Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.     

Diving behaviour of dugongs, Dugong dugon

The diving behaviour of 15 dugongs (Dugong dugon) was documented using time-depth recorders (TDRs), which logged a total of 39,507 dives. The TDRs were deployed on dugongs caught at three study sites in northern Australia: Shark Bay, the Gulf of Carpentaria and Shoalwater Bay. The average time for which the dive data were collected per dugong was 10.4±1.1 (S.E.) days. Overall, these dugongs spent 47% of their daily activities within 1.5 m of the sea surface and 72% less than 3 m from the sea surface. Their mean maximum dive depth was 4.8±0.4 m (S.E.), mean dive duration was 2.7±0.17 min and the number of dives per hour averaged 11.8±1.2. The maximum dive depth recorded was 20.5 m; the maximum dive time in water >1.5 m deep was 12.3 min. The effects of dugong sex, location (study site), time of day and tidal cycle on diving rates (dives per hour), mean maximum dive depths, durations of dives, and time spent ≤1.5 m from the surface were investigated using weighted split-plot analysis of variance. The dugongs exhibited substantial interindividual variation in all dive parameters. The interaction between location and time of day was significant for diving rates, mean maximum dive depths and time spent within 1.5 m of the surface. In all these cases, there was substantial variation among individuals within locations among times of day. Thus, it was the variation among individuals that dominated all other effects. Dives were categorised into five types based on the shape of the time-depth profile. Of these, 67% of dives were interpreted as feeding dives (square and U-shaped), 8% as exploratory dives (V-shaped), 22% as travelling dives (shallow-erratic) and 3% as shallow resting dives. There was systematic variation in the distribution of dive types among the factors examined. Most of this variation was among individuals, but this differed across both time of day and tidal state. Not surprisingly, there was a positive relationship between dive duration and depth and a negative relationship between the number of dives per hour and the time spent within 1.5 m of the surface after a dive.     

Surgical implantation techniques for electronic tags in fish

Intracoelomic implantation of transmitters into fish requires making a surgical incision, incision closure, and other surgery related techniques; however, the tools and techniques used in the surgical process vary widely. We review the available literature and focus on tools and techniques used for conducting surgery on juvenile salmonids because of the large amount of research that is conducted on them. The use of sterilized surgical instruments properly selected for a given size of fish will minimize tissue damage and infection rates, and speed the wound healing of fish implanted with transmitters. For the implantation of transmitters into small fish, the optimal surgical methods include making an incision on the ventral midline along the linea alba (for studies under 1 month), protecting the viscera (by lifting the skin with forceps while creating the incision), and using absorbable monofilament suture with a small-swaged-on swaged-on tapered or reverse-cutting needle. Standardizing the implantation techniques to be used in a study involving particular species and age classes of fish will improve survival and transmitter retention while allowing for comparisons to be made among studies and across multiple years. This review should be useful for researchers working on juvenile salmonids and other sizes and species of fish.     

The dispersal patterns and behaviour of North Sea cod (Gadus morhua) studied using electronic data storage tags*

The migratory behaviour of adult cod was studied using electronic data storage tags (DSTs). Fifty-eight cod were externally tagged with LOTEK LTD-100 DSTs and released off Lowestoft in March and April 1999. Twenty-two tags were returned by March 2000. Individuals demonstrated significant shifts in behaviour patterns that were associated with migration and residence on feeding grounds. During migration, individuals were very active and utilised a large vertical range. In contrast, individuals demonstrated low activity and a much reduced vertical range whilst on summer feeding grounds. Use of the Tidal Location Method showed that individuals dispersed to feeding grounds in the central and southern North Sea. Our results demonstrate that individual-based studies of fish behaviour can provide information relevant to fisheries management.     

Tracking invasive animals with electronic tags to assess risks and develop management strategies

Invasive species alter ecosystem structure and function when they establish in new habitats. Although preventing or managing invasions is extremely important for maintaining biodiversity, doing so is difficult and requires efficient intervention. Remote monitoring of free-living animals with electronic tags (i.e. tags that transmit data remotely or log them for future retrieval) can contribute important knowledge about invasive animal biology. A quantitative literature review identified instances in which electronic tagging has contributed to studying invasions. Electronic tags were generally used for one of four purposes: (1) characterize spatial ecology; (2) identify interactions; (3) assess risk potential; or (4) evaluate management options. Overall, electronic tags have considerable potential for developing, refining, and evaluating invasion management strategies that contribute to conservation efforts. We explore the role of electronic tags as a component of integrated control program design and implementation for invasive animals.     

