Primary cell culture from fish gills and kidney using fish serum

A novel, cost effective and time saving technique for primary cell culture from gills and kidney using fish serum has been developed. Single cell suspension of gills and kidney of Clarias gariepinus was obtained by trypsinization. Minimum essential medium supplemented with 10% fish serum in place of 10% foetal calf serum and 20% fish muscle extract, yielded confluent monolayer on 6th and 8th day in gill and kidney tissue respectively at 28 degrees C. Fish serum can be successfully used as media supplement for cultivation and maintenance of primary cell culture from fishes.     

Size-assortativeness in multi-species fish shoals

Theory predicts that fish should assort in shoals on the basis of similar phenotypic traits to minimize predation risk and to maximize foraging efficiency. A single phenotypic character, body size, was considered and the hypothesis tested that free-ranging fish shoals are sizeassorted. Furthermore, a second test investigated whether fish within multi-species shoals are more strongly size-assorted with conspecifics than with heterospecifics. Twelve fish shoals, each comprising two different species (golden shiner Notemigonus crysoleucas , and banded killifish Fundulus diaphanus ) were caught in the littoral zone of a north temperate lake using a beach seine. Shoal membership size ranged from 36 to 776 fish, and mean standard body length of members ranged from 18 to 34 mm. Fish were assorted by body size at two different levels, namely, between shoals and at the level of species within shoals. Body sizes of shiners and killifish within shoals were significantly different in seven out of 12 shoals, with killifish being the larger species in five cases and shiners being the larger one in two cases. Because there is considerable overlap in body size between the two species in the population, the observed species-related size-assortativeness within shoals was not just the by-product of a directional size difference (between species) in the population. These findings provide strong quantitative evidence for size-sorting in free-ranging fish shoals and raise questions concerning the formation of multi-species fish shoals.     

The ornamental fish trade and fish conservation

The global trade in ornamental fish and associated aquarium and pond accessories is in excess of U.S. $7 × 10⁹ each year. Although the majority of freshwater fish involved in the trade are from captive-bred sources, significant numbers are still removed from the wild. In addition, almost all of the marine fish in the trade are wild-caught. Whilst habitat destruction, pollution and overfishing for food are the main threats facing fish populations in the wild, the ornamental fish trade may have adverse effects as a result of the introduction of nonnative organisms, and by the direct depletion of wild stocks. The ornamental fish trade is ideally placed to raise public awareness of, and actively support, fish conservation efforts, and must maintain a close liaison with legislative-forming organizations seeking to impose further trade restrictions.     

