The early life history stages of riverine fish: ecophysiological and environmental bottlenecks

Fritz Schiemer, Ewa Kamler, Hubert Keckeis

Institute of Ecology & Conservation Biology, Department of Limnology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria

Fish are good indicators of environmental health of rivers and their catchments as well as important conservation targets. Bioindication has to be based on an understanding of the requirement of charcteristic species: The fitness response of fish towards environmental changes has to be addressed with regard to 3 aspects:

• The match-mismatch between reproductive strategies and the environmental conditions.
• The niche dimensions of the critical stages vis-avis the key environmental conditions.
• The availability of microhabitats along the ontogentic niche profiles, i.e. the connectivity from spawning substrates to early life history microhabitats.

The main environmental conditions for the embryonic phase are temperature and oxygen supply which will be responsible for egg mortality, the duration of the embryonic stage, and size and condition of fry.
The main fitness dimensions of 0+ stage can be described by the functional response of food acquisition, growth and bioenergetics to the 3 environmental axis temperature, food and current velocity.
Results are presented on Chondrostoma nasus, a target species for monitoring and conservation in large European river systems. Results on experimental studies egg quality and embryogenesis and on feeding performances and growth of externally feeding larvae and early juveniles are compared with field studies in order to evaluate the match-mismatch between performances and micrrohabitat choice and popualtion dynamics in the field.
Results of C. nasus will be compared with performances of characteristic representatives for the sequence of fish assemblages along the course of rivers - from headwater reaches to the downstream floodplain zones – i.e. the salmonid region and the lower cyprinid region.

Development of the cardiovascular system in fish: Shape and performance

Bernd Pelster

Dept. of Zoology, University of Innsbruck, Austria

During development the circulatory system of vertebrates typically starts operating earlier than any other organ. In these early stages, however, blood flow is not yet linked to metabolic requirements of tissues, as it is well established for adults. While the autonomic nervous system becomes functional only quite late during development, in the early stages control of blood flow is possible by blood born and/or local hormones. This allows for at least some adaptational responses to environmental perturbations like hypoxia, for example. Very little, however, is known about a possible flexibility in tissue vascularization during early development. While in adult vertebrates the vascular bed of various organs and tumors can be significantly modified by local signals, during early development at least the formation of major blood vessels appears to be driven by genetic information. This study presents methods based on video-imaging techniques and fluorescence microscopy to visualize the vascular bed of developing lower vertebrates and to tests the idea that environmental factors like hypoxia or chronic application of vasoactive hormones may modify the early formation of blood vessels in embryos and larvae. The results show that in zebrafish the formation of some blood vessels is enhanced under chronic hypoxia, and that chronic application of NO can stimulate blood vessel formation. In consequence, during early development of fish blood vessel formation is not only controlled by genetic information.

Supported by the Austrian Science Foundation (FWF)

Ontogeny and Vertebrate Design: Lessons from the Fish Gill

Peter Rombough

Zoology Department, Brandon University, Manitoba Canada R7A 6A9, Rombough@brandonu.ca

Evolutionary theory tells us that organ systems evolve incrementally as a result of natural selection. Natural selection, however, operates within certain constraints, the most obvious of which are phylogenetic. The manner and extent to which an organ of an adult animal can be modified is, to a large extent, determined by its pre-existing structure. Less well appreciated but equally important are ontogenetic constraints. Development is a sequential process. The design of adult structures, therefore, is also circumscribed by the morphology of the homologous embryonic and larval structures the adult structure replaces. While obvious on reflection, this fact is frequently overlooked when biologists discuss the design of adult organs. A case in point is the fish gill.

Most biologists consider that the fish gill is designed to accommodate the demands of gas exchange. In adult fish, the critical function of the gill is indeed gas exchange. When the gill first forms, however, its primary critical function is not gas exchange but rather ion balance. Ablation studies indicate that zebrafish (Danio rerio) larvae need gills for ion balance by 7 days postfertilization (dpf) but do not require gills for gas exchange until about 16 dpf 1. Morphological examination reveals that zebrafish do not even begin to form gill lamellae, the definitive adult respiratory structure, until 14dpf by which time the basic structure of the gill is already well established. Morphological evidence indicates the situation is similar in an unrelated species, the rainbow trout (Oncorhynchus mykiss) 2, suggesting that the functional sequence of ion balance followed by gas exchange probably applies to the fish gill generally. Knowledge of this sequence can! help us better understand gill design. For example, the reason chloride cells are located mainly on filaments rather than on lamellae is probably simply because when chloride cells first appear on the gill there are no lamellae. More speculatively, one might expect the secondary circulation of the gill to play a role in ionoregulation since it is located mainly within the filament, the ionoregulatory component of the gill.

