Transgenic Zebrafish for Studying Nervous System Development and Regeneration

1 tubulin gene expression is induced in the developing and regenerating CNS of vertebrates. Therefore, 1 tubulin gene expression may serve as a good probe for mechanisms underlying CNS development and regeneration. One approach to identify these mechanisms is to work backwards from the genome. This requires identification of 1 tubulin DNA sequences that mediate its developmental and regeneration-dependent expression pattern. Therefore, we generated transgenic zebrafish harboring a fragment of the 1 tubulin gene driving green fluorescent protein expression (GFP). In these fish, and similar to the endogenous gene, transgene expression was dramatically induced in the developing and regenerating nervous system. Although transgene expression generally declined during maturation of the nervous system, robust GFP expression was maintained in progenitor cells in the retinal periphery, lining brain ventricles and surrounding the central canal of the spinal cord. When these cells were cultured in vitro they divided and gave rise to new neurons. We also show that optic nerve crush in adult fish re-induced transgene expression in retinal ganglion cells. These studies identified a relatively small region of the 1 tubulin promoter that mediates its regulated expression pattern in developing and adult fish. This promoter will be extremely useful to investigators interested in targeting gene expression to the developing or regenerating nervous system. As adult transgenic fish maintain transgene expression in neural progenitors, these fish also provide a valuable resource of labeled adult neural progenitor cells that can be studied in vivo or in vitro. Finally, these fish should provide a unique in vivo system for investigating mechanisms mediating CNS development and regeneration.     

Fish, Flows and Flood Plains: Links between Freshwater Fishes and their Environment in the Murray-Darling River System, Australia

Knowledge of the biology of native fishes of the Murray-Darling Basin is based largely on studies conducted under hatchery conditions and on a limited number of recreationally important species. From observations that increases in water level in aquaculture ponds initiate spawning in some species, and from limited studies of wild fishes and studies in overseas floodplain river systems, a perception has emerged of the importance of flooding and the flood plain in the life cycles of Murray-Darling fishes in general. However, there is little confirmatory evidence of the use of temporary floodplain habitats by larvae, juveniles or adults. The significance of in-channel habitats, especially for rearing, has received little attention. Murray-Darling fish species can be placed into three life history modes, based mainly on spawning style and time and developmental intervals of larvae at first feeding. Fish in each group may be able to take advantage of floods if the timing is right and prey are plentiful, however, the larvae of some species are able to recruit under non-flood conditions within the main river channel. This forms the basis of the low flow recruitment hypothesis, which attempts to explain why some species spawn during the warmest months and lowest flows and how they are able to recruit under these conditions. This hypothesis is then placed in the context of the current state of knowledge of the relationships between flow and the biology of Murray-Darling fishes, specifically cues for spawning, movement and recruitment. The lack of widespread evidence for floodplain use by any life history interval of fish may be due to a paucity of study, however, there are some fundamental factors, such as the predictability of timing and duration of high flow events as well as the lack of coincidence of high flows and high temperatures in some regions of the Basin, which may be important in determining the use of floodplain habitats by fish.     

An empirical model for predicting microhabitat of 0+ juvenile fishes in a lowland river catchment

Summary Species-habitat relationships are an important aspect of fish life history, particularly in early ontogeny. To address the lack of information on the microhabitat use/requirements of European coarse fishes, particularly 0-group fishes in riverine systems, an empirical model was generated from data collected in the River Great Ouse catchment (UK). During 7 weeks in autumn 1990, data on young-of-the-year (0+) juvenile fishes and 15 environmental variables were collected at 2800 stratified, random point samples within 130 study sites (streams, rivers, side-channels, backwaters). Of 24 species of 0+ fish, only 10 were captured in 3% of non-null samples. Association analysis and canonical correspondence analysis of the samples-by-species (967×10) and samples-by-variables (967×15) data matrices revealed that the shallow, narrow, lotic, stony-pebbly channel microhabitat of riffles and runs was preferred by the progeny of substrate-spawning, substrate/plant-spawning and nest-guarding fishes. Moderately deeper and wider, sinuous channels, with slowto-moderate water velocities and medium-sized substrata were also favourable to progeny of substrate spawners. Assemblages of 0+ fish in deeper, wider, silted, trapezoidal-shaped channels with slow-to-lentic flowing water, i.e. channelised and regulated, were dominated by ubiquitous (plant/substrate) spawners.     