Current issues in fish welfare

Human beings may affect the welfare of fish through fisheries, aquaculture and a number of other activities. There is no agreement on just how to weigh the concern for welfare of fish against the human interests involved, but ethical frameworks exist that suggest how this might be approached. Different definitions of animal welfare focus on an animal's condition, on its subjective experience of that condition and/or on whether it can lead a natural life. These provide different, legitimate, perspectives, but the approach taken in this paper is to focus on welfare as the absence of suffering. An unresolved and controversial issue in discussions about animal welfare is whether non‐human animals exposed to adverse experiences such as physical injury or confinement experience what humans would call suffering. The neocortex, which in humans is an important part of the neural mechanism that generates the subjective experience of suffering, is lacking in fish and non‐mammalian animals, and it has been argued that its absence in fish indicates that fish cannot suffer. A strong alternative view, however, is that complex animals with sophisticated behaviour, such as fish, probably have the capacity for suffering, though this may be different in degree and kind from the human experience of this state. Recent empirical studies support this view and show that painful stimuli are, at least, strongly aversive to fish. Consequently, injury or experience of other harmful conditions is a cause for concern in terms of welfare of individual fish. There is also growing evidence that fish can experience fear‐like states and that they avoid situations in which they have experienced adverse conditions. Human activities that potentially compromise fish welfare include anthropogenic changes to the environment, commercial fisheries, recreational angling, aquaculture, ornamental fish keeping and scientific research. The resulting harm to fish welfare is a cost that must be minimized and weighed against the benefits of the activity concerned. Wild fish naturally experience a variety of adverse conditions, from attack by predators or conspecifics to starvation or exposure to poor environmental conditions. This does not make it acceptable for humans to impose such conditions on fish, but it does suggest that fish will have mechanisms to cope with these conditions and reminds us that pain responses are in some cases adaptive (for example, suppressing feeding when injured). In common with all vertebrates, fish respond to environmental challenges with a series of adaptive neuro‐endocrine adjustments that are collectively termed the stress response. These in turn induce reversible metabolic and behavioural changes that make the fish better able to overcome or avoid the challenge and are undoubtedly beneficial, in the short‐term at least. In contrast, prolonged activation of the stress response is damaging and leads to immuno‐suppression, reduced growth and reproductive dysfunction. Indicators associated with the response to chronic stress (physiological endpoints, disease status and behaviour) provide a potential source of information on the welfare status of a fish. The most reliable assessment of well‐being will be obtained by examining a range of informative measures and statistical techniques are available that enable several such measures to be combined objectively. A growing body of evidence tells us that many human activities can harm fish welfare, but that the effects depend on the species and life‐history stage concerned and are also context‐dependent. For example, in aquaculture, adverse effects related to stocking density may be eliminated if good water quality is maintained. At low densities, bad water quality may be less likely to arise whereas social interactions may cause greater welfare problems. A number of key differences between fish and birds and mammals have important implications for their welfare. Fish do not need to fuel a high body temperature, so the effects of food deprivation on welfare are not so marked. For species that live naturally in large shoals, low rather than high densities may be harmful. On the other hand, fish are in intimate contact with their environment through the huge surface area of their gills, so they are vulnerable to poor water quality and water borne pollutants. Extrapolation between taxa is dangerous and general frameworks for ensuring welfare in other vertebrate animals need to be modified before they can be usefully applied to fish. The scientific study of fish welfare is at an early stage compared with work on other vertebrates and a great deal of what we need to know is yet to be discovered. It is clearly the case that fish, though different from birds and mammals, however, are sophisticated animals, far removed from unfeeling creatures with a 15 s memory of popular misconception. A heightened appreciation of these points in those who exploit fish and in those who seek to protect them would go a long way towards improving fish welfare.     

Space-time organization of riverine fish communities in French Guiana

Synopsis A riverine fish assemblage in French Guiana was examined over several consecutive years. Fish distribution was highly structured on at least 3 different scales: between rivers, between the different zones of the rivers and between the different biotopes studied in sampling areas. A temporal organisation of fish communities was also found, superimposed on the spatial structure. The latter was due to the seasonal variations of the water level, and allowed temporary access to flooded areas. The different space-time factors of organisation of fish communities presented here are discussed in regard to their contribution to the maintenance of the observed high species diversity.     

The restoration of fish ponds in agricultural landscapes

Fish ponds in central and eastern Europe are artificial lakes of less than 1 ha to several 100 ha in size. Some of them were created around 900 years ago. These fish ponds form an important part of the hydrological system in the landscape; they also serve as water purification systems and sediment traps, provide habitat for many plant and animal species, as well as being used for recreation purposes—but above all, they are dedicated to fish production. The original oligo- or mesotrophic character of these fish ponds has been altered to one of eutrophic or even hypertrophic conditions due to the nature of the intensive management for high fish production, as well as agriculture practices within the catchment area. It can also be due to other uses of fish ponds, such as wastewater recipients. A thick layer of black anaerobic sediment invariably develops and serves as an uncontrolled internal source of nutrients. In order to stop the development of water blooms and restore the lake to reasonable healthy conditions, both the external and internal loading has to be reduced. An example of such a fish pond restoration within the Czech Republic is described. The phosphorus budget of the Vajgar fish pond (40 ha, S Bohemia) was evaluated and the dry matter, organic matter, nutrients, heavy metals and other metals content were estimated in the vertical profile of the fish pond's sediment. A precision suction dredger was designed, constructed, and used for the selective removal of 330 000 m³ black sediment from the Vajgar fish pond. The sediment was pumped to settling ponds at a distance of 2.5 km. Later, the suction dredger was used for sediment removal from other fish ponds. In one case, 40 000 m³ of eutrophic sediment was directly applied on an agriculture field. In another case, sediment containing oil products was decontaminated by using a biocatalyst and bacterial culture. Sediment removal from Vajgar fish pond resulted in a temporary negative phosphorus budget (lower concentrations in the outflow than the inflow). The temporary absence of cyanobacteria (Microcystis sp.) blooms was the result of the removal of the black sediment layer. The improvement of water quality after sediment removal was, however, temporary. A more holistic approach, aimed at the reduction of matter losses from the fish pond's catchment area, is therefore being adopted. The principles of such an holistic approach are described.     

