined for amphibian egg jelly (Seymour 1994). This value was used as there is no directly measured comparable estimate for sh egg jelly. GO2 was calculated using the measurements taken for each quarter sphere of the egg and then averaged. Effect of temperature on capsule GO2 and perivitelline PO2 In November 2008, N. forsteri eggs collected from the Macquarie University breeding pond were placed individually in sterilised plastic containers (33 mm diameter, with a perforated screw top lid) in 15 mm of autoclaved pond water. The eggs were randomly assigned to one of three incubation temperatures: 15, 20 and 25C when they were at stages 1518 (2 days old). A constant temperature room was set to 15C, while two temperature control cabinets in the room were set at 20 and 25C. The use of one chamber per treatment may be a form of pseudoreplication, but due to equipment this was unavoidable. Target temperatures were maintained at 1C. The inner and outer diameter of the egg capsule was measured through the equator of randomly selected eggs

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throughout development using the horizontal microscope described above. Eggs were exposed to air-equilibrated water in the chamber, using a small aquarium bubbler, for a minimum of 1 h. Perivitelline PO2 was then measured using a needle-mounted bre-optic microprobe (\50 lm diameter tip), connected to an optical oxygen sensor (Microx TX3, Precision Sensing, Regensburg, Germany). The optode was zeroed with an anoxic solution of reverse osmosis water with sodium sulphite and spanned with the air-equilibrated water of the chamber. A thermal probe measured the chamber water temperature and adjusted measurements accordingly. The optode was attached to a micromanipulator positioned above the egg chamber. While viewing the egg through the horizontal dissecting microscope, the optode tip was extended beyond the needle, inserted through the capsule and placed in the approximate centre of the perivitelline space with no contact with the embryo. Measurements were taken for 3060 s. To assess if PO2 varied within the perivitelline space, further PO2 measurements were made at four locations within stage 3839 eggs at 20C. Using the micrometer, measurements were taken at 4.4, 3.1, 1.7 and 0.3 mm from the embryo. To assess the role of convection in mixing perivitelline PO2 , the rate of movements of particles were measured within stage 3839 eggs at 20C. The micrometer in the microscope was aligned with a particles trajectory and the time taken to travel a certain distance was recorded. Analyses Statistical analyses were performed in JMP IN (SAS, Version 4.0.4). All data met the parametric assumptions of normality and equal variances, as assessed by the ShapiroWilk and OBriens tests, respectively. Statistics included one-way analysis of variance (ANOVA) for single factor analyses and two-way ANOVA with an interaction term for two factor analyses. When signicance was found, a TukeyKramer HSD for means comparisons was performed. Survival analysis, using a log-rank test, examined survival between PO2 treatments, and an analysis of covariance (ANCOVA) of log_ O2 regressions assessed differences between transformed M treatments. The signicance value was set at 0.05. All other data were expressed as mean 95% condence interval.