Diving behaviour and respiration in Blethisa multipunctata in comparison with two other ground beetles

ABSTRACT. The diving behaviour of Blethisa multipunctata (L.), a carabid species living on shores, is compared with that of two other hygrophilous carabid species. B. multipunctata enters the water spontaneously and is able to stay more than 1 h beneath the surface without renewing its respiratory air, In flooding experiments, c . 50% of submerged beetles do not leave the water after 2h. These animals emerge from time to time for a few seconds and then descend beneath the water again. Measurements of oxygen consumption and the volume of the respiratory air show that the underwater air-supply is sufficient for only a few minutes. B. multipunctata achieves maximum diving times of up to 97 min by using its air storage as a physical gill. In comparison with other Carabidae it shows neither morphological nor physiological adaptations, but only behavioural adaptations for its amphibious mode of life.     

Diving behaviour and diet of the blue-eyed shag at South Georgia

Summary This paper describes a concurrent investigation of individual variation in diet, diving patterns and performance of blue-eyed shags Phalacrocorax atriceps breeding at South Georgia. Within one day individual shags exhibited one of three foraging strategies: short diving (4 birds, all dives 120 s) and mixed diving (15 birds, predominantly long but with a few short dives). The mean number of dives per day was significantly higher in shags that only made short dives (mean=172.0, SE=43.2) than birds with a mixed diving strategy (mean=40.5, SE=4.7) and birds that made only long dives (mean=30.8, SE=1.8). Diet was assessed using hard remains recovered from pellets regurgitated by the shags. Small nototheniid fish (c. 10 kJ per item) were by far the commonest prey but most pellets contained additional items. The frequency of pellets with additional items of higher energy value than nototheniid fish (10.c. 900 kJ per item), lower energy value (>1–10 kJ per item) and both higher and lower energy items was strikingly similar to the frequency of shags making long, short and both long and short dives respectively. Predicted aerobic dive limits suggested that during long dives, blue-eyed shags were probably sustained by anaerobic metabolism. Models of prey capture rates demonstrated that for both long and short diving, many items must be caught per dive when birds are feeding on prey at the lower end of the energy range. Predicted capture rates for the commonest recorded prey (small fish) differ markedly between the two diving strategies.     

Diving behaviour and foraging location of female southern elephant seals from Patagonia

Our aim was to describe the free-ranging diving pattern and to determine the location of foraging of pregnant female southern elephant seals, Mirounga leonina , from Peninsula Valdes, Argentina. This colony is unusual in two respects: it is removed from deep water by a broad shallow shelf (345–630 km wide), and colony numbers have been increasing in recent years in contrast to numbers from other southern hemisphere colonies that are stable or in decline. Microprocessor controlled, geolocation-time-depth recorders were deployed on four females, recording a total of 15,836 dives (270 dive days) during the period February to April, 1992. Departing seals crossed the continental shelf quickly (54–5–62–1 h) and did not show signs of foraging until reaching deep water, due east of the colony in the South Atlantic Ocean. Diving was virtually continuous (93% of the time underwater) with overall mean (±S.D.) rates of 2.5±0.2 dives/h, mean dive durations of 22.8 ± 7.1 min (maximum dive duration = 79 min) with 1.6±0.6min surface intervals between dives, and dive depths of 431±193m (maximum dive depth = 1,072 m). The diving pattern of females from Patagonia is similar to that of seals from colonies where numbers are decreasing (Macquarie stock) or are stable (South Georgia Island). Our subjects did not, however, feed in or south of the Antarctic Polar Front, or in cold waters along the Antarctic coast, where seals from declining or stable colonies forage.     

Diving behaviour of white-chinned petrels and its relevance for mitigating longline bycatch

The white-chinned petrel (Procellaria aequinoctialis) is the seabird species most commonly killed by Southern Hemisphere longline fisheries. Despite the importance of diving ability for mitigating longline bycatch, little is known of this species’ diving behaviour. We obtained data from temperature–depth recorders from nine white-chinned petrels breeding on Marion Island, southwestern Indian Ocean, during the late incubation and chick-rearing period. Maximum dive depth (16 m) was slightly deeper than the previous estimate (13 m), but varied considerably among individuals (range 2–16 m). Males dived deeper than females, and birds feeding chicks dived deeper than incubating birds, but dive rate did not differ between the sexes. Time of day had no significant effect on dive depth or rate. Our findings will help to improve the design and performance of mitigation measures aimed at reducing seabird bycatch in longline fisheries, such as the calculation of minimum line sink rates and optimum aerial coverage of bird-scaring lines.     

