The Energy Cost of Embryonic Development in Fishes And

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The Energy Cost of Embryonic Development in Fishes And J Comp Physiol B (2011) 181:43–52 DOI 10.1007/s00360-010-0501-y ORIGINAL PAPER The energy cost of embryonic development in fishes and amphibians, with emphasis on new data from the Australian lungfish, Neoceratodus forsteri Casey A. Mueller • Jean M. P. Joss • Roger S. Seymour Received: 10 March 2010 / Revised: 14 July 2010 / Accepted: 16 July 2010 / Published online: 30 July 2010 Ó Springer-Verlag 2010 Abstract The rate of oxygen consumption throughout genome size. The low cost of development may be asso- embryonic development is used to indirectly determine the ciated with construction of a rather sluggish fish with a low ‘cost’ of development, which includes both differentiation capacity for aerobic metabolism. The metabolic rate is and growth. This cost is affected by temperature and the lower in N. forsteri hatchlings than in any other fishes or duration of incubation in anamniote fish and amphibian amphibians at the same temperature, which matches the embryos. The influences of temperature on embryonic extremely low aerobic metabolic scope of the juveniles. development rate, respiration rate and energetics were investigated in the Australian lungfish, Neoceratodus Keywords Fishes Á Amphibians Á Neoceratodus forsteri Á forsteri, and compared with published data. Developmental Embryonic development Á Oxygen consumption stage and oxygen consumption rate were measured until hatching, upon which wet and dry gut-free masses were determined. A measure of the cost of development, the Introduction total oxygen required to produce 1 mg of embryonic dry tissue, increased as temperature decreased. The relation- Embryos are formed by differentiation (the specification of ship between the oxygen cost of development (C, cell types) and growth (the proliferation of cells). Together, ml mg-1) and dry hatchling mass (M, mg) in fishes and these two processes constitute development. During amphibians is described by C = 0.30 M0.22 ± 0.13 (95% CI), development in the anamniote eggs of fishes and amphib- r2 = 0.52. The scaling exponent indicates that the cost of ians, oxygen is consumed and used to convert yolk into embryonic development increases disproportionally with embryonic tissue. The embryonic respiration of fishes and increasing hatchling mass. At 15 and 20°C, N. forsteri cost amphibians can be measured throughout the incubation of development is significantly lower than the regression period to provide a means of estimating the energy ‘cost’ of mean for all species, and at 25°C is lower than the allo- development. The term ‘cost of development’ is used, metrically scaled data set. Unexpectedly, incubation of rather than the ‘cost of growth’ as stated in some studies N. forsteri is long, despite natural development under (Conceic¸a˜o et al. 1998; Wieser 1994; Wieser and Medgyesy relatively warm conditions, and may be related to a large 1990), as it encompasses all the energetic processes that occur during embryogenesis, including differentiation, growth and tissue maintenance. ‘Cost of development’ has Communicated by I. D. Hume. also been used in previous embryonic bird and reptile studies (Vleck and Hoyt 1991; Vleck and Vleck 1987), and C. A. Mueller (&) Á R. S. Seymour Ecology and Evolutionary Biology, University of Adelaide, therefore the term is used for consistency across embryonic Adelaide, SA 5005, Australia literature. e-mail: [email protected] The total amount of oxygen consumed during develop- ment divided by the resulting gut-free hatchling mass J. M. P. Joss Biological Sciences, Macquarie University, determines the respiratory cost. This cost of development Sydney, NSW 2109, Australia (C,mlmg-1) can be altered by temperature. An increase in 123 44 J Comp Physiol B (2011) 181:43–52 temperature increases both development and respiration feeding embryos is an area of research that has been rates in fish and amphibian embryos (Bradford 1984; Das neglected until now. Developmental cost of numerous et al. 2006; Kuramoto 1975; Mitchell and Seymour 2000; species is assessed in terms of temperature and develop- Rombough 1994; Seymour et al. 1991). This can have a mental duration in an effort to understand the factors that significant impact on the energy used throughout incuba- influence the cost of development in fishes and amphibians. tion, which in turn can influence hatchling morphology, but The results for N. forsteri are placed in the context of these with inconsistent results: higher temperatures producing findings. smaller, larger or equal-sized hatchlings (see Table 10 in Kamler 2008 for examples). The Australian lungfish, Neoceratodus forsteri, provides Methods a unique opportunity to examine such embryonic physiol- ogy in a fish with very large, yolk-rich eggs. Neoceratodus Egg collection and incubation forsteri, a sarcopterygian dipnoan fish, is now almost uni- versally believed to be the closest living relative to the A hundred eggs of Neoceratodus forsteri were collected