P = 0.94). Therefore, the combined pre-dawn pond oxygen level averaged 14.21 0.19 kPa and PO2 increased to an average of 19.91 0.25 kPa by 15:00 h. Likewise, temperature did not vary with depth at pre-dawn (F2,9 = 0.38, P = 0.69) or 15:00 h (F2,9 = 0.48, P = 0.46). Minimum pond temperature was 16.8 0.1C at pre-dawn, increasing to 20.1 0.1C at 15:00 h. Effect of environmental PO2 on development, _ O2 and hatching M Embryonic survival until 20 days of age increased with higher PO2 (Fig. 1). Survival analysis indicates that the curves presented in Fig. 1 are signicantly different (v2 = 419.98, df = 4, P \ 0.0001). At 20.9 and 15 kPa, survival was 60 and 45%, respectively. This compares to only 15 and 5% survival at 10 and 5 kPa (Fig. 1). All the embryos placed in the 2 kPa treatment died within 4 days of the start of treatments. The development rate did not vary between PO2 treatments until approximately 13 days old, at which development slowed at 5 and 10 kPa (Fig. 2). After this point, development continued to diverge between all four treatments until hatching. Experimental year (hypoxia exposure time) had no effect on hatching stage or age, and there was no interaction between PO2 treatment and year for stage or age (Table 1). There was a signicant difference in hatching stage between PO2 treatments (Table 1). Embryos hatched at progressively later stages as PO2 increased (P \ 0.05, TukeyKramer HSD, Table 2). Likewise, the age of hatching signicantly increased as PO2 increased (Tables 1, 2). Experimental year had no effect on wet mass, dry mass and water content of the body or residual yolk; also, there was no signicant interaction between year and PO2 treatment for any hatching mass attributes (Table 1). However, PO2 treatment did affect certain hatching mass attributes (Table 1). Wet gut-free body mass was signicantly higher at 15 and 20.9 kPa compared to 5 and 10 kPa (P \ 0.05, TukeyKramer HSD, Table 2). Likewise, dry gut-free body mass was signicantly different between treatments (Table 1), with dry mass of 15 kPa hatchings greater than at both 5 and 10 kPa, whilst the mass of hatchlings at 20.9 kPa was signicantly greater than 5 kPa (P \ 0.05, TukeyKramer HSD, Table 2). Water content of the gutfree body did not vary with treatment (Tables 1, 2). Residual yolk did not differ signicantly between treatments, in terms of wet or dry mass (Tables 1, 2). However, water content of residual yolk was signicantly different (Table 1), due to higher water content at 20.9 kPa compared to 5 kPa (P \ 0.05, TukeyKramer HSD, Table 2). Using a fresh ovum dry mass of 4.73 mg

Results Pond measurements Pond oxygen levels did not vary with depth at pre-dawn (F2,9 = 0.059, P = 0.94) or 15:00 h (F2,9 = 0.066,

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J Comp Physiol B (2011) 181:941952 Fig. 1 Survival of Neoceratodus forsteri embryos at different PO2 treatments at 20C in 2010. Initial sample sizes were 20 embryos per treatment. Treatments were started when embryos were approximately 2 days old

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Fig. 2 Stage of development of Neoceratodus forsteri as a function of age under selected PO2 treatments at 20C in 2010. Symbols are means of 25 individuals. Bars represent 95% CI. The relationship between age and stage at each PO2 is described by the following equations: 20 kPa: stage = -0.019age2 ? 1.36age ? 20.50, r2 = 0.99; 15 kPa: stage = -0.020age2 ? 1.37age ? 20.27, r2 = 0.99; 10 kPa: stage = -0.021age2 ? 1.27age ? 20.72, r2 = 0.99; 5 kPa: stage = -0.031age2 ? 1.57age ? 18.45, r2 = 0.99

(Mueller et al. 2011), yolk to hatching mass conversion was 22% at 5 kPa, 25% at 10 kPa, 34% at 15 kPa and 32% at 20.9 kPa. _ O2 was most strongly affected by PO2 treatment at M the later stages of embryogenesis (Fig. 3). The log_ O2 against stage showed transformed regressions of M signicant differences across PO2 treatments (ANCOVA, _ O2 diverged during developF3,92 = 2.78, P = 0.043). M ment, increasing at 15 and 20 kPa above 5 and 10 kPa (Fig. 3). Just prior to hatching, embryos consumed a _ O2 as PO2 decreased signicantly lower maximum M (F3,9 = 15.28, P = 0.0007).

Effect of environmental PO2 on capsule GO2 The mean diameter of young ova (stage 24) was 3.48 0.20 mm with a mean volume of 22.07 1.17 mm3. Whole eggs, including the capsule, had a mean diameter of 6.74 0.10 mm and a volume of 160.44 7.30 mm3. Neither mean ova diameter (F4,35 = 1.07, P = 0.38) nor whole egg diameter (F4,35 = 0.30, P = 0.88) varied at the start of PO2 treatments. Capsule dimensions were constant until 10 days old, when embryos reached Kemp (1982) stage 32 (embryo longer, tail bud) and the vitelline membrane began to break down, which