Transgenic fish

Transgenic fish are produced by the artificial transfer of rearranged genes into newly fertilized eggs. Currently microinjection is the preferred method, although the integration rates of transgenes are generally low. A number of fusion genes, containing retrovirus sequences which direct integration, have been developed to enhance integration of transgenes. Mass gene transfer methods are also being developed. These include lipofection, particle bombardment, and electroporation of embryos and sperm cells. These methods are potentially useful for marine organisms such as crustaceans and molluscs as well as fish. In contrast to microinjection, which treats single cells individually, these methods can transfer genes into a large number of eggs at once. There is some evidence to indicate successful integration and expression of transgenes transferred by the electroporation of embryos and sperm cells. Germline transmission of transgenes has been observed through mating studies, and in some cases the progeny express the new phenotype consistently. However, germline transmission does not necessarily confirm stable integration of the transgene. There is evidence that transgenes may exist extrachromosomally. Transgenic fish are viewed as a useful model for the study of complex biological phenomena such as growth and differentiation, and as a fast track to the production of broodstock for the aquaculture industry. Current research focuses on the elucidation of the mechanisms controlling the regulation of gene expression. The use of transgenic fish for the isolation of developmental genes has just begun. Applications of transgenesis to broodstock development have been focused on the development of fish with accelerated growth, tolerance to low temperature, and disease resistance. However, before the release of transgenic fish into the environment, the possible impact on the environment must be assessed. There must be safeguards to protect the genetic diversities of the natural populations, and to conserve the natural habitats     

Biotechnology and fish culture

Biotechnology can currently be considered of importance in aquaculture. The increase in the production of aquatic organisms over the last two decades through the use of biotechnology indicates that in a few generations biotechnology may overtake conventional techniques, at least for the commercially more valuable species. In the last few years, genetics has contributed greatly to fish culture through the application of the more recent techniques developed in biotechnology and in genetic engineering. At present, the most commonly used methods in fish biotechnology are chromosome manipulation and hormonal treatments, which can be used to produce triploid, tetraploid, haploid, gynogenetic and androgenetic fish. These result in the production of individuals and lineages of sterile, monosex or highly endogamic fish. The use of such strategies in fish culture has as a practical objective the control of precocious sexual maturation in certain species; other uses are the production of larger specimens by control of the reproductive process and the attainment of monosex lines containing only those individuals of greater commercial value. The use of new technologies, such as those involved in gene transfer in many species, can result in modified individuals of great interest to aquaculturists and play important roles in specific programmes of fish production in the near future.     

Interaction between fish spoilage bacteria Pseudomonas sp. and Shewanella putrefaciens in fish extracts and on fish tissue

The interaction between fish spoilage bacteria, Pseudomonas sp. and Shewanella putrefaciens , was investigated using fish extract and fish tissue as model systems. Isolates of Pseudomonas that produced iron chelators, siderophores, inhibited growth of S. putrefaciens in a fish-extract-agar diffusion assay but no, or only weak, antagonistic activity was seen when the medium was supplemented with iron. Sterile-filtered supernatant fluid from a siderophore-producing Pseudomonas grown in fish extract was inhibitory to S. putrefaciens if the number of Pseudomonas was above 10⁸ cfu ml⁻¹. In contrast, supernatant fluids from siderophore-negative Pseudomonas isolates did not inhibit growth of S. putrefaciens. The inhibitory effect was, except for one strain of Pseudomonas , not seen in supernatant fluids from iron-enriched cultures of Pseudomonas sp. Finally, siderophore-producing Pseudomonas sp. lowered the maximum cell level of S. putrefaciens 1–2 log units from 10⁹ to 10¹⁰ cfu g⁻¹ when the strains were grown on fish muscle blocks at 0°C but the growth rate of S. putrefaciens was not affected.     