The role of learning in fish behaviour

Summary The behavioural patterns of fish are the result of innate (built-in) patterns of maturation (developmental changes) and of learning processes (imprinting and trial-and-error learning). Innate behavioural patterns are considered to be hard-wired and inflexible. However, through learning, fish can adapt to environmental change. For instance, the homing behaviour of fish may be partly the result of the development of specific parts of the brain and partly because of changes in behaviour with experience. Similarly, one can assume that the feeding mode of fish involving snap-responses is innate, but learning enables fish to modify their foraging behaviour in response to a fluctuating environment. By reviewing these and other examples, such as the role of recognition learning and socially transmitted behaviour, one can illustrate the importance of learning in the everyday life of fishes. Although learning plays a large role in the behaviour of fishes, the learning capacity of fishes may also be useful to fisheries research and hatchery operations.     

Appetitive conditioning technique reveals behavioural limits to passage performance in fishes

Numerous environmental features have the potential to act as barriers to fishes in the field. Passage of these barriers depends on two main properties: willingness (behavioural) and physiological capacity. The physiological swimming capacity of fishes has been well studied. However, because most barrier research has focused on the population level effects of man – made barriers, little attention has been given to the behavioural aspects of fish passage. We used appetitive conditioning to create a simple laboratory based protocol to estimate behavioural limits to fish passage. We tested our protocol using barrier height (or passage space) as a model. Our protocol successfully identified behavioural limits in two species, juvenile rainbow trout, Oncorhynchus mykiss, and fathead minnows, Pimephales promelas. Our most interesting observation was that even a partial, submerged barrier had the potential to block fish movements. Our results suggest a potential use for conditioning techniques in assessing factors that constitute behaviour barriers to fishes. This information may help us to better predict fish movements in the field and may help us to design barriers that are more efficient at passing fishes.     

Species diversity and abundance of freshwater fishes in irrigation ditches around rice fields

The relationships between environmental variables, distribution of freshwater fishes and their diet were investigated at 40 sites on irrigation ditches for rice fields in central Japan. Multivariate analyses showed that fish species richness and diversity in ditches was high when fishes were able to easily invade the rice fields. Natural stream beds not covered by concrete had greater total number and biomass of fishes in irrigation ditches. The connection between ditches and rice fields was of secondary importance. Canonical correlation analysis indicated that the two principal fish community variables, abundance and diversity, were respectively represented by the presence of the minnow, Gnathopogon elongatus elongatus, and the absence of the loach, Misgurnus anguillicaudatus. Most fishes in the irrigation ditches predominantly preyed upon aquatic insects such as ephemeropteran nymphs, chironomid larvae and trichopteran larvae, but aerial insects and benthic algae were also fed upon by a few species. Small fishes (<4cm SL) preyed more on chironomid and less on trichopteran larvae than large (4cm SL) fishes. The diet overlap between species was extremely high for small fishes in ditches weakly connected with rice fields. The recent changes in rice fields and irrigation ditches to increase production efficiency has led to a decrease in fish diversity. For the coexistence of rice production and fish diversity in rice areas, a new irrigation system, where fishes can easily invade rice fields and where plenty of invertebrates are available for fishes, should be developed.     

Ecological versus phylogenetic determinants of helminth parasite community richness

Summary We examine patterns of community richness among intestinal parasitic helminth communities in fishes, herptiles, birds and mammals with respect to the comparative number of component species in a host population. We show that terrestrial hosts have, on average, fewer component species than aquatic hosts. We also show that the mean number of component species in aquatic hosts increases from fishes through herptiles to birds before declining slightly in mammals. For terrestrial hosts, the mean number of component species increases from herptiles, through birds, reaching a maximum in mammals. We conclude that: (i) habitat of the host is more important in determining community richness than is host phylogeny; (ii) the phenomenon of host capture may be largely responsible for increased species richness in some host groups; (iii) aquatic birds harbour the richest intestinal helminth communities; and (iv) as we interpret them, our data refute the time hypothesis, which would predict that fishes as the oldest lifestyle should have the richest helminth communities.Order of authorship determined by random draw and does not imply seniority.     

Have fishes had their chips? The dilemma of threatened fishes

Synopsis The conservation status and factors threatening fishes worldwide are reviewed in order to introduce a series of one-page articles on Threatened fishes of the world, and to encourage the incorporation of information on threatened fishes into international conservation programmes. Information on fish extinction and threat rates are compared with those of other animal groups, and the unique characteristics of fish conservation problems are highlighted. At present 979 species of fishes are listed as threatened in the IUCN Red List and at least 36 species and three subspecies are listed as recently extinct. It is argued that these figures are probably gross underestimates and that they may mislead conservation authorities and resource users about the seriousness of the situation. Freshwater fishes may be the most threatened group of vertebrates after the Amphibia. Urgent action is required to save many narrowly endemic, stenotopic species from extinction, especially in Africa, Asia and South America. The conservation of common species that drive essential ecological processes is also important. Anthropogenic pressures, especially habitat degradation, the introduction of invasive species and pollution, on inland and coastal waters are particularly severe and many major fish communities are threatened with elimination throughout the world. The conservation of marine fishes is complicated by the fact that it is difficult to ascertain their rarity. The importance of the retention of genetic variation is highlighted, and both orthodox and innovative conservation measures are encouraged. Further research on minimum viable populations, genetics, and the factors that cause fishes to become vulnerable to extinction, is urgently required.Invited editorial     