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Table 1 Results of a two-way ANOVA testing the factors of PO2 treatment, year (hypoxia exposure time) and an interaction between PO2 treatment and year for Neoceratodus forsteri hatching attributes PO2 treatment Hatching stage Hatching age Hatching mass Wet gut-free body Dry gut-free body Water content of body (%) Wet residual yolk Dry residual yolk Water content of yolk (%) F3,24 = 4.66, P = 0.01 F3,24 = 5.55, P = 0.0046 F3,24 = 1.77, P = 0.18 F3,24 = 1.83, P = 0.17 F3,24 = 3.06, P = 0.052 F3,24 = 3.32, P = 0.033 F1,24 = 0.02, P = 0.90 F1,24 = 0.88, P = 0.36 F1,24 = 0.65, P = 0.43 F1,24 = 0.23, P = 0.64 F1,24 = 3.21, P = 0.09 F1,24= 1.15, P = 0.29 F3,24 = 0.51, P = 0.68 F3,24 = 1.22, P = 0.32 F3,24 = 0.25, P = 0.86 F3,24 = 0.40, P = 0.75 F3,24 = 0.73, P = 0.54 F3,24 = 0.22, P = 0.88 F3,32 = 17.67, P < 0.0001 F3,32 = 9.46, P = 0.0001 Year F1,32 = 1.47, P = 0.23 F1,32 = 2.13, P = 0.15 PO2 treatment 9 year F3,32 = 1.36, P = 0.27 F3,32 = 0.82, P = 0.49

Signicance set at 0.05. Bold type indicates signicant results

Table 2 Neoceratodus forsteri hatching stage, age and wet and dry hatchling masses incubated at four PO2 treatments at 20C PO2 treatment (kPa) 5 Hatching stage Hatching age (days) Hatchling mass Wet gut-free body (mg) Dry gut-free body (mg) Water content of body (%) Wet residual yolk (mg) Dry residual yolk (mg) Water content of yolk (%) 6.33 1.13(5) 1.04 0.09 (5) 82.8 4.2 (5) 8.77 0.92 (5) 4.72 0.48 (5) 45.9 5.6 (5) 7.35 1.32(6) 1.18 0.13 (6) 83.6 2.1 (6) 9.44 0.50 (6) 4.77 0.29 (6) 49.3 3.6 (6) 10.78 1.89(11) 1.63 0.24(11) 84.5 1.3(11) 8.82 0.32(11) 4.17 0.28(11) 52.8 2.5(11) 11.29 1.74(11) 1.50 0.12(11) 86.1 1.7(11) 9.63 0.63(11) 4.28 0.31(11) 55.1 4.4(11) 37.2 0.4(5) 17.6 2.5(5) 10 38.8 1.2(6) 20.0 2.2(6) 15 41.6 0.7(14) 23.9 1.6(14) 20.9 42.8 0.8(16) 26.0 1.6(16)

Data are means from 2 years. Data are presented as means 95% CI (n)

Fig. 3 Oxygen consumption rate of Neoceratodus forsteri during embryonic development as a function of stage under selected PO2 treatments at 20C. Symbols are means of 26 individuals. Bars represent 95% CI. The relationship between stage and oxygen consumption _ O2 ) at each PO2 is described (M by the following equations: _ O2 0:14e0:126stage , 20.9 kPa: M r2 = 0.92, 15 kPa: _ O2 0:10e0:129stage , r2 = 0.84, M _ O2 0:13e0:110stage ; 10 kPa: M _ O2 r2 = 0.77, 5 kPa: M 0:39e0:070stage ; r2 = 0.46