Knowledge of functional sequences also provides information about the evolutionary history of the vertebrates. While ontogeny clearly does not recapitulate phylogeny, there is ample evidence that features that appear earlier in development often tend to be older phylogenetically 3. Applied to the gills, this would suggest that the vertebrate gill evolved in response to ionoregulatory rather than respiratory pressures. The fact that that protovertebrates apparently needed a gill for ionoregulation as well as a kidney for water excretion would appear to support the hypothesis that vertebrates originated in freshwater rather than in sea water 4.

1 Rombough, P. J. 2000. Why zebrafish develop gills. J. Exp. Biol. (in review).
2 Rombough, P. J. 1999. The gill of fish larvae. Is it primarily a respiratory or an ionoregulatory structure? Journal of Fish Biology 55 (Supplement A): 186-204.
3 Romer, A. S. & Parsons, T. S. 1986. The Vertebrate Body 6th ed. Saunders, Philadelphia.
4Griffith, R. W. 1985. Habitat, phylogeny and the evolution of osmoregulatory strategies in primitive fishes. In.Evolutionary Biology of Primitive Fishes. Foreman, R. E., Gorbman, A., Dodd, J. M. & Olsson, R., eds. Plenum, New York.

Ontogeny of digestive function of fish larvae

Ivar Rønnestad

Department of Zoology, University of Bergen, Norway,
ivar.ronnestad@zoo.uib.no

The digestive tract is rudimentary developed at the onset of first feeding in marine fish larvae and many species lack a stomach and pyloric caeca limiting the total digestive capacity. A fully functional digestive tract is acquired during metamorphosis, when the stomach develops and consequently the protein digestion capacity increases. Due to their rapid growth rate fish larvae require a full complement of all amino acids necessary for efficient protein synthesis. Amino acids also serve as an important energy source. Consequently, there is a critical demand for considerable levels of amino acids in the diet. Using an in vivo set-up for controlled tube feeding of fish larvae together with metabolic tracer studies, we have shown that fish larvae absorb free amino acids up to 10 times faster than protein from the digestive tract. Taken together these findings suggest that the availability of amino acids from intestinal digestion of complex proteins may be insufficient to satisfy the metabolic demands of the rapidly developing fish larvae. The second part of the presentation deals with peristaltic activity and control of intestinal function in fish larvae.

Control of gut motility in larval fish and amphibians

Susanne Holmgren1, Anna Holmberg1, Regina Fritsche1, Bernd Pelster2 and Thorsten Schwerte2

1University of Göteborg, Department of Zoology/Zoophysiology, Box 463, SE-405 30, Göteborg, Sweden
2University of Innsbruck, Department of Zoology, A-6020 Innsbruck, Austria

Corresponding author: S.Holmgren@zool.gu.se

The enteric nervous system develops from neural crest cells, which colonize the gut at early embryonic stages. There are several studies of this development in mammals and birds, but only a few studies in other vertebrates. In zebrafish, a ret-receptor homologue, ret1, and its expression pattern during embryogenesis has been described and in some other fish species neurotrophin receptor distribution in the adult gut has been investigated. The first occurrence of some enteric neurotransmitters have been described e.g. in the urodele amphibian Ambystoma mexicanum (Maake et al. Gen Comp Endocrinol 2001.121.74-83), and in some teleost species (Reinecke et al. Anat Embryol, 1997.195.87-102). We aim to investigate when neurotransmitters first have a function in the developing gut of zebrafish, Danio rerio, and the South African clawed frog, Xenopus laevis, and in which order. Of special interest is to correlate the development of the gut to the onset of external feeding. We have found several transmitter substances before the onset of feeding in Xenopus, and around the onset of feeding in zebrafish, and conclude that the control systems are developed when food is first processed. In contrast to the finding in Ambystoma of Maake et al. (2001), our results suggest that neurohormones are expressed earlier in enteric nerves than in endocrine cells of the gut. To study the effects of these transmitters on gut motility we are analysing microscopic video-recordings of spontaneous gut movements in developing larvae (which are transparent), using the method of Schwerte and Pelster (J Exp Biol 2000.203.1659-1669).