Diving behaviour and energetics in breeding little penguins (Eudyptula minor)

We present data on the diving behaviour and the energetics of breeding little penguins in Tasmania, Australia. Using an 18 m long still water canal in conjunction with respirometry, we determined the energy requirements while diving. Using electronic devices measuring dive depth or swimming speed, we investigated the foraging behaviour at sea. Cost of Transport was calculated to be minimal at the speed the birds prefer at sea (1.8 m/s) and averaged 11.1 J/kg/m (power requirements at that speed: 20.0 W/kg). Metabolic rate of little penguins resting in water was found to be 8.5 W/kg. The externally-attached devices had no significant influence on the energy expenditure.
Foraging trips can be divided into four distinct phases with different diving behaviours. A mean of 500 dives was executed per foraging trip lasting about 18 hours with 60% of this time being spent swimming. The total distance travelled averaged 73 km per day, although foraging range was about 12km. Mean swimming speed of little penguins at sea was 1.8 m/s, maximum swimming speed was 3.3 m/s. More than 50% of all dives had maxima not exceeding 2 m. Maximum depth reached was 27 m. Mean dive duration was 21 s. There were inter-sex differences in diving behaviour as well as changes in foraging behaviour over the breeding period. Aerobic dive limits (ADL) in the wild were estimated between 42 and 50 s. From the swim canal experiments we derived an ADL of 44 s. Total oxygen stores were calculated to be 45 ml O₂/kg. Only 2% of all dives exceeded the ADL. FMRs at sea were calculated to be between 1280 and 1500 kJ/kg/d according to chick size. The yearly food requirements of a breeding little penguin amount to 114 kg.     

Advances in tagging syngnathids, with the effects of dummy tags on behaviour of Hippocampus guttulatus

Artificial marking and tagging techniques have been used to study movement, population dynamics, behaviour, ecology, survival and growth of at least 25 syngnathid species. External necklace-style tags and injection of visible implant elastomer have been the most used techniques, uniquely identifying hundreds of individual syngnathids to study population dynamics, mortality, behaviour, ecology and growth in at least 13 and 12 species, respectively. Only two studies, both on larger syngnathid species, have tested the use of internal or electronic tags. This new case study reveals that dummy tags, weighing up to 6% of individual body mass, have minimal effect on normal ex situ behaviour of the long-snouted seahorse Hippocampus guttulatus, a smaller syngnathid. In paired aquarium trials, tags did not affect movement, holdfast use or general behavioural state, and only had a short-term effect (1 day) on vertical orientation. Tagged H. guttulatus gained more mass during the 5 day trials, a result which warrants further exploration but indicates that tags did not reduce feeding. This study shows promise for using electronic tagging to study H. guttulatus and similarly sized syngnathids in the wild.     

Diving at the shallow end: Green turtle behaviour in near-shore foraging habitat

Green turtles Chelonia mydas of immature and adult size (n = 19, curved carapace length 49 to 118 cm) were equipped with time-depth recorders for short periods (≤ 7 d) to investigate diel and seasonal variation in diving behaviour. Research sessions were distributed over 2 years to cover seasonal variation in sea temperature from 14 °C to 30 °C. Diurnal dives were shallower and shorter than nocturnal dives, with diel patterns also evident in dawn and dusk peaks in occupation of depths within 1 m of the surface, elevated diurnal occupation of depths 1 to 2 m below the surface and elevated nocturnal occupation of depths > 2 m. Dive duration increased as sea temperature decreased, showing strong negative correlation by day and by night. Study turtles made resting dives that were 3 to 4 times longer in median duration, and six times longer in maximum duration, at cool temperatures than they were at warm temperatures, but there was no evidence of winter diapause or location shift to avoid cold water. The large majority of turtles spent 89 to 100% of their time at depths ≤ 5 m below the surface, three individuals did not exceed 3 m and the maximum depth recorded by any turtle was 7.9 m, although deeper water was available. Furthermore, the dive data indicated that study turtles collectively spent more than 80% of their time at charted (low tide) depths of 3 m or less, indicating that they consistently used the shallow margins of the bay where human activities tend to be concentrated, thereby potentially increasing their exposure to anthropogenic threats.     

