Description: Improving the understanding of digestive physiology in first feeding larvae is a prerequisite for advancing diet formulations and feeding protocols. In marine fish larvae that lack a stomach at first-feeding trypsin represents the main proteolytic enzyme. CCK is one of the key regulators of digestive enzyme secretion in adult vertebrates and current knowledge suggests that it is also involved in early stages of teleosts, although this may vary between species. Here, we investigated the influence of Artemia and a commercial microdiet on the ontogenetic development of tryptic enzyme activity as an indicator for digestive capacity in first-feeding sea bass. In order to examine the regulation and feedback mechanisms in the digestive tract we followed the response of gut CCK and tryptic enzyme activity during a one-day observation depending on the feeding regime at 23 days post hatch. Larvae fed the microdiet showed a higher tryptic enzyme activity, probably as an adaptation to the higher content in complex protein in the diet. The plant protein phytohemagglutinin (PHA), added to the microdiet as a potential stimulator for the digestive system, did not induce elevated tryptic enzyme activity nor was it beneficial for growth. This was possibly due to adverse effects of too high doses. We observed an endogenous rhythm of CCK over the day, independent of the dietary treatment or short-term fasting. Higher tryptic enzyme activity in larvae fed Artemia during the day might indicate a better stimulation by live prey in the digestive tract or the superiority of a discontinuous feeding schedule in this group. We suggest that a reduction in tryptic enzyme activity after several feeding events indicates a limit in diurnal digestive capacity. Sea bass larvae are apparently able to adapt to the feeding schedule by synchronizing the tryptic enzyme activity like adult fish.

Description: In order to maximize protein digestion, the release of enzymes into the gut lumen is closely controlled by a regulatory loop. Cholecystokinin (CCK) is among the enteric hormones that play a key role in the control of digestive enzyme secretion, but its role in first-feeding larvae is still unclear and may differ between species. However, in all marine fish larvae that have not developed a stomach by first-feeding, trypsin is the most important proteolytic enzyme. In order to examine the regulation and feedback mechanisms in the gut of larval cod, we therefore studied the interactions between cholecystokinin and tryptic enzyme activity following the administration of solutions containing test substances directly into the gut. We tube-fed a single dose of physiological saline solution containing either CCK, CCK antagonist, trypsin inhibitor, phytohemagglutinin (PHA; a possible trigger for the digestive response) or physiological saline alone, while a further control group was left untreated. We then followed the response in CCK and tryptic enzyme activity for 0.5–8 h after the administration. We performed the experiment on larvae at 26 day post first-feeding, which is before the stomach has evolved and the size of the larvae allows easier handling. Individual larvae were analyzed for CCK and tryptic enzyme activity using radioimmunoassay and fluorimetric techniques respectively. Both factors varied over time in the untreated control group, possibly due to an endogenous daily rhythm. The higher CCK levels at 4 h and 8 h in the saline-injected group may be caused by reflexes initiated by distension of the gut. An increase in tryptic enzyme activity after injection of CCK supports the hypothesis that this hormone plays a part in the release of pancreatic enzymes in larval cod at this developmental stage. However, administration of a CCK antagonist and a trypsin inhibitor did not reveal conclusive results, probably due to the relatively low concentrations used. The response in tryptic activity in the PHA group was similar to the administration of CCK, pointing towards a stimulatory effect of PHA on the proteolytic enzyme capacity of cod larvae.