Energy and nutrient utilisation by embryonic reptiles

Michael B. Thompson

School of Biological Sciences (A08), University of Sydney, NSW 2006, Australia

Most reptiles are oviparous, with the developing embryos relying on the contents of the yolk to sustain development until hatching (lecithotrophy). The yolk is composed primarily of lipid and protein, which act as an energy source and the essential components to build embryonic tissue. Nevertheless, yolk and the resulting embryos contain many other nutrients, including inorganic ions, vitamins, carotenoids, water, cholesterol and hormones. Apart from water and oxygen, which may be taken up by eggs, and some inorganic ions that can come from the eggshell or even from outside the egg, everything required by the embryo must be in the egg when it is laid. About 20% of squamate reptiles are viviparous, exhibiting a variety of placental complexities. Species with complex placentae have reduced yolk volumes, with the mother augmenting embryonic nutrition by provision across the placenta (placentotrophy). Despite assumed advantages of placentotrophy, only 5 out of about 100 lineages of viviparous squamates exhibit substantial placentotrophy. This paper reviews available and recent information on the yolk contents of a variety of squamate reptiles to ask the question, how are nutrients transported from the yolk to the embryo or across the placenta? Although, current available data suggest that, in broad terms, yolk is taken up by embryos without discrimination of the nutrients, there are some apparent exceptions, including the very long chain polyunsaturated fatty acids. In addition, fundamental differences in the patterns of energy utilisation in lizards and snakes suggest fundamental differences in lipid profiles in these taxa which appear to reflect the differences between placentotrophic and lecithotrophic viviparous lizards.

Functional Cardiovascular Development in Xenopus and Zebra fish

Regina Fritsche

University of Göteborg, Department of Zoophysiology, Göteborg, Sweden

Independent of species, the cardiovasular system is the first functioning component of the developing embryo and the increase in embryonic demand is matched by increasing cardiovascular function. Since diverse species exhibit both functional and structural similarities in their cardiovascular development, “model” animals can be used for these studies. We are using two animal models; the larval form of the African clawed frog, Xenopus laevis and embryos of zebra fish, Danio rerio. The larval forms of both these animals allow us for the first time to study cardiovascular functions immediately after the onset of heart beat with minimal disturbance to the animal. Furthermore, the rapidly increasing knowledge of the genome of Xenopus and Zebrafish makes them ideal models for studies of functional genomics. In the mature animal, the autonomic nervous system and the endocrine systems are vital for maintenance of the homeostasis, but both systems mature and begin to function at late stages of organogenesis or even after birth. It has been suggested that before these systems are fully mature, the cardiovascular system is controlled exclusively by intrinsic mechanisms of the heart and vessels. However, many different peptides and amines have been found in the vasculature and heart of embryos before the nerves or adrenal glands appear. Thus, because many hormones and peptides that have potent cardiovascular effects in the adult animal are found in embryonic tissues, it is important to know if these substances play a role in cardiovascular control during early developmental stages. By using microtechniques such as dual servo-null micropressure recordings, nanoliter injections and videotechniques we are now able to investigate the functional significance of these regulatory substances.

Blood pressure regulation prior to cardiac innervation in Xenopus laevis.

S.J. Warburton1) and R. Fritsche2)

1) Dept. Biology, New Mexico State University, Las Cruces, NM, USA
2) Dept. Zoophysiology, Göteborg University, Göteborg, Sweden.

Despite the apparent lack of cardiac innervation, blood pressure during development is quite predictable in vertebrates, suggesting regulation. We investigated hypertensive and hypotensive responses in larval Xenopus laevis lacking a baroreflex. Hypertension (120% control pressure) was induced by volume-loading; hypotension (80% control pressure) was induced by blood removal. Larvae were able to correct hypertension within 30 minutes using mechanisms that were stage-dependent. NF stage 51-53 larvae relied on nitric oxide (NO) to combat hypertension via vasodilatation, although with blockade of NO synthase, larvae also restored blood pressure but at the expense of cardiac output, suggested fluid movement may have been the operative component. Earlier stages (NF 49-50), also corrected hypertension but without NO. Overcompensation was not uncommon, especially in later stage animals. Paradoxically, the primary response to hypertension in all animals was an increase in! peripheral resistance (13-37% increase at 30 min.), which was reversed in later stage animals with functional NOS (13% decrease at 30 min). These data, together with the relatively slow onset of NO effects (15-30 minutes) suggests that NO effects may be secondary to endothelin release. The response to hypotension was quickly and completely in place. A smaller withdrawal volume was required to drop blood pressure in animals pretreated with the angiotensin II (ANG II) antagonist saralasin, 0.53 ± 0.06% BW versus 1.00 ± 0.20% BW in controls. Animals pretreated with saralasin also had lower resistances (-8.3 ± 6.1% versus -22.1 ± 11.6% control). Supported by NSF IBN-0078094 and Swedish Natural Research Council NFR.