Diving behaviour and prey of the Humboldt Penguin (Spheniscus humboldti)

Summary Humboldt Penguins diving in clear water in Chile had two distinct behaviours; short dives where the birds remained within a metre of the surface and bounce dives, where the penguins descended the water column to the sea bed where they immediately returned to the surface. Here, dive time was correlated with water depth. Stomach pumped penguins had eaten predominantly pelagic school fish,Scomberesox, Engraulis andSardinops. Most prey (94 %) had been seized from below. We suggest that short, shallow dives are typical of travelling penguins. Bounce dives, however, would enable foraging penguins, which have an enhanced binocular field above the line of the bill, to perceive surface-swimming prey more easily because silvery clupeid fish appear as silhouettes.

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Zusammenfassung In klaren Gewässern in Chile tauchende Humboldt-Pinguine zeigten zwei deutlich verschiedene Verhaltensweisen. Während kurzer Tauchgänge blieben die Tiere im Bereich von 1 m unter der Oberfläche. Tauchgänge durch unmittelbares Abtauchen in einem Winkel von 45° eingeleitet, führten direkt zum Meeresboden; die Vögel kehrten sofort wieder an die Oberfläche zurück. In diesem Fall war die Tauchzeit von der Wassertiefe abhängig. Pinguine, deren Magen durch Auspumpen untersucht wurde, hatten vorwiegend pelagische Schwarmfische gefressen:Scomberesox, Engraulis undSardinops. Die Mehrzahl der Fische (94 %) wurde von unten kommend erfaßt. Nach unserer Auffassung sind die kurzen und flachen Tauchgänge typisch für nicht jagende Pinguine, die längere horizontale Strecken zurücklegen. Das tiefe Abtauchen dagegen ermöglicht jagenden Pinguinen, die ein besonders gutes Fernsichtvermögen oberhalb des Schnabels besitzen, nahe der Oberfläche schwimmende Beute leichter zu erfassen. Die sonst silbrigen clupeiden Fische sind als dunkle Silhouetten gegen die Wasseroberfläche besser zu erkennen.

     

Diving behaviour, dive cycles and aerobic dive limit in the platypus Ornithorhynchus anatinus

We investigated the diving behaviour, the time allocation of the dive cycle and the behavioural aerobic dive limit (ADL) of platypuses (Ornithorhynchus anatinus) living at a sub-alpine Tasmanian lake. Individual platypuses were equipped with combined data logger-transmitter packages measuring dive depth. Mean dive duration was 31.3 s with 72% of all dives lasting between 18 and 40 s. Mean surface duration was 10.1 s. Mean dive depth was 1.28 m with a maximum of 8.77 m. Platypuses performed up to 1600 dives per foraging trip with a mean of 75 dives per hour. ADL was estimated by consideration of post-dive surface intervals vs. dive durations. Only 15% of all dives were found to exceed the estimated ADL of 40 s, indicating mainly aerobic diving in the species. Foraging platypuses followed a model of optimised recovery time, the optimal breathing theory. Total bottom duration or total foraging duration per day is proposed as a useful indicator of foraging efficiency and hence habitat quality in the species.     

Holding of juvenile salmonids for surgical implantation of electronic tags: a review and recommendations

Many telemetry-based studies require that fish be sampled from the wild and then held both prior to and after the implantation of an electronic tag. However, the effects of such holding (or the lack thereof) have yet to be studied intensively. Pre-surgical holding often occurs to facilitate logistical needs of research projects and as an attempt to minimize negative physiological effects due to capture and handling stress. Further, post-surgical holding time and conditions greatly influence the physiological state of fish prior to being returned to the wild. This paper reviews pertinent studies pertaining to the effects of surgical holding on the behavior, physiology, and survival of fishes, with particular emphasis on juvenile salmonids. The effects of individual aspects of surgical holding such as handling, water quality, light conditions, holding density, metabolic scope, and duration of holding are reviewed. Recommendations regarding certain aspects of surgical holding are offered with a goal of reducing bias related to the surgical process.