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occurred at this stage irrespective of PO2 treatment. Membrane remnants that settled at the bottom of the perivitelline space increased capsule thickness at the bottom of the egg, but average capsule wall thickness decreased slightly (Fig. 4a). Effective surface area increased, approximately doubling throughout incubation (Fig. 4b). Perivitelline volume increased throughout development in a similar manner in all treatments, with the maximum volume reaching approximately 120 mm3 at 5, 10 and 15 kPa and a slightly higher 130 mm3 at 20.9 kPa (Fig. 4c). The capsule wall volume also increased from approximately 130 mm3 to 260 mm3 prior to hatching (Fig. 4d). These changes resulted in an increase in capsule GO2 throughout development that was similar across PO2 treatments (Fig. 5). A maximum GO2 of approximately 10 nmol kPa-1 h-1 was reached prior to hatching at 5, 10 and 15 kPa, slightly lower than 12 nmol kPa-1 h-1 reached at 20.9 kPa. Effect of temperature on capsule GO2 and perivitelline PO2 Capsule GO2 increased during development at all temperatures (Fig. 6). The increase was stage specic, with the membrane breakdown beginning at Kemp (1982) stage 32 at all temperatures. The magnitude of the increase in GO2 at later stages differed with temperature (F2,14 = 14.25, P = 0.0004), with a greater GO2 at stage 40 at 15C compared to 20 and 25C (P \ 0.05, Tukey Kramer HSD). Average measured perivitelline PO2 levels varied with temperature and stage of development (Fig. 7). Perivitelline PO2 declined initially at 20 and 25C, before steadying from stage 32 at 13 and 10 kPa, respectively. Perivitelline PO2 decreased only slightly to 17.5 kPa at 15C. Convection rate within the perivitelline uid averaged 0.18 0.04 mm s-1 (n = 10) at stage 39 at 20C. Despite this, the perivitelline uid was not completely mixed, with differences in PO2 with depth (F3,36 = 4.8212, P = 0.0064, Fig. 8). PO2 was signicantly higher just inside the capsule (16.7 1.2 kPa at 4.4 mm from the embryo) compared to the other three depths in the egg (P \ 0.05, TukeyKramer HSD, Fig. 8). At 3.1, 1.7 and 0.3 mm from the embryo, PO2 was not signicantly different (P [ 0.05, TukeyKramer HSD). The PO2 of 16.7 kPa just under the membrane corresponds well to the calculated PO2 of 17.0 kPa at the same stage and temper_ O2 and ature using the Fick equation (1) and measured M _ O2 , GO2 . Likewise, using measured perivitelline PO2 and M -1 -1 GO2 is estimated at 7 nmol h kPa , similar to 9 nmol h-1 kPa-1, determined from egg geometry and KO2 (Fig. 5).

Discussion Effect of environmental PO2 on development, _ O2 and hatching M Embryonic survival is highest at 20.9 and 15 kPa, indicating that these levels of oxygen are optimal for N. forsteri development. Below 15 kPa, there is a progressive decrease in survival until none at 2 kPa (Fig. 1). A study by Kemp (1984) on spawning in the Brisbane River found that adults lay most eggs in PO2 around 1218 kPa, similar to the PO2 of 1420 kPa measured in the breeding pond in this study. However, a few eggs were found in PO2 as low as 2 kPa, but it is not known whether they survived. The nature of spawning, in which eggs are laid individually in water plant beds (Kemp 1986), would enhance survival as eggs do not compete with each other for oxygen, eggs are kept off the substrate where oxygen decreases, and photosynthesis can potentially augment overall oxygen levels. The development rate of embryos at the different PO2 treatments slowly diverges so that, once hatching begins, embryos at 5 and 10 kPa are two to three stages behind embryos at 15 and 20.9 kPa (Fig. 2). The slowing of development with decreasing oxygen is not unique to N. forsteri, with similar declines in development rate under hypoxia in other shes (Alderdice et al. 1958; Garside 1959; Hamor and Garside 1976) and amphibians (Bradford and Seymour 1988; Mills and Barnhart 1999). Embryos also respond to hypoxia by hatching at both an earlier age and stage of development (Table 2). This occurs whether embryos have been exposed to hypoxia for all of development (2010 embryos) or from 15 days of age (2009 embryos). Premature hatching under hypoxic conditions has been found in shes (Alderdice et al. 1958; Czerkies et al. 2001; DiMichele and Powers 1984; Latham and Just 1989; Oppen-Bernsten et al. 1990) and amphibians (Bradford and Seymour 1988; Mills and Barnhart 1999; Petranka et al. 1982; Warkentin 2002) and is considered a respiratory response. The earlier stage of hatching under hypoxia is evidenced by lower wet and dry gut-free masses of hatchlings at 5 and 10 kPa (Table 2). While yolk to body tissue conversion decreased at lower PO2 , residual yolk mass did not increase signicantly. However, whether hypoxia affects conversion efciency cannot be assessed without energy content data from yolk and hatchlings. _ O2 throughout development The pattern of embryonic M matches closely with that of development (Fig. 3). The _ O2 between PO2 treatments becomes more difference in M _ O2 pronounced at later developmental stages. Pre-hatch M _ O2 decreases signicantly with PO2 due to an already low M in combination with the earlier hatching stage and age. The _ O2 with PO2 at most stages of development drop in M

