Are bold-shy personalities of European perch (Perca fluviatilis) linked to stress tolerance and immunity? A scope of harnessing fish behavior in aquaculture.

The sensitivity to stress and its impact on immunity are supposedly related to a fish's personality. In the present study, European perch (Perca fluviatilis) were exposed to an open-field and a novel-object test to identify distinctive shy and bold individuals. This series of cognitive tests revealed clear differences between proactive individuals with pronounced exploration behavior (bold personality) and reactive individuals that took a freeze-hide position (shy personality). A cohort of shy and bold perch was then exposed to elevated stocking density. Frozen activity and lower explorative behavior were related to higher basal and stocking-induced cortisol levels compared to proactive individuals. Since cortisol is a well-known modulator of immune-gene expression, we used multiplex real-time PCR to profile the differential immune responses to the intraperitoneal injection of Aeromonas hydrophila in the head kidney and peritoneal cells of bold and shy perch individuals. These expression differences between stimulated bold and shy perch were generally modest, except for the genes encoding the complement component c3 and the matrix metallopeptidase mmp9. The strong differential expression of these two bactericidal and inflammatory genes in the context of the modestly regulated features suggests that a fish's personality is linked to a particular immune-defense strategy. In conclusion, our approach, based on behavioral video observations, phagocytosis and enzyme assays, immunogene-expression profiling, and quantification of stress-relevant metabolites, revealed indications for divergent coping styles in cohorts of bold or shy European perch. This divergence could be exploited in future selective breeding programs.

Data for non-invasive (photo) individual fish identification of multiple species.

This paper describes data from five studies focused on the individual fish identification of the same species. The lateral images of five fish species are present in the dataset. The dataset's primary purpose is to provide a data to develop a non-invasive and remote method of individual fish identification using fish skin patterns, which can serve as a substitute for the common invasive fish tagging. The lateral images of the whole fish body on the homogenous background for Sumatra barb, Atlantic salmon, Sea bass, Common carp and Rainbow trout are available with automatically extracted parts of the fish with skin patterns. A different number of individuals (Sumatra barb - 43, Atlantic salmon - 330, Sea bass - 300, Common carp - 32, Rainbow trout - 1849) were photographed by the digital camera Nikon D60 under controlled conditions. The photographs of only one side of the fish with several (from 3 to 20) repetitions were taken. Common carp, Rainbow trout and Sea bass were photographed out of the water. Atlantic salmon was photographed underwater, out of the water, and the eye of the fish was photographed by the microscope camera. Sumatra barb was photographed under the water only. For all species, except Rainbow trout, the data collection was repeated after a different period (Sumatra barb - four months, Atlantic salmon - six months, Sea bass - one month, Common carp - four months) to collect the data for a study of skin patter changes (ageing). The development of the method for photo-based individual fish identification was performed on all datasets. The identification accuracy for all species for all periods was 100% using the nearest neighbour classification. Different methods for skin pattern parametrization were used. The dataset can be used to develop remote and non-invasive individual fish identification methods. The studies focused on the discrimination power of the skin pattern can benefit from it. The changes of skin patterns due to fish ageing can be explored from the dataset.

Environmentally relevant concentration of caffeine-effect on activity and circadian rhythm in wild perch.

We studied the ecological consequences of widespread caffeine contamination by conducting an experiment focused on changes in the behavioral traits of wild perch (Perca fluviatilis) after waterborne exposure to 10 µg L-1 of caffeine. We monitored fish swimming performance during both light and dark conditions to study the effect of caffeine on fish activity and circadian rhythm, using a novel three-dimensional tracking system that enabled positioning even in complete darkness. All individuals underwent three behavioral trials-before exposure, after 24 h of exposure, and after 5 days of exposure. We did not observe any effect of the given caffeine concentration on fish activity under light or dark conditions. Regardless of caffeine exposure, fish swimming performance was significantly affected by both the light-dark conditions and repeating of behavioral trials. Individuals in both treatments swam significantly more during the light condition and their activity increased with time as follows: before exposure < after 24 h of exposure < after 5 days of exposure. We confirmed that the three-dimensional automated tracking system based on infrared sensors was highly effective for conducting behavioral experiments under completely dark conditions.

The Effect of Different Feeding Applications on the Swimming Behaviour of Siberian Sturgeon: A Method for Improving Restocking Programmes.

Restocking programmes of different fish species have been implemented worldwide. However, the survival of hatchery-reared fish after release to riverine ecosystems is at a very low level. One of the reasons for the high mortality rate of post-released fish is their modified swimming behaviour due to the hatchery rearing practice. To investigate one of the possible causes for modified swimming behaviour, Acipenser baerii larvae were exposed to surface- and bottom-feeding applications with day and night light regimes in a factorial design. We also analysed the effect of 5 and 10 days of starvation after different feeding applications on sturgeon swimming behaviour. The surface-feeding application was previously expected to promote the frequent Siberian sturgeon swim up to the mid- and top-water layers in our rearing facilities. However, our results indicated that the modified behaviour of the Siberian sturgeon in our study was caused by fish starvation and a possible predator-free environment rather than by the method of feed application or the day/night light regimes. These results may be used to improve the implementation of restocking programmes either through modified hatchery rearing practice or the training of foraging skills with predator stimuli.

Computer vision based individual fish identification using skin dot pattern.

Precision fish farming is an emerging concept in aquaculture research and industry, which combines new technologies and data processing methods to enable data-based decision making in fish farming. The concept is based on the automated monitoring of fish, infrastructure, and the environment ideally by contactless methods. The identification of individual fish of the same species within the cultivated group is critical for individualized treatment, biomass estimation and fish state determination. A few studies have shown that fish body patterns can be used for individual identification, but no system for the automation of this exists. We introduced a methodology for fully automatic Atlantic salmon (Salmo salar) individual identification according to the dot patterns on the skin. The method was tested for 328 individuals, with identification accuracy of 100%. We also studied the long-term stability of the patterns (aging) for individual identification over a period of 6 months. The identification accuracy was 100% for 30 fish (out of water images). The methodology can be adapted to any fish species with dot skin patterns. We proved that the methodology can be used as a non-invasive substitute for invasive fish tagging. The non-invasive fish identification opens new posiblities to maintain the fish individually and not as a fish school which is impossible with current invasive fish tagging.

Cardiac and Locomotor Responses to Acute Stress in Signal Crayfish Pacifastacus leniusculus Exposed to Methