Targeted gene expression by the Gal4-UAS system in zebrafish

Targeted gene expression by the Gal4-UAS system is a powerful methodology for analyzing function of genes and cells in vivo and has been extensively used in genetic studies in Drosophila . On the other hand, the Gal4-UAS system had not been applied effectively to vertebrate systems for a long time mainly due to the lack of an efficient transgenesis method. Recently, a highly efficient transgenesis method using the medaka fish Tol2 transposable element was developed in zebrafish. Taking advantage of the Tol2 transposon system, we and other groups developed the Gal4 gene trap and enhancer trap methods and established various transgenic fish expressing Gal4 in specific cells. By crossing such Gal4 lines with transgenic fish lines harboring various reporter genes and effector genes downstream of UAS (upstream activating sequence), specific cells can be visualized and manipulated in vivo by targeted gene expression. Thus, the Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrates.     

The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.     

High-throughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germ line-transmissible insertions in zebra fish

Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fish blastulae and efficiently infects embryonic cells that give rise to the future germ line. Using GT virus preparations we generated roughly 500,000 germ line-transmissible proviral insertions in a population of 25,000 founder fish in about 2 months. The GT virus contains a gene trap, and trap events can be detected in the offspring of almost every founder fish. We discuss potential applications of this highly efficient method for generating germ line-transmissible insertions in a vertebrate     

A note on a possible influence of traps on assessment of the diet of Jasus lalandii (H. Milne-Edwards)

Dietary analyses are important components of ecological studies. However, some methods of collecting organisms may expose them to exceptionally high densities of prey items, leading to inaccurate dietary assessments. These methods include the use of baited traps. We hypothesised that such a "trap effect" occurred during work on the diet of the rock lobster Jasus lalandii, because baited traps attracted isopods, which were then eaten opportunistically by trapped J. lalandii. To test this hypothesis, rock lobsters were collected at two sites using both baited-traps and Scuba diving. Results showed that large numbers of isopods were attracted to, and consumed, the trap-bait. Analyses of the stomach contents of trap-caught J. lalandii from both localities reflected a larger occurrence and significantly greater abundance of isopods in stomach samples from trap- rather than Scuba-caught rock lobsters. For probably similar reasons, small fish were significantly over-represented in the gut contents of trap-caught rock lobsters, although the evidence is less clear-cut and there may be other explanations for the high proportions of fish in the diet. Although isopods and fish may be naturally consumed at a low rate, predation on them is greatly inflated in traps, and the dietary analyses of trap-caught J. lalandii. All assessments of the diets of animals traditionally caught with traps should consider and account for such "trap effects", by calibrating the data for trap-caught animals against an alternative sampling method.     

Shoal size as a key determinant of vulnerability to capture under a simulated fishery scenario

Group living is widespread among animals and has a range of positive effects on individual foraging and predator avoidance. For fishes, capture by humans constitutes a major source of mortality, and the ecological effects of group living could carry‐over to harvest scenarios if fish are more likely to interact with fishing gears when in social groups. Furthermore, individual metabolic rate can affect both foraging requirements and social behaviors, and could, therefore, have an additional influence on which fish are most vulnerable to capture by fishing. Here, we studied whether social environment (i.e., social group size) and metabolic rate exert independent or interactive effects on the vulnerability of wild zebrafish (Danio rerio) to capture by a baited passive trap gear. Using video analysis, we observed the tendency for individual fish to enter a deployed trap when in different shoal sizes. Fish in larger groups were more vulnerable to capture than fish tested individually or at smaller group sizes. Specifically, focal fish in larger groups entered traps sooner, spent more total time within the trap, and were more likely to re‐enter the trap after an escape. Contrary to expectations, there was evidence that fish with a higher SMR took longer to enter traps, possibly due to a reduced tendency to follow groupmates or attraction to conspecifics already within the trap. Overall, however, social influences appeared to largely overwhelm any link between vulnerability and metabolic rate. The results suggest that group behavior, which in a natural predation setting is beneficial for avoiding predators, could be maladaptive under a trap harvest scenario and be an important mediator of which traits are under harvest associated selection.     

The scaling of locomotor performance in predator-prey encounters: from fish to killer whales.