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948 Fig. 4 Capsule thickness (a), capsule effective surface area (b), perivitelline space volume (c) and capsule wall volume (d) of Neoceratodus forsteri eggs in relation to stage during development under four PO2 treatments at 20C. Symbols are means of 26 individuals. Bars represent 95% CI

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J Comp Physiol B (2011) 181:941952 Fig. 5 Oxygen conductance (GO2 ) of the egg capsule of Neoceratodus forsteri in relation to stage throughout development under four PO2 treatments at 20C. Symbols are means of 26 individuals. Bars represent 95% CI

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Fig. 6 Oxygen conductance (GO2 ) of the egg capsule of Neoceratodus forsteri in relation to embryonic stage at three incubation temperatures. Symbols are means of 314 individuals. Bars represent 95% CI

indicates that N. forsteri is an oxyconformer rather than an _ O2 in oxyregulator (Prosser 1973). A lack of regulation in M response to hypoxia has been found for other embryonic shes (Davenport 1983; Gruber and Wieser 1983; Hamor and Garside 1979), and is most likely due to the presence of the capsule (Rombough 1988). Environmental effects on capsule GO2 The vitelline membrane breakdown in N. forsteri egg capsules represents the beginning of a gradual hatching process, but it also has signicant respiratory implications. Upon membrane breakdown, which occurs at Kemp (1982) stage 32, regardless of oxygen level or temperature,

capsule geometry changes, allowing capsule GO2 to increase (Figs. 5, 6). Unlike amphibian eggs, in which water is slowly absorbed into the perivitelline space, increasing the volume and decreasing the capsule thickness evenly, the membrane breakdown in N. forsteri causes egg capsule geometry to become uneven. While membrane and jelly remnants increase thickness at the bottom of the egg, overall capsule thickness decreases slightly (Fig. 4a), which, together with an increase in effective surface area (Fig. 4b), substantially increases capsule GO2 (Eq. 2, Fig. 5). Water absorption into the egg, which only occurs after membrane breakdown, is evidenced by the increase in perivitelline volume and capsule wall volume from stage 32 (Fig. 4c, d).

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950 Fig. 7 Perivitelline PO2 of Neoceratodus forsteri eggs in relation to stage throughout development at three incubation temperatures. Symbols are means of 38 eggs. Bars represent 95% CI

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Fig. 8 Oxygen partial pressure (PO2 ) at different depths in stage 3839 of Neoceratodus forsteri eggs under normoxia at 20C, presented as mean 95% CI (n = 10). PV perivitelline

Changes in capsule GO2 remain stage specic, irrespective of incubation temperature, but maximum GO2 prior to hatching is slightly greater at cooler temperatures (Fig. 6). This is likely due to a greater loss of capsule structural integrity, resulting in a larger expansion of the egg, due to a longer incubation time. Furthermore, at 15C, oxygen demand is at its lowest (Mueller et al. 2011), so an increase in GO2 at this temperature is the opposite to what would be a regulatory response. The greater increase in GO2 under the temperature treatments compared to the PO2

treatments is due to egg geometry measurements being taken through the equator of the egg only. This does not account for the greater thickness at the bottom of the egg due to the membrane remnants, and is therefore an overestimation of GO2 . This difference in measurement also overestimates the continual increase in GO2 after stage 32 (Fig. 6). Measurements through the equator of eggs for both the temperature and PO2 treatments were similar in both experiments. It is clear that capsule GO2 of N. forsteri eggs is dependent upon developmental stage and therefore controlled by the embryo, but unresponsive to the environmental factors of PO2 or temperature. Not only is the initial membrane breakdown hard-wired to occur at stage 32, but it also appears that the magnitude of water absorption into the perivitelline space does not alter signicantly with environmental conditions. Such a correlation between stage of development and capsule GO2 , and a lack of response to ambient PO2 and temperature, was also found for the eggs of the terrestrial amphibian Pseudophryne bibronii (Seymour et al. 1991). This contrasts with the capsule GO2 of Ambystoma, which do increase when eggs are exposed to lower PO2 (Mills et al. 2001). The response of GO2 in Ambystoma was attributed to their aquatic reproduction, in which any response that reduces the exposure of embryos to low oxygen levels is a selective adaptation. The ability of GO2 to respond to hypoxia is probably less important in the eggs of P. bibronii, which develop in an oxygen-rich terrestrial environment. Under this hypothesis, the capsule GO2 of aquatic N. forsteri eggs should respond to the environment. However, it is possible that the increase in GO2 that occurs throughout development is adequate under most natural conditions. In the wild, the large eggs of

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951 comments on the manuscript. We acknowledge funding from the University of Adelaide and Australian Geographic Society.

N. forsteri are laid individually, rather than in a mass, maximising oxygen availability (Seymour 1994). This, together with the preferential spawning of adults at PO2 around 15 kPa (Kemp 1984; this study) and the inherently low oxygen demand of the embryos (Mueller et al. 2011), may eliminate the selective advantage of a hypoxic response in GO2 . Perivitelline PO2 Perivitelline PO2 declines initially at 20 and 25C, but stabilises with membrane breakdown (Fig. 7). However, the increase in perivitelline volume, while increasing capsule diffusive GO2 , potentially decreases the effectiveness of the cutaneous cilia-driven mixing of the perivitelline uid, thereby reducing convective GO2 . This is evidenced by an oxygen gradient present in the uid late in development at 20C (Fig. 8). Using the PO2 measured just inside the capsule and next to the embryo, the resistance exhibited by the capsule and perivitelline uid can be determined. Of the total resistance, which drops PO2 to 13.6 kPa next to the embryo (Fig. 8), the capsule contributes 58% and the perivitelline uid 42%. The high resistance of the perivitelline uid is a contradictory result to other sh and amphibian eggs in which the uid is well mixed (Burggren 1985; Green 2004; Mueller and Seymour 2011) but similar to that found in Misgurnis fossilis eggs (Berezovsky et al. 1979 cited in Rombough 1988). The perivitelline PO2 gradient occurs despite a convective current of 0.18 mm s-1. This current was measured at the centre of the egg and it is likely that velocity increased at the embryos surface and, hence, the lack of a boundary layer, but the current was too weak to mix uid near the capsule. Burggren (1985) measured velocities of 0.1 mm s-1 at 5C to nearly 0.3 mm s-1 at 25C in the eggs of Rana palustris. Using a Q10 of 2.52 between 15 and 25C calculated by Burggren (1985), a predicted convection rate of 0.25 mm s-1 at 20C is 1.4 times faster than the rate observed in N. forsteri. Complete mixing is further hindered by the large perivitelline volume of approximately 120 mm3 in late-stage N. forsteri eggs. This is much larger than, for example, P. bibronii, an amphibian of similar embryonic mass with a volume of approximately 30 mm3 (Bradford and Seymour 1985; Seymour and Bradford 1987). Additionally, N. forsteri embryos lie at the bottom of the perivitelline space, resulting in greater distances over which currents created by cilia need to travel. The embryos are also mostly quiescent, which limits the muscular movements that stir the perivitelline uid.
Acknowledgments We thank Rolf Ericsson from the Australian lungsh laboratory, Macquarie University for his expertise and help in the collection of eggs and the two anonymous reviewers for helpful

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