Ontogeny and paleophysiology of the gill: new insights from larval and air-breathing fish.

There are large changes in gill function during development associated with ionoregulation and gas exchange in both larval and air-breathing fish. Physiological studies of larvae indicate that, contrary to accepted dogma but consistent with morphology, the initial function of the gill is primarily ionoregulatory and only secondarily respiratory. In air-breathing fish, as the gill becomes progressively less important in terms of O(2) uptake with expansion of the air-breathing organ, it retains its roles in CO(2) excretion, ion exchange and acid-base balance. The observation that gill morphology and function is strongly influenced by ionoregulatory needs in both larval and air-breathing fish may have evolutionary implications. In particular, it suggests that the inability of the skin to maintain ion and acid-base balance as protovertebrates increased in size and became more active may have been more important in driving gill development than O(2) insufficiency.

The energetics of embryonic growth.

Embryos typically operate under much tighter energy constraints than older animals. This has had a profound impact on how energy is stored, mobilized and partitioned. The result is sometimes quite different ways of doing things. Growth, in particular, is a much more important activity during development. Compared with adults, specific growth rates (g) are extremely high (≥150%day(-1) for some fish). Production efficiencies are also much higher, particularly for early stages where values of 80-90% are not uncommon. Higher production efficiencies are possible, in part, because of lower unit costs at high g. Unlike in adults, the unit cost of growth does not appear to be fixed during early life. Energy also tends to be partitioned in a different manner, with compensatory partitioning being much more important during early life. Other differences include much higher routine metabolic intensities, smaller aerobic scopes and approximately isometric scaling of routine metabolism. The implications for ontogenetic growth models are discussed.

Functional plasticity of the developing cardiovascular system: examples from different vertebrates.

Technical advances that have made it possible to perform physiological measurements on very small organisms, including those in embryonic and larval stages, have resulted in the formation of the discipline of developmental physiology. The transparency and size of developing organisms in some areas permit insights into physiological processes that cannot be obtained with opaque, adult organisms. On the other hand, it is widely accepted that without eggs, there are no chickens, so physiological adaptations during early life are just as important to species survival as those manifested by adults. Physiological adaptations of early developmental stages, however, are not always the same as patterns known in adults; they often follow their own rules. The adaptability of early developmental stages demonstrates that development is not stereotyped and a phenotype is not just the result of genetic information and the expression of a certain series of genes. Environmental factors influence phenotype production, and this in turn results in flexibility and plasticity in physiological processes. This article comprises exemplary studies presented at the Fourth International Conference in Africa for Comparative Physiology and Biochemistry (Maasai Mara, Kenya, 2008). It includes a brief introduction into technical advances, discusses the developing cardiovascular system of various vertebrates, and demonstrates the flexibility and plasticity of early developmental stages. Fluid forces, oxygen availability, ionic homeostasis, and the chemical environment (including, e.g., hormone concentrations or cholesterol levels) all contribute to the shaping and performance of the cardiovascular system.

Ions first: Na+ uptake shifts from the skin to the gills before O2 uptake in developing rainbow trout, Oncorhynchus mykiss.

This is the first direct physiological evidence in support of the ionoregulatory hypothesis, challenging the long-held assumption that teleost gills develop initially for gas exchange. Resting unidirectional sodium (Na(+)) uptake and oxygen (O(2)) uptake across the skin and gills were measured simultaneously in larval rainbow trout, Oncorhynchus mykiss, during development. In soft and hard water, Na(+) uptake shifted to the gills by 15 and 16 days post-hatch (dph) while O(2) uptake took 50-80% longer and shifted by 23 and 28 dph, respectively. This suggests that gills are required for ionoregulation prior to gas exchange in developing rainbow trout. The age of transition for Na(+) uptake, gill Na(+), K(+)-ATPase (NKA) alpha-subunit protein expression and gill NKA enzyme activity were not significantly different between soft and hard water-reared groups, which suggests a lack of plasticity in gill ionoregulatory development. In rainbow trout, the gills assume a dominant role in ionoregulation before gas exchange, suggesting that ionoregulation may be the initial driving force for gill development. Further investigation is required to determine whether this pattern is consistent with other teleosts and more basal fishes during early development to gain insight into the role of ionoregulation in vertebrate gill evolution.

Hemoglobin enhances oxygen uptake in larval zebrafish (Danio rerio) but only under conditions of extreme hypoxia.

The role of hemoglobin (Hb) in O(2) uptake by zebrafish larvae ranging in age from 5 to 42 days postfertilization was assessed under conditions of normoxia, moderate hypoxia and extreme hypoxia. This was achieved by exposing larvae with and without functional Hb to continuously declining oxygen levels (P(O(2))) in closed-system respirometers. Exposure to 5% CO for 2-4 h was used to render Hb effectively non-functional in terms of its ability to transport O(2). Routine metabolic rate (rM(O(2))), critical dissolved oxygen level (P(c)) and residual oxygen level (P(r)) were determined and used, respectively, as indicators of response in normoxia, moderate hypoxia and extreme hypoxia. rM(O(2)) was defined as the average rate of O(2) uptake before O(2) became limiting (i.e. at high P(O(2))s). P(c) is the P(O(2)) at which rM(O(2)) first becomes O(2)-limited and P(r) is the P(O(2)) below which larvae are no longer able to extract O(2) from the ambient medium. CO poisoning had no significant impact on rM(O(2)) or P(c) at any age, indicating that the lack of functional Hb does not impair routine O(2) usage in normoxia or at moderate levels of hypoxia [down to at least 25-50 torr (1 torr approximately 0.133 kPa), depending on age]. P(r), however, was significantly lower overall for control larvae (6.7+/-1.1 torr; mean +/- 95%CI) than for CO-poisoned larvae (11.2+/-2.1 torr). It would appear that the presence of functional Hb allows zebrafish larvae to extract O(2) from water down to lower P(O(2))s under conditions of extreme hypoxia. This is the first documented (as opposed to inferred) benefit of Hb in developing zebrafish. However, given the relatively small magnitude of the effect it is unclear if this benefit on its own is sufficient to balance the costs associated with Hb production and maintenance.