Taxonomic and Distributional Databases of Fishes in Taiwan

The fishes of Taiwan comprise total 2450 species in 250 families, about 1/10 of the world's fishes. Curatorial and distributional data of Taiwanese fish have been integrated into a database that can be accessed interactively on the Internet at http://fishdb.sinica.edu.tw. The database includes the following items: basic information and specimen photo of each species, distributional database, bibliographic database, curatorial database, inquiring system for Chinese fish names of the world fishes, and new version of erratum of Fishes of Taiwan. The above regional database of Taiwanese fishes provides hyperlinks to fish data for each species in the global fish data, FishBase, of ICLARM (http://www.fishbase.org). Through the collaboration with FishBase and other global networks for taxonomic purposes, like Species 2000, BioNet, or GBIF (Global Biodiversity Information Facilities), users in the world can also obtain updated data provided by Taiwan. We hope our experience with database establishment can be used as a model to help other countries to develop their own regional fish databases or even to build up databases of other groups of organisms. There should be one good approach for all people in the world for sharing complete and updated biodiversity information via the Internet.     

Buffering capacity of vertebrate muscle: Correlations with potentials for anaerobic function

Summary Buffering capacities (), measured in slykes (moles of base required to titrate the pH of one gram wet weight of muscle by one pH unit, over the pH range of pH 6 to pH 7) due to non-bicarbonate buffers were measured in locomotory muscles from a variety of terrestrial and marine mammals and teleost fishes (Tables 1 and 2). The highest buffering capacities were found in muscles capable of either intense, burst glycolytic function or prolonged, low-level anaerobic function. Marine mammals had higher muscle buffering capacity on the average than terrestrial mammals. Among the fishes studied, warm-bodied species had the greatest values of all animals examined (Table 2). Deep-sea fishes and shallow-living fishes with sluggish locomotory abilities had low values. Fish white muscle displayed higher buffering capacity than red muscle (Fig. 1; Table 2), in keeping with the more aerobic poise and higher capillary density of the latter type of muscle. Strong correlations were found between (1) and muscle myoglobin concentrations in the mammalian species (Table 1; Fig. 2), and (2) and muscle lactate dehydrogenase (LDH) activities in both the mammals and the fishes (Tables 1 and 2; Fig. 3). No correlation was found between and the activity of a citric acid cycle indicator enzyme, citrate synthase, in the mammalian species. While strongly correlated with buffering capacity, the amounts of myoglobin and LDH in a muscle are not the principal determinants of . The results indicate that muscle intracellular buffering capacity is especially critical in locomotory muscles which must function under conditions (burst locomotion and prolonged, low-level anaerobic function) where circulatory perfusion is inadequate to rapidly remove the acidic end-products such as lactic acid that are produced by anaerobic glycolysis. In this respect, the locomotory muscle of diving mammals and the white skeletal muscles of teleost fishes face a common acid-base regulatory problem and utilize a common biochemical strategy to resolve it.     

Gene Expression in Tilapia Following Oral Delivery of Chitosan-Encapsulated Plasmid DNA Incorporated into Fish Feeds

DNA delivery into fish is important for transient gene expression, (e.g., DNA vaccination). Previous studies have generally focused on intramuscular injection of DNA vaccines into fish. However, this method is obviously impractical and laborious for injecting large numbers of fishes. This study reports oral delivery of a construct expressing the β-galactosidase reporter gene into fish by encapsulating the DNA in chitosan and incorporating it into fish feeds. We found that β-galactosidase expression could be observed in the stomachs, spleens, and gills of fishes fed with flakes containing the chitosan-DNA complex. These results suggest that DNA vaccines and other constructs can be easily and cheaply delivered into fishes orally by use of carriers and incorporation into fish feeds.     

Copy Number Related Transgene Expression and Mosaic Somatic Expression in Hemizygous and Homozygous Transgenic Tilapia (Oreochromis Niloticus)

Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcontaining a lacZ reporter gene spliced to a 4.7kb 5 regulatory region of a carp beta actin gene. All these three lines contain different copy numbers oftransgenes and the levels of lacZ expressionwere found to be related to transgene copy number.Mosaic patterns of somatic lacZ expression wereobserved in these three lines which differed between linesbut were consistent within a line. We also observedthat expression of the reporter gene in homozygoustransgenic fish was approximately two-fold greater thanin the hemizygous transgenics. Analysis of expressionof the reporter gene on a tissue-to-tissue basisdemonstrated that lacZ expression of thereporter gene in stably transformed fish occured withvariable intensity in different organs and tissues andwas also sometimes variable in different cells of thesame tissue in G1and G2 generations of the transgenic lines.     