Cleaner fish drives local fish diversity on coral reefs

Coral reefs are one of the most diverse habitats in the world, yet our understanding of the processes affecting their biodiversity is limited. At the local scale, cleaner fish are thought to have a disproportionate effect, in relation to their abundance and size, on the activity of many other fish species, but confirmation of this species' effect on local fish diversity has proved elusive. The cleaner fish Labroides dimidiatus has major effects on fish activity patterns and may indirectly affect fish demography through the removal of large numbers of parasites. Here we show that small reefs where L. dimidiatus had been experimentally excluded for 18 months had half the species diversity of fish and one-fourth the abundance of individuals. Only fish that move among reefs, however, were affected. These fish include large species that themselves can affect other reef organisms. In contrast, the distribution of resident fish was not affected by cleaner fish. Thus, many fish appear to choose reefs based on the presence of cleaner fish. Our findings indicate that a single small and not very abundant fish has a strong influence on the movement patterns, habitat choice, activity, and local diversity and abundance of a wide variety of reef fish species.     

Erythrocyte eicosapentaenoic acid versus docosahexaenoic acid as a marker for fish and fish oil consumption.

The fatty acid composition of erythrocyte membranes was investigated in 21 healthy men after 6 wk of varying intakes of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In one experiment, 12 subjects were fed three diets in a 3 x 3 crossover design: an essentially fish-free control diet, a fish diet (0.15 g EPA/d, 0.41 g DHA/d) and the same fish-based diet supplemented with 5 g/d fish oil (Fish + Oil: 0.99 g EPA/d, 0.99 g DHA/d). A 6 wk wash-out period was allowed between each diet. In another experiment, 11 subjects were supplemented with 5 g/d fish oil alone for 6 wk (0.84 g EPA/d, 0.48 g DHA/d). After fish or fish oil feeding, the percent proportion of EPA and DHA in the erythrocyte membranes rose at the expense of linoleic and arachidonic acids. After 6 wk on the fish-based diets, EPA incorporation approached saturation, with the incremental increases being proportional to the amounts supplied by the diets. In contrast, parallel increases were observed for erythrocyte DHA even though the Fish + Oil diet was supplying twice as much DHA as the fish alone diet. These observations imply different metabolic rates for EPA and DHA and their importance is discussed in terms of the value of erythrocyte EPA versus DHA as markers for fish and fish oil consumption.     

Diurnal variation in the abundance of juvenile parasitic gnathiid isopods on coral reef fish: implications for parasite-cleaner fish interactions

 The dynamics of parasitic gnathiid isopod infestation on the fish Hemigymnus melapterus were examined at Heron Island, Great Barrier Reef, by measuring the abundance and feeding state of gnathiids on fish collected between dawn and sunset and by estimating the time required for gnathiids to become engorged on host fluids. A model was developed to estimate gnathiid abundance on fish for any given time of day and host size. Fish at dawn had 2.4 times as many gnathiids compared with fish at sunset, indicating that some gnathiids infest fish overnight. Most gnathiids had engorged guts (72–86%); the proportion of empty guts and engorged guts did not differ in three time periods of collection (<0800 h, 0800 to 1100 h, and >1100 h). In the laboratory, gnathiids fed quickly with 75% of gnathiids exposed to fish for 4 h having engorged guts. The short time required for gnathiids to become engorged and the presence of gnathiids with empty guts throughout the day suggests that gnathiids also infest fish during the day. Thus gnathiids eaten by cleaner fish during the day may be replaced by other gnathiids during the day or night suggesting that interactions between gnathiids and cleaner fish are highly dynamic. Accepted: 15 April 1999     

"Instantaneous" growth rates of fish scales and their use in studies of fish populations

A technique has been devised for measuring the incorporation of ¹⁴C-glycine into fish scales. This provides an indication of the rate of growth of the scale at the time of sampling. The growth of fish scales is classically considered to be related to the growth of the whole fish' hence the method devised has been used to investigate aspects of fish growth in a number of environmental situations.     