Ontogenetic changes in the toxicity and efficacy of the anaesthetic MS222 (tricaine methanesulfonate) in zebrafish (Danio rerio) larvae.

Median lethal (LC(50)) and effective (EC(50)) concentrations for 1-h and 24-h exposures to the anaesthetic MS222 (tricaine methanesulfonate) were determined for zebrafish Danio rerio larvae ranging in age from 3 days postfertilization (dpf) to 9 dpf. Cessation of heart beat was used as the indicator of death (LC(50)) while failure to respond to direct mechanical stimulation of the head region was taken as an indication of deep anaesthesia (EC(50)). 1-h LC(50)s, 1-h EC(50)s and 24-h EC(50)s all decreased gradually but significantly (all P<0.01) with age. Mean values for 1-h LC(50)s were 1633 mg L(-1) and 730 mg L(-1), respectively, for 3 dpf and 9 dpf larvae. Mean value for 1-h and 24-h EC(50)s were 106 mg L(-1) and 100 mg L(-1), respectively, at 3 dpf and 65 mg L(-1) and 31 mg L(-1), respectively, at 9 dpf. The gradual increase with age in sensitivity to the anaesthetic implied by these indicators is probably a reflection of ontogenetic changes in the activity of detoxification pathways. Mean values for the 24-h LC(50) also decreased significantly (P<0.001) with age, from 566 mg L(-1) at 3 dpf to 64 mg L(-1) at 9 dpf. However, unlike the other indicators, the decrease was not gradual but occurred in a step-like fashion with virtually all of the change occurring between 4 dpf and 7 dpf. This sharp increase in sensitivity coincides with the shift in the major site of systemic ionoregulatory activity from the skin to the gills. The implications of these ontogenetic changes in lethal and effective levels for researchers or others intending to use the anaesthetic with fish larvae are discussed.

The functional ontogeny of the teleost gill: which comes first, gas or ion exchange?

For most of the last century, the need to obtain sufficient oxygen to meet the respiratory requirements of the tissues was viewed as the primary selective pressure driving gill development in teleost fish. Recently, however, it has been suggested that ionoregulatory pressures may actually be more important. This manuscript reviews the theoretical and empirical evidence dealing with the functional ontogeny of the gill in the context of the oxygen and ionoregulatory hypotheses. Gas and ion exchange are subject to similar geometric constraints in developing fish. Both initially are exclusively cutaneous but shift to the gill with tissue growth because of declining surface-to-volume ratios. Based on the appearance of mitochondria-rich cells (MRCs), ionoregulatory activity shifts to the gill in advance of gas exchange. In every species examined to date, MRCs appear on the developing gill in advance of secondary lamellae, the definitive gas exchange structure of the adult gill. Biochemical and histochemical studies indicate that these early branchial MRCs are actively involved in ion exchange. In some cases, the specific activity is many times greater than in the adult gill. In contrast, O2 microelectrode and hemoglobin ablation experiments suggest that the early gill contributes little O2 to the general systemic circulation. Any oxygen taken up appears to be consumed locally. Functional ablation experiments with zebrafish indicated that the larval gill became essential for ion balance well before it was needed for O2 uptake. Similar experiments with rainbow trout, however, found that the gill became essential in terms of gas and ion exchange at about the same time. On balance, the evidence appears to favour the ionoregulatory hypothesis but the oxygen hypothesis cannot be absolutely rejected without more information. Some of the major deficiencies in our knowledge regarding the transition from cutaneous to branchial gas and ion exchange are highlighted and potential implications of the ionoregulatory hypothesis are discussed.

Gills are needed for ionoregulation before they are needed for O(2) uptake in developing zebrafish, Danio rerio.

A variation on the classic ablation method was used to determine whether O(2) uptake or ionoregulation is the first to shift from the skin to the gills in developing zebrafish, Danio rerio. Zebrafish larvae, ranging in age from 3 to 21 days postfertilization, were prevented from ventilating their gills and forced to rely on cutaneous processes by exposing them to one of two anaesthetics (tricaine methanesulphonate or phenoxyethanol) or by embedding their gills in agar. They were then placed in solutions designed to compensate selectively for impaired O(2) uptake (42% O(2)), impaired ionoregulatory capacity (50% physiological saline) or impairment of both functions (42% O(2)+50% physiological saline). Survival under these conditions was compared with that in normoxic (21% O(2)) fresh water. Neither hyperoxia nor 50% physiological saline had any significant effect on the survival of newly hatched larvae (3 days postfertilization), suggesting that at this stage cutaneous exchange was sufficient to satisfy both ionoregulatory and respiratory requirements. At 7 days postfertilization, the skin still appeared capable of satisfying the O(2) requirements of larvae but not their ionoregulatory requirements. Physiological saline significantly improved survival at 7 days postfertilization; hyperoxia did not. At 14 days postfertilization, both hyperoxia and 50% saline significantly improved survival, indicating that at this stage gills were required for O(2) uptake as well as for ionoregulation. At 21 days postfertilization, only hyperoxia significantly improved survival. By this stage, larvae apparently are so dependent on gills for O(2) uptake that they suffocate before the effects of ionoregulatory impairment become apparent. Thus, it would appear that in zebrafish it is the ionoregulatory capacity of the skin not its ability to take up O(2) that first becomes limiting. This raises the possibility that ionoregulatory pressures may play a more important role in gill development than is generally appreciated.