Description: Highlights • We describe the ontogenetic development of CCK and tryptic enzyme activity in larval cod • CCK is known to play a key role in regulating digestive processes • CCK concentrations increased during ontogeny suggesting a growing role in regulating digestive processes • A short-term experiment reveals a feedback mechanism between CCK and tryptic enzyme activity • Cod larvae have limited regulatory and digestive capacity to handle several meals in a short period The ontogenetic development of the gut hormone cholecystokinin (CCK) and the key proteolytic enzyme trypsin was described in Atlantic cod larvae (Gadus morhua) from first-feeding until 38 days post first-feeding (dpff). CCK is known to play a major role in the endocrine control of digestive processes in mammals and adult fish, but its regulatory role in the larval stages of marine fish is largely unknown. Only small amounts of CCK were found in the body (excluding head) in cod larvae at first-feeding, but CCK levels increased exponentially with development, suggesting a more pronounced role of CCK during ontogeny. Tryptic enzyme activity increased slightly until a standard length of ca. 8 mm (approx. 33 days dpff) with a significant increase in larvae larger than 8 mm standard length, indicating limited digestive capacity in the early stages. To entangle the short-term feedback mechanism between CCK and tryptic enzyme activity, we conducted a 12 hour feeding experiment at 21 dpff. Cod larvae receiving only algae revealed a noticeable response in tryptic enzyme activity within two hours in the morning, whereas larvae fed algae and rotifers at the same time showed a slightly delayed response up to four hours. Tryptic enzyme activity remained low in the group receiving only algae as well as the two fed groups in the afternoon. No reaction in tryptic enzyme activity was observed in larvae that received a second meal of rotifers in the afternoon, indicating limited regulatory and digestive capacity to handle several meals in a short period. CCK levels remained relatively constant throughout the day but increased in the afternoon in all three groups when tryptic enzyme activity was low, suggesting that a negative feedback mechanism between CCK and tryptic enzyme activity is present in larval cod at least from 21 dpff.

Description: A protocol for combined analysis of cholecystokinin (CCK) levels based on radioimmunoassay (RIA) and fluorescence tryptic enzyme activity (FTA) was developed in order to accomplish a sensitive analysis of individual bodies and gut segments of fish larvae. Methanol was used for CCK extraction. The gut of herring larvae contained 8.9±1.2 fmol CCK/mg dry weight and in the post-larval Atlantic halibut the CCK levels varied significantly (p〈0.05) from 20.9±15.6 to 101.8±56.7 fmol/mg dry weight for separated intestinal and pyloric segments, respectively. Acid solution, 0.02 mol/l HCl–CaCl2 (pH 1.8), and alkaline solution, 0.1 mol/l Tris–0.02 mol/l CaCl2 (pH 8.0) were tested to prepare crude trypsin extracts from Coregonids and Atlantic halibut larvae. The tryptic activity of crude extracts prepared with acid solution was enhanced by a factor of 3.19±0.52 compared to the tryptic activity of crude extracts prepared with alkaline solution. The larval trypsins (from yolk sac larvae) were stable in methanol, preserving 88% of its starting activity after 6 days of storage. Based on the results, the method of extraction using methanol and acid solution (pH 1.8) was suitable for the combined analysis for CCK levels and FTA in gut segments or single larvae. The potential application of these analytical tools may allow a better understanding of the individual variability of gut functionality, nutritional condition and the feeding activity of developing fish based on their content of CCK and tryptic activity.

Description: Distributed collaborative applications for supporting complex use cases in mobile environments have to provide contextual information (e.g. presence and group awareness) via their user interface. Social interaction and data sharing — being essential aspects of distributed collaborative applications — typically result in conflicting goals, primarily awareness vs. privacy. Preserving the end users' privacy especially in mobile collaborative settings is the most often-cited point of critique of mobile and ubiquitous computing. Since usability is a prerequisite for privacy and awareness mechanisms especially for mobile applications, we report in this paper on how to balance usability, privacy, and awareness trade-offs when building mobile collaborative applications. This is complemented by new approaches for preserving privacy tailored to the needs of respective communities in the domain of decentralized group-centric solutions. The requirements were gathered through an analysis of user's needs as well as first evaluations of prototypes. Those were built for different case studies focusing on privacy, trust, and identity management in real-life communities. We report on the outcomes of our work and show this exemplary with the help of a mobile prototype application to support an Angling Community with privacy and collaboration needs related to location-based services.