Ribonucleases and angiogenins from fish

For the first time fish RNases have been isolated and characterized. Their functional and structural properties indicate that they belong to the RNase A superfamily (or tetrapod RNase superfamily), now more appropriately described as the vertebrate RNase superfamily. Our findings suggest why previously repeated efforts to isolate RNases from fish tissues have met with no success; fish RNases have a very low ribonucleolytic activity, and their genes have a low sequence identity with those of mammalian RNases. The investigated RNases are from the bony fish Danio rerio (or zebrafish). Their cDNAs have been cloned and expressed, and the three recombinant proteins have been purified to homogeneity. Their characterization has revealed that they have indeed a very low RNA-degrading activity, when compared with that of RNase A, the superfamily prototype, but comparable with that of mammalian angiogenins; that two of them have angiogenic activity that is inhibited by the cytosolic RNase inhibitor. These data and a phylogenetic analysis indicate that angiogenic fish RNases are the earliest diverging members of the vertebrate superfamily, suggesting that ribonucleases with angiogenic activity were the ancestors of all ribonucleases in the superfamily. They later evolved into both mammalian angiogenins and, through a successful phylogenesis, RNases endowed with digestive features or with diverse bioactivities.     

Size-selective predation by the cleaner fish Labroides dimidiatus

A comparison of the size-frequency distribution of parasitic gnathiid isopod larvae in the diet of the cleaner fish Labroides dimidiatus and on six host fish species ( Chlorurus sordidus, Ctenochaetus striatus, Hemigymnus melapterus, Scolopsis bilineatus, Siganus doliatus, Thalassoma lunare ) was made on one occasion. The comparison was repeated with Hemigymnus melapterus on three occasions and between two islands in Australia. L. dimidiatus selected larger gnathiids at all times at Lizard Island but not at Heron Island. Size-selective predation by L. dimidiatus suggests any potential effect of cleaner fish on parasites may vary according to the size of parasite. However, this effect appears to vary spatially.     

Resource allocation in yolk-feeding fish

An insight is made into the main processes that occur in fish during endogenous feeding period. The ways in which yolk absorption rate can be measured are evaluated. Essential amino acids and polyunsaturated fatty acids are preferentially retained for incorporation into body tissue. Profound physiological and anatomical changes in yolk and a sequence of slow, fast, and a second period of slow absorption occur during the endogenous feeding period. Attempts to quantify the ontogenetic sequence are reviewed. Various methods of body size assessment are compared, and sources of bias in individual and population growth estimates are discussed. Several calorimetric methods are compared of which direct calorimetry using an oxygen bomb is the reference method. An advanced elemental analysis (CHNS) is a reliable technique that is adequate for early stages. Indices of growth potential are reviewed including a comparison of different measures, models and approaches used to estimate growth. Changes in body hydration, caloric value, content of lipids, protein, free amino acids (FAA) and minerals, and in content of RNA and DNA occur in early ontogeny. Ways to quantify metabolic rate are identified. Mean relative respiration rate of initial egg before activation is very low, about 20 mm³ g⁻¹ h⁻¹. Ontogenetic sequence in absolute metabolic rate of fish embryos and yolk-feeding larvae involves an increase through hatching to a peak at the time of first feeding ability, and a decrease under starvation. Models predicting the relationship between oxygen consumption and age in yolk-feeding fish are reviewed. Sequence of metabolic fuels begins with use of small molecules as carbohydrates, soon switched to FAA. Later lipids are progressively used, they provide energy for swimming activity. After yolk depletion body protein-bound amino acids are mobilised. In this review I focused on the major environmental variables as temperature, oxygen, salinity, pH, toxic xenobiotics, light, UV radiation, magnetic field and substrate, along with intrinsic factors as egg or body size, sex and genetic factors. A question was posed on how the extrinsic and intrinsic factors determine yolk absorption, growth and metabolic rates in yolk-feeding fish. Special attention is devoted to fish body size attained exclusively on yolk. A considerable variety of body size responses to temperature was found, for which several explanations are forwarded. Methodological progress made recently is characterised and the most conspicious advances in understanding of fish early life history are highlighted. Information derived from these studies can be used in management of fish populations in the field and to optimise activities in aquaculture.     