tetrapods (land vertebrates, including amphibians) (Brink- from breeding ponds at Macquarie University, Sydney in mann et al. 2004; Yokobori et al. 1994 and references November, 2008. The eggs were either laid the previous therein). At approximately 1 cm in diameter, including a evening or were no more than 2 days old, judged by their thick jelly capsule, N. forsteri eggs are closer in size to stage of development. Each egg was placed in its own ster- those of large-egged amphibians than other fish. The eggs ilised plastic container (33 mm diameter, with a perforated are laid from August to December (Kemp 1986) in tem- screw top lid) in 15 mm of autoclaved pond water and peratures ranging from 16 to 26°C (Kemp 1984) and are transported by air to the University of Adelaide. The eggs individually deposited, adhering to water plants by an were randomly assigned to one of three incubation temper- initially sticky outer jelly layer (Kemp 1994). Spawning atures: 15 (n = 34), 20 (n = 34) and 25°C(n = 32). These sites are usually characterised by a slow or moderate water temperatures were chosen as they represent the range of current to a depth of 1.5 m (Kemp 1984). midday temperatures recorded within water plant beds used The amphibian-like development of N. forsteri embryos for spawning during the breeding season (Kemp 1984). A is well documented morphologically (Kemp 1982), but constant temperature room was set to 15°C whilst two tem- there is a dearth of information about their physiology and perature control cabinets in the room were set at 20 and 25°C. environmental influences upon it. Only one study has Thermometers placed inside each cabinet verified that target examined the effect of temperature on N. forsteri embry- temperatures were maintained ±1°C. The water of each egg onic development (Kemp 1981). Development rates did not container was replaced with fresh autoclaved and filtered vary within the temperature range of 18–22°C, a 5°C range. rainwater at least once a week. Care was taken not to cross Embryos incubated at 10°C did not survive to gastrulation, contaminate the water in containers, as the embryos are whilst those above 30°C died within hours of being placed highly susceptible to bacterial infections (Kemp 1994). This at the high temperature (Kemp 1981). This indicates both technique of incubation was very successful, with approxi- 10 and 30°C are beyond the survival limits of the embryos. mately 90% of the eggs surviving to hatching. Such results are not unexpected, considering that 10 and To associate stage with age, some embryos had to be 30°C are outside the natural spawning temperatures visible. The jelly layers of four eggs from each temperature recorded. This study uses temperatures over a broader treatment were removed by placing the egg in a 0.05 M range, yet encompassing natural conditions, to examine the solution of sodium hypochlorite. The water was swirled effect of temperature on development rate of N. forsteri gently until the jelly dissolved down to the vitelline embryos. membrane. The eggs were then rinsed in water several This study also examines the effect of temperature on times until the smell of the bleach had disappeared. These N. forsteri respiration, yolk utilisation and the size of eggs were examined at the same time each day throughout hatchlings produced. From this, a measure of the cost of development and their stage of development was recorded. development, the amount of oxygen required to build a Staging was based on Kemp (1982) and photographs from hatchling, can be calculated. This study aims to use pre- the Macquarie University lungfish laboratory website (http:// vious literature on embryonic respiration in other fishes and mac-0170.bio.mq.edu.au/*gjoss/lungfish_development/ amphibians to calculate and examine the embryonic cost of lungfishSQL.php?g=1). Embryos and larvae were staged development in this group of animals. Respiratory costs according to this system whenever measurements were have been compared in larval fish species (Conceic¸a˜o et al. undertaken. 1998; Wieser and Medgyesy 1990), however, the com- A selection of fresh ova were randomly chosen, parison of the cost of development in endogenously removed from the egg capsule, killed by chilling, weighed 123 J Comp Physiol B (2011) 181:43–52 45 to 0.01 mg, dried over silica gel and reweighed on tared consumption of water-filled chambers was also measured foil to 0.01 mg with an electronic balance (Mettler AE183, to control for respiration of microorganisms in the water Greifensee, Switzerland). Hatchlings were randomly and oxygen uptake by the electrode, and the M_ O2 of cap- selected from each temperature, killed by chilling and sules removed from eggs was measured separately. placed in Tyler’s preservative (Tyler 1962) for 24 h. The Chamber and capsular M_ O2 values were subtracted from residual yolk, which pulled away from the body tissue M_ O2 measurements to calculate average embryonic M_ O2 cleanly due to the preservative, was dissected under a of relevant experiments.
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