During predator-prey encounters, a high locomotor performance in unsteady manoeuvres (i.e. acceleration, turning) is desirable for both predators and prey. While speed increases with size in fish and other aquatic vertebrates in continuous swimming, the speed achieved within a given time, a relevant parameter in predator-prey encounters, is size independent. In addition, most parameters indicating high performance in unsteady swimming decrease with size. Both theoretical considerations and data on acceleration suggest a decrease with body size. Small turning radii and high turning rates are indices of maneuverability in space and in time, respectively. Maneuverability decreases with body length, as minimum turning radii and maximum turning rates increase and decrease with body length, respectively. In addition, the scaling of linear performance in fish locomotion may be modulated by turning behaviour, which is an essential component of the escape response. In angelfish, for example, the speed of large fish is inversely related to their turning angle, i.e. fish escaping at large turning angles show lower speed than fish escaping at small turning angles. The scaling of unsteady locomotor performance makes it difficult for large aquatic vertebrates to capture elusive prey by using whole-body attacks, since the overall maneuverability and acceleration of small prey is likely to be superior to that of large predators. Feeding strategies in vertebrate predators can be related to the predator-prey length ratios. At prey-predator ratios higher than approximately 10(-2), vertebrate predators are particulate feeders, while at smaller ratios, they tend to be filter feeders. At intermediate ratios, large aquatic predators may use a variety of feeding methods that aid, or do not involve, whole body attacks. Among these are bubble curtains used by humpback whales to trap fish schools, and tail-slapping of fish by delphinids. Tail slapping by killer whales is discussed as an example of these strategies. The speed and acceleration achieved by the flukes of killer whales during tail slaps are higher and comparable, respectively, to those that can be expected in their prey, making tail-slapping an effective predator behaviour.     

Baiting improves CPUE in nine‐spined stickleback (Pungitius pungitius) minnow trap fishery

Whether or not baiting influences stickleback catch per unit effort (CPUE) remains a matter of debate among stickleback researchers: While the opinions about the impact of baiting on CPUE differ, supporting quantitative data are scarce. The effect of baiting and trap type on nine‐spined stickleback (Pungitius pungitius) CPUE was studied in a field experiment conducted over four consecutive days in a small pond in northeastern Finland. The results show that baited traps yielded better (mean CPUE = 1.24 fish/trap/d) catches than unbaited traps (mean CPUE = 0.66); however, there were also differences in CPUE depending on the type of collapsible trap that was used. The trap type effect on CPUE seemed to differ among age classes – the finer meshed trap caught more young‐of‐the‐year fish than the coarse‐meshed one, whereas the opposite was true for the older and larger individuals. The results agree with those of an earlier more restricted study conducted in the same locality: Together, these results provide strong evidence for the positive impact of baiting on nine‐spined stickleback CPUE.     

Software for minimalistic data management in large camera trap studies

The use of camera traps is now widespread and their importance in wildlife studies is well understood. Camera trap studies can produce millions of photographs and there is a need for a software to help manage photographs efficiently. In this paper, we describe a software system that was built to successfully manage a large behavioral camera trap study that produced more than a million photographs. We describe the software architecture and the design decisions that shaped the evolution of the program over the study's three year period. The software system has the ability to automatically extract metadata from images, and add customized metadata to the images in a standardized format. The software system can be installed as a standalone application on popular operating systems. It is minimalistic, scalable and extendable so that it can be used by small teams or individual researchers for a broad variety of camera trap studies.     

The expanding role of fish models in understanding non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a condition in which excessive fat accumulates in the liver of an individual who has not consumed excessive alcohol. Non-alcoholic steatohepatitis (NASH), a severe form of NAFLD, can progress to hepatic cirrhosis and/or hepatocellular carcinoma (HCC). NAFLD is considered to be a hepatic manifestation of metabolic syndrome, and its incidence has risen worldwide in lockstep with the increased global prevalence of obesity. Over the last decade, rodent studies have yielded an impressive list of molecules associated with NAFLD and NASH pathogenesis. However, the identification of currently unknown metabolic factors using mammalian model organisms is inefficient and expensive compared with studies using fish models such as zebrafish (Danio rerio) and medaka (Oryzias latipes). Substantial advances in unraveling the molecular pathogenesis of NAFLD have recently been achieved through unbiased forward genetic screens using small fish models. Furthermore, these easily manipulated organisms have been used to great advantage to evaluate the therapeutic effectiveness of various chemical compounds for the treatment of NAFLD. In this Review, we summarize aspects of NAFLD (specifically focusing on NASH) pathogenesis that have been previously revealed by rodent models, and discuss how small fish are increasingly being used to uncover factors that contribute to normal hepatic lipid metabolism. We describe the various types of fish models in use for this purpose, including those generated by mutation, transgenesis, or dietary or chemical treatment, and contrast them with rodent models. The use of small fish in identifying novel potential therapeutic agents for the treatment of NAFLD and NASH is also addressed.     

Feeding and trapping fish withPiper auritum

The “natives” at Rio Indio, on the central Caribbean slope of Panama, trap the river fishBrycon chagrensis using leaves ofPiper auritum as bait in feeders. The people use this technique because the fish tastes like the leaf after it has fed regularly on the leaf. This technique has potential use in aquaculture. The plant could also be cultivated for marketing as a food-flavoring condiment and fish food.     