The development of the lymphocyte repertoires in the larvae of the South African frog Xenopus

Louis Du Pasquier, Erika Meier, Rainer Mußmann

Basel Institute for Immunology PO Box CH- 4005 Basel Switzerland

Xenopus shares with mammals the organization and the usage of its immunoglobulin and T-Cell receptor gene loci with combinatorial joining of V, D, and J elements. The immune system of Xenopus develops under the double pressure to develop early and to produce a heterogeneous repertoire when lymphocyte numbers are or of the order of 5000 and imposes a limitation to the build up of B- and T cell population.
B- cells.
In a first phase of differentiation spanning day 5-12 after fertilization in the liver, before immunological competence and before the appearance of secondary lymphoid organs, the heavy (H) chain locus starts rearranging followed by the light (L) chain locus 3 days later. By immunohistology the first B cells expressing H and L chain are detectable on day 10. Despite the small number of cells available and the lack of external antigen selection at these early stages, the repertoire is heterogeneous. The VH families are used stepwise although their genes are interspersed in the genome.
In the second phase, from day 12-13 onwards the spleen differentiates and the animal becomes immunologically competent. The V, D, and J usage is similar to that of adults although VDJ junctions lack N nucleotides until metamorphosis.
T-Cells.
When compared to mammals Xenopus provides an interesting variation on the theme of repertoire selection. Only one classical MHC class I locus is present. Furthermore unlike adults tadpoles do not express MHC class Ia or Ib in the thymus. Both adults and tadpoles express class II although with a different tissue distribution. These differences may influence the development of peripheral T-cell repertoire. The V, D, J, usage and C of Xenopus T-cells have been compared in larvae and adults in the thymus and in the periphery of MHC identical or MHC disparate clonal individuals. The first rearrangements in the T-Cell occur on day 6 whereas cell-expressing CD3e can be detected one day earlier. Unlike in mammals the larval and adult differ significantly in the frequency of each V b usage. Of the 19 V b genes about half can be used from day 9 the remaining being progressively rearranged until day 24. The trend is therefore the same as for B-cells: Irrespective of the small number of lymphocytes there is a pressure to express early as diverse a repertoire of T-cells as possible.

The control of development of pulmonary surfactant in egg-laying amniotes

Lucy C. Sullivan, Sandra Orgeig and Christopher B. Daniels

Dept of Environmental Biology, University of Adelaide, Adelaide SA 5005, AUSTRALIA

Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar type II cells in the lungs of all air-breathing vertebrates. Pulmonary surfactant functions to reduce the surface tension in the lungs and therefore reduce the work of breathing. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones and autonomic neurotransmitters. Whether the difference in birthing strategy between mammals and egg-laying amniotes leads to differences in the mechanisms of control of the surfactant system is unknown. We have used a co-culture system of embryonic type II cells and lung fibroblasts to investigate the ability of adrenaline, dexamethasone and tri-iodothyronine to stimulate the cellular secretion of phosphatidylcholine during the final 25% of incubation in the chicken (Gallus gallus domestica), bearded dragon (Pogona vitticeps), green sea turtle (Chelonia mydas) and salt-water crocodile (Crocodylus porosus). Adrenaline stimulates surfactant secretion at every stage examined, including post-hatching. Glucocorticoids and thyroid hormones are stimulators during development and lose their efficacy closer to hatching. The action of glucocorticoids is dependent upon a fibroblast-type II cell interaction, as is the case in mammals. Furthermore, there appears to be a synergistic effect between glucocorticoids and thyroid hormones. Hence, the factors controlling the development of the surfactant system appear to be conserved amongst the amniotes. However, the relative timing of cellular surfactant secretion and the timing over which the hormones act appears to differ between species, and presumably relates to the specific biology of the animal.

Supported by the Australian Research Council

A change of heart: cardiovascular development in the shrimp Metapenaeus ensis

B. R. McMahon, K. Tanaka , J. E. Doyle and K-H. Chu.

Department of Biological Sciences, University of Calgary and Biology Department Chinese University of Hong Kong.