Fish can be First – Advances in Fish Transgenesis for Commercial Applications

Over the past 15 years researchers have generated stable lines of several species of transgenic fish important for aquaculture. All-fish growth hormone (GH) gene constructs and antifreeze protein (AFP) genes have been successfully introduced into the fish genome resulting in a significant acceleration of growth rate and an increase in cold and freeze tolerance. However, neither gene modification is completely understood; there are still questions to be resolved. Expression rates are still low, producing variable growth enhancement rates and less than desired levels of freeze resistance. Transgene strategies are also being developed to provide improved pathogen resistance and modified metabolism for better utilization of the diet. Additional challenges are to tailor the genetically modified fish strains to prevent release of the modified genes into the environment.     

Coexistence of coral reef fishes — a lottery for living space

Synopsis Data are summarised from studies of two reef fish communities — pomacentrids territorial on rubble patches, and fishes resident in small isolated colonies of coral. In each case there is evidence that availability of living sites limits numbers of fishes, and that similar species of fish use the same kinds of spaces. Priority of arrival as recruits, rather than subtle differences in requirements or competitive abilities of adults, appears to determine which species holds each site. Faced with a limited and patchy supply of living space, most reef fishes are sedentary as adults, and produce frequent clutches of pelagic larvae over extended breeding seasons In this way they maximise their chances of getting offspring into suitable living sites as such sites appear. It is argued that by adopting this strategy, reef fishes are preadapted for forming inter-specific lotteries for living space if several species with similar requirements occur together. Such lotteries among similar species may be a feature common to many reef fish communities, and may explain the typically high within-site diversity found in them.This paper forms part of the proceedings of a mini-symposium convened at Cornell University, Ithaca, N.Y., 18–19 May 1976, entitled Patterns of Community Structure in Fishes (G. S. Helfman, ed.).     

Scientists Prefer them Blind: The History of Hypogean Fish Research

The history of hypogean fish research has been strongly influenced by neo-Lamarckism (including orthogenesis) and typological thinking. Only in the last few decades neo-Darwinism has made any inroads in the research approach to this subject. The majority of the most distinguished and productive hypogean fish researchers have used their research subjects to confirm their own views on evolution rather than to use those subjects as a spring of knowledge to enrich mainstream biological thought. Of these views, I found that the most perversive of all is the notion of evolutionary progress that has led many researchers to envision hypogean fishes as prime examples of regressive evolution. I propose that the utilization of hypogean fishes for the study of convergent evolution should catapult these subjects of research into prime objects of evolutionary ideas.     

A repetitive DNA sequence in the salmonid fishes similar to a retroviral long terminal repeat

Summary We describe the characteristics of a repetitive DNA sequence from the rainbow trout and related salmonid fishes that is similar to a retroviral long terminal repeat (LTR). The repeat is 160 bp long and contains a region of homology to the LTR of the avian sarcoma virus. Two clones with this repeat from the chum salmon also have a polypurine tract and tRNA binding site, respectively, and these clones may represent the two LTRs of a retrovirus or retroviral-like repetitive element. Copies of the repeat are also adjacent to rainbow trout and chum salmon protamine genes. These repeats may be solo LTRs. There appears to be some polymorphism in restriction sites between individual rainbow trout and considerable differences between salmonid fish species when the repeat is used as a probe.     

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5-Srglb3-uidA-3nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.     

Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants

Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene. Transformants were directly selected and rooted on medium containing cadmium and kanamycin. A total of 49 individual transgenic tobacco plants were regenerated. Among them 20% showed a very high expression level and their growth was unaffected by up to 200 M cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 M cadmium. The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting. In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.     

Circadian rhythms and feeding time in fishes

Synopsis Although several studies have described effects of meal frequency and timing of meals on growth performance and body composition of different species of fishes, the mechanisms by which such variables influence the energy partitioning processes is not known. They may interact with the natural feeding rhythm of the fish, or with various behavioural and physiological parameters that exhibit circadian-like patterns; however, in most cases, the endogenous character of such rhythms is not clear. The time of feeding, perse, can act as a Zeitgeber and override the effect of the light/dark alternation. Fish that are fed always at the same time of day show typical pre-feeding activity. Blood levels of some nutrients and hormones also show peaks or troughs at the time of feeding, but whether these are pre-feeding or post-prandial events is not clear. These results from fish are compared with similar studies with mammals. The existence and location of an endogenous multi-oscillator system is also discussed.