The influence of trap density and sampling duration on the detection of small mammal species richness

Assessing species richness of small mammal communities is an important research objective for many live-trapping studies designed to assess or monitor biological diversity. We tested the effectiveness and efficiency of various trap densities for determining estimates and counts of small mammal species richness. Trapping was conducted in grassland habitats in northeastern Kansas during spring and fall of 2002 and 2003. Estimates and counts of species richness were higher at increased trap densities. This effect appeared to be primarily due to the higher number of individuals sampled at higher trap densities. At least 3 nights duration was needed to produce a stable estimate of species richness for the range of trap densities tested (9–144 trap stations/ha). Higher trap densities generally reached stable richness estimates in fewer nights than low density trapping arrangements. Given that counts and estimates of species richness were influenced by trap density and sampling duration, it is critical that these parameters are selected to most effectively meet research objectives.     

Effective generation of transgenic reporter and gene trap lines of the medaka (Oryzias latipes) using the Ac/Ds transposon system

In model teleost fishes like the medaka and the zebrafish many genes which have been identified in genome sequencing projects await their functional characterization. Techniques for the effective generation of transgenic animals are a prerequisite for this challenging task, and, due to their transparency, fish offer the possibility to combine the use of fluorescent proteins and developmental analysis in vivo. Here we describe the application of the Ac/Ds transposon system to generate transgenic medaka reporter and gene trap lines. We determined a germline transmission rate of 30% in our experiments using constructs ranging in size from 1.8 to 6 kilobase pairs. The genomic integration site of the Ds-elements can be easily identified which is an important feature for gene trap mutagenesis experiments and similar approaches. We constructed gene trap vectors with functional elements of medaka sequences that produce in frame fusions of the endogenous sequence to EGFP. These vectors mimic endogenous expression of the trapped allele in transgenic animals and are capable to interfere with the expression of the wild type allele in the homozygous individuals.     

Development of a new shape of pheromone trap for the bean bug, <Emphasis Type="Italic">Riptortus pedestris</Emphasis> (Hemiptera: Alydidae)

Riptortus pedestris F. [=R. clavatus (Thunberg)] is one of the most serious pests affecting the cultivation of sweet persimmon, which is one of the most important export fruits in Korea. A pheromone-baited trap has been used in attempts to control this pest. In this study, the development of a more efficient pheromone-baited trap for R. pedestris was pursued by investigating three different shapes of traps: the fish trap, the pyramidal trap, and the symmetry trap. The fish trap, which is the only one commercially available, captured the lowest number of R. pedestris, and the pyramidal trap showed moderate capture efficacy. The symmetry trap captured significantly greater numbers of R. pedestris adults (ca. 6 and 28 times more than the pyramidal and fish trap, respectively). Significantly increased trapping efficacy of the symmetry trap would be useful for mass trapping of R. pedestris.     

A noninvasive method to determine fat content in small fish based on swim bladder size estimation

The presence of fat stores in fish is widely used as a correlate of fish health and fitness. Techniques to measure fat content with some accuracy are available for medium-sized and large fish, but apart from morphometric indices, a noninvasive method to determine fat content in small fish has hitherto been lacking. In this study, we introduce a novel method to measure the fat content in live fish that can be applied also to small fish of less than 0.5?g of body mass. This approach relies on a precise measurement of the swim bladder volume, from which fat content can subsequently be deduced. As fat is positively buoyant, fish with larger fat stores require a smaller swim bladder to attain neutral buoyancy. To determine swim bladder volume, we developed a measuring device, which makes use of the differential compressibility of air and water. A fish is placed in a pressure-tight chamber to which a standardized amount of water is added. The resulting change in pressure Δp is inversely proportional to the volume of the swim bladder. Using juveniles and adults of Simochromis pleurospilus (Nelissen, '78; Pisces: Tropheini) a small cichlid fish, we show that Δp is tightly related to structural size, mass, and body condition. Most importantly, this approach allows to predict the visceral fat content of small fish more precisely than the six most commonly used morphometric body indices.     

Ecology of the introduced snapper Lutjanus kasmiva (Forsskal) in the reef fish assemblage of a Hawaiian bay