The larval development of penaeid shrimp is among the most complicated in crustaceans. In Metapenaeus ensis, there are 6 naupliar (N), 3 protozoeal (PZ) and 3 mysid (M) larval instars, followed by postlarval development. Irregular heartbeat begins late in naupliar stage 6. Coordinated beating at 400-600 bts.min^-1 commences in PZ1 and continues throughout larval life. Initially the heart pump is located in the anterior cephalothorax, has a single pair of ostia and arterial distribution from the heart is limited to a single anterior vessel. In later mysid instars a second cardiac pumping site develops posterior to but connected with the original. This extension is more muscular, contains additional ostia and has additional distribution vessels supplying the cephalothorax and abdominal areas. The original site is gradually merged into the new extension and only small refinements in the circulation occur in postlarval and juvenile life. Changes in physiological responses of the heart also occur throughout development. Responses to intra-pericardial micro-injection of 5-hydroxytryptamine change drastically during development as do cardiac responses to ambient hypoxia. Similarly heartbeat of later juvenile instars are inhibited by injection of tetrodotoxin, while heartbeat of larval and early juvenile instars are not, suggesting that neurogenic regulation via the cardiac ganglion arises later in development. Our present studies attempt to integrate the anatomical and physiological changes in the crustacean heart.

The enteric nervous system in developing Xenopus and zebrafish

Anna Holmberg, Regina Fritsche, and Susanne Holmgren

University of Göteborg, Department of Zoology/Zoophysiology, Box 463, SE-405 30
Göteborg, Sweden

Corresponding author: anna.holmberg@zool.gu.se

The information about the ontogeny of the enteric nervous system (ENS) in non-mammalian and non-avian vertebrates is sparse. We have studied the development of ENS in relation to onset of exogenous feeding in developing South African clawed frog, Xenopus laevis and zebrafish, Danio rerio. Xenopus enteric neurons are present before the larvae start to feed (Epperlein, Krotoski, Halfter, Frey. Anatomy & Embryology 1990.182.53-67). By using immunohistochemistry, we studied the first occurrence of structural and functional nerves in Xenopus. In agreement with Epperlein et al. 1990, we found that neurons are present at NF stage 38-39, before the onset of feeding (stage 45). Shortly after the first occurrence of these neurons, they were shown to contain the neurotransmitters VIP (stage 40), PACAP (41), NOS, substance P, or NKA (stage 42). CGRP was demonstrated at stage 47, after the onset of feeding. However, the fact that transmitters are present in these nerves does not necessarily mean that they have a function on the gut motility i.e. the smooth muscles cells may not yet express the proper receptors. Neurotransmitters such as VIP can exhibit a trophic effect. A parallel study on developing zebrafish is performed, and enteric neurons are present from day 3 post fertilization. We conclude that several types of neurons are present and contain transmitters before the onset of feeding.

Adrenergic cardiac control during development of the African clawed frog, Xenopus laevis.

Angélica Jacobsson Kloberg and Regina Fritsche

Göteborg University, Department of Zoophysiology. Box 463 SE 405 30 Göteborg Sweden
angelica.kloberg@zool.gu.se

In embryos of the African clawed frog, Xenopus laevis, administration of isoprenaline on the heart causes an increase in heart rate four days post fertilisation. Three days post fertilisation an adrenergic tonus is active on the heart. This tonus increases during early development up to a peak at an age of four to seven days post fertilisation, and then decreases again. Similarly, the embryonic heart beats at its highest rate at day four to seven, suggesting that at least part of the high heart rate at these stages is due to a high adrenergic tonus. Earlier studies have not been able to show any adrenergic nerves in the heart at these early stages, suggesting that adrenergic cardiac control is due to either blood circulating catecholamines or catecholamines endogenous to the heart (or both). In fact, in recent studies catecholamines have been detected in the larval heart tissues from day three post fertilization and further on. A peak in adrenaline concentration, at an age of four to seven days post fertilisation, coincides with the peak in adrenergic tonus. In addition, cells immunoreactive to enzymes involved in the catecholamine synthesis have been found in the heart of Xenopus larvae already from the third day post fertilisation. We propose that adrenergic cardiac control can be achieved by catecholamines, produced and stored in specialised cells in the heart and acting upon b-like adrenoreceptors.
All animal experiments have been approved by the local ethical committee.