Echo-sounding can discriminate between fish and macroinvertebrates in fresh water

1. Acoustic scattering from fish and macroinvertebrates was studied in a boreal Finnish lake at three echosounder frequencies (38, 120 and 200 kHz). Split-beam transducers with partly overlapping 7° beams were employed. Acoustic, fish and invertebrate sampling were undertaken simultaneously. Vertical gradients of temperature and oxygen concentration were measured during the exercise.
2. At all frequencies, a narrow scattering layer coincided with the thermocline. At 38 kHz, fish were detected well with practically no reverberation from invertebrates while 200 kHz detected both fish and invertebrates.
3. Minor differences in the magnitude of acoustic scattering from fish were found between frequencies and between depth layers, but scattering at different frequencies was correlated at all depths. Acoustic scattering and fish density indices from trawl catches, consisting mostly of smelt ( Osmerus eperlanus ) (97%) and vendace ( Coregonus albula ) (3%), were significantly correlated.
4. Acoustic scattering from invertebrates increased with sound frequency. Correlation analysis suggested that the invertebrate scattering was mostly induced by Chaoborus flavicans . A low frequency is recommended for estimating fish abundance without bias from reverberation induced by invertebrate scattering. Although fish and invertebrates can also be successfully discriminated at a single frequency by thresholding and cross filtering, the combination of a low and a high frequency is a more robust tool for effective fish-invertebrate discrimination.     

Microbiological quality of frozen breaded fish and shellfish products.

A survey was made of the microbiological quality of seven frozen, breaded, precooked fish and shellfish products and of frozen, breaded, uncooked shrimp at the retail level. Geometric mean aerobic plate counts per gram (and number of units examined) were as follows: fish sticks, 8,300 (1,539); fish cakes, 5,600 (1,378); crab cakes, 4,900 (1,226); scallops, 1,700 (1,392); clams, 450 (1,384); haddock, 15,000 (1,306); fish in fish and chips dinner, 7,200 (1,485); and uncooked shrimp, 220,000 (1,462). Geometric mean coliform, Escherichia coli, and Staphylococcus aureus counts for all eight products ranged from 1 to 10/g.     

Interactions among xerophilic fungi associated with dried salted fish

Interactions were investigated among five xerophilic fungi, Polypaecilum pisce, Basipetospora halophila, Eurotium rubrum, Aspergillus wentii and A. penicillioides, isolated from Indonesian dried salted fish. A range of water activities ( a _w) (0·98, 0·95, 0·90 and 0·84) and temperatures (15°, 25° and 30°C) were studied on agar media in Petri dishes, and with dried fish as a substrate at 0·90 and 0·84 a _w at 30°C. Generally, the fungi exhibited one of two interaction types: mutual inhibition on contact, or inhibition of one or both species on contact, with the inhibited species continuing to grow at a significantly reduced rate. On glucose-based agar media A. wentii and E. rubrum were most competitive at all a _w values and temperatures studied, while on NaCl media P. pisce and B. halophila were usually most competitive. The Petri dish system was a useful model, but did not completely simulate the interactions observed on dried fish. Polypaecilum pisce and B. halophila were able to compete more strongly on fish than on agar media, especially at 0·90 a _w. This study provides some evidence that each species examined had a niche in which it was dominant, and that species interactions as well as environmental factors are important in determining the dominant fungal species on dried salted fish.