Since its first introduction in 1955, the snapper Lutjanus kasmira (taape) has developed large populations in shallow coastal waters of Hawaii. Visual abundance estimates and trap catches of taape were different between the main habitat types of the reef. Overall, taape was the second most abundant species by numbers and biomass over a hard substratum and was patchily distributed, even within habitat type. The largest individuals occurred singly or in small groups on shallow reef slopes; on the deep slopes and in spur-and-groove habitat, taape occurred in larger groups of smaller sized fish. Catches in fish traps showed a negative relationship with the complexity of the adjacent natural substratum, suggesting that traps are more productive where they offer additional cover in areas with less natural cover. In all areas in which traps were used, from fringing reef to open sand, the catch was dominated by taape. Tagging confirmed the theory that taape tend to maintain a limited range over long periods. Tagging results also indicated occasional long-range movements among major habitats and provided data confirming patterns of movement between reef habitat by day and sand habitat by night. Taape collected over open sand substratum by trap and by hook-and-line were smaller than those collected by the same means at the reef. Observations and collections suggested an ontogenetic trend in habitat use by taape from initial settlement in fringe areas toward ultimate residence on the main reef. Other common fish species in the reef assemblages were tested for statistical associations with taape. Although several positive species associations were found, multivariate analysis did not reveal patterns that indicated strong ecological relationships.     

Fish habitat rehabilitation using wood in the world

To provide river managers and researchers with practical knowledge about fish rehabilitation, various studies of fish habitat rehabilitation that used wood were reviewed. The review focuses on fish responses, wood installation methods, and geomorphic features of the rehabilitation sites. Most studies were conducted in moderately sized (small and medium) streams with relatively high bed gradients and aimed to improve the habitats of salmonid species. In this stream type, structures spanning the full (log dam) and partial (log deflector) width of the river were most common, and wood structures that created pools and covers were successful in improving fish habitat. Some projects were conducted in moderately sized low-gradient streams, in which wooden devices used to create instream cover were effective for fish assemblages. There were few studies in other aquatic ecosystems. However, well-designed large wood structures, known as engineered log jams, were used in rehabilitation projects for large rivers. In slack-water or lentic systems such as side-channels, estuaries, and reservoirs, small and large wood structures that created cover were used to improve habitat for many fish species. For successful fish habitat rehabilitation projects, the hydrogeomorphic conditions of rehabilitation sites should be carefully examined to avoid physical failure of wood structures. Although artificial wood structures can be used to improve fish habitat in various aquatic ecosystems, they should be considered to be a complementary or interim habitat enhancement technique. The recovery of natural dynamic processes at the watershed scale is the ultimate target of restoration programs.     

Intensive trapping and increased fish predation cause massive population decline of an invasive crayfish

1. Invasive species frequently have adverse impacts on native communities and ecosystems. Management options are often limited. Our goal is to evaluate the effect of intensive trapping and fish predation on the population dynamics of an invasive crayfish. 2. From 2001 to 2005, we removed invasive rusty crayfish (Orconectes rusticus) by trapping in Sparkling Lake in northern WI. In addition, the Wisconsin Department of Natural Resources restricted harvest of fish species known to consume crayfish, thereby increasing predation on crayfish that are too small to trap. 3. After an initial increase, catch rates of rusty crayfish declined by approximately 95%, from 11 crayfish per trap per day in 2002 to 0.65 in 2004. The catch rate in 2005 remained low at 0.5 crayfish per trap. Females comprised nearly 50% of the catch from 2002 to 2004. Unlike rusty crayfish in Sparkling Lake, catch rates of O. rusticus and Orconectes propinquus in three nearby lakes increased or remained relatively constant over the 5‐year removal period. 4. We also examined the influence of habitat and temperature on crayfish catch rates. Catch rates were highest at water temperatures between 20 and 25 °C and on cobble, log or macrophyte habitats that may serve as refuge from fish predation. 5. Five summers of intensive trapping and fisheries management practices reduced abundances, but did not extirpate rusty crayfish in Sparkling Lake. To determine the potential of trapping as a management option for invasive crayfishes, these methods must be tested in other systems.     

Investigating the behavioural responses of trapped fishes using underwater video surveillance

An underwater video surveillance system known as TrapCam was used to continuously record (15 ×c. 24 h periods) fish behaviour within and immediately surrounding an experimental fish trap situated in a coral reef ecosystem in the United States Virgin Islands. Of the 100 fishes (18 species, 12 families) trapped, surgeonfishes (Acanthuridae) and snappers (Lutjanidae) were most common. Thirteen distinctively identifiable behaviours were observed for trapped fishes. Species did not differ significantly in the proportion of time allocated to different behaviours (ANOSIM, R = 0·142). Doctorfish Acanthurus chirurgus and grey angelfish Pomacanthus arcuatus allocated the largest proportion of their recorded time to enter and exit the trap. Fishes spent an average of 15 min in the trap before escaping. Sixty‐seven per cent of trap approaches consisted of an individual of the same species as one already trapped suggesting that conspecific attraction may have occurred. Fifteen per cent of trapped species were observed with abrasions to the head and 70% were observed approaching the trap corners. The results of this study provide a greater understanding of the behavioural interactions between fishes and traps that can help explain patterns of catch composition, the physical condition of fishes in traps and inform design of gear modifications to optimize by‐catch reduction in the trap fishery.