Description: When evaluating live prey for the successful rearing of fish larvae, general availability and nutritional quality of the prey are mainly in the major focus. However, the digestibility of prey items is also crucial as it directly affects the accessibility of the nutrients inside the prey item. Harpacticoid copepods, as well as nematodes, are considered as potential live feed for fish larvae but their digestibility has not yet been identified. Therefore, a comparative in-vitro evaluation of the digestibility of several prey organisms for larval fish was conducted under specific consideration of the efficiency of the proteolytic enzyme trypsin. This endoprotease plays a major role in the digestion process in the early stages of marine species. Common (Artemia sp., Brachionus plicatilis) and candidate prey organisms for commercial larval rearing (Acartia tonsa (Calanoida), Tachidius discipes, Tisbe sp. (both Harpacticoida), Panagrolaimus sp. (Nematoda)) were exposed to a trypsin solution. Photos of trypsin-treated prey organisms were taken and compared with controls to quantify the effect of trypsin on the inner body disintegration. Additionally, the effects of the larval ring muscles in the gut and the pharyngeal teeth were imitated by mechanical treatment. While Artemia sp. showed the highest digestibility, Panagrolaimus sp. was the opposite with very low degradation. The calanoid copepod A. tonsa and the harpacticoid copepod Tisbe sp. were more digestible than T. discipes and the nematode Panagrolaimus sp.

Description: As food protein digestion is instrumental for promoting growth, the main protease in young marine fish larvae, trypsin, was studied in pre-leptocephalus larvae of Anguilla japonica. Tryptic enzyme activity was monitored until day 24 and rates of growth until day 36 after hatch in larval A. japonica derived from artificially matured parent fish. Tryptic activity increased with larval age and developmental stage until day 16, after which tryptic levels stabilized. In start-feeding larvae, tryptic activities increased after ingestion of an artificial diet, but elevated tryptic activities could also be found in larvae with guts void of prey, possibly due to intestinal retention of trypsin secreted in response to ingestion of a previous meal. Gut retention time for trypsin was estimated to be at least 15 h. The gut evacuation time for the artificial diet depended on the meal size and was in the range of 1-5.5 h. Rates of larval growth in length were ca. 5% day-1 during the yolk-sac stage, declining to ca. 1% day-1 in older larvae. Vision was highly important for initiation of feeding. Results provide the first quantitative information on aspects of protein digestion in eel larvae. 2003 Elsevier Science B.V. All rights reserved.

Description: This study determined the effects of two test diets — a dry-phytoplankton and a trout-fry feed — and a control diet (Artemia nauplii) on tryptic activity, growth and survival rates during early life stages of Nile tilapia (Oreochromis niloticus) larvae. In addition, during a second experimental series, the interaction between trypsin and CCK (cholecystokinin) secretion was monitored in larvae fed with a PHA (phytohemagglutinin, a protein extract from the red kidney bean) enriched commercial trout-diet and compared with the data resulting from the use of the same but non-enriched feed. Subgroups were taken from the experimental units and kept under starvation. Oreochromis niloticus was chosen as a model species, since the larvae are able to intake artificial diets by the time of first feeding, and thus featuring the experiments with a manipulated micro-diet. The results demonstrated that larval mortality and growth are affected by the diet given and this was mostly observed in the group fed on dry-phytoplankton (12.2% mortality, 1.45 mg/d; control group: 2.9% mortality, 3.19 mg/d). The same larval group showed also a higher tryptic activity compared with all the other groups, which in combination with the bad survival and poor growth performance gives evidence for inadequate nutritional quality of the dry-phytoplankton feed for larvae aged more than two weeks after hatching. Every other feeding group showed good growth rates (trout-fry feed: 3.04–3.19 mg/d, with PHA enriched trout-fry feed: 2.85 mg/d), similar to the larvae fed with live prey (3.19–3.35 mg/d). A reduction of tryptic activity characterised the starvation process. These results confirm the usefulness of monitoring the individual tryptic activity as an indicator for evaluating the quality of a diet and the nutritional condition of fish larvae, but also the necessity of combining data of tryptic activity with growth and survival data for a correct interpretation. An interaction between trypsin and CCK secretion was also confirmed with this experimental approach, since induction and reduction of tryptic activity followed a reverse pattern compared with the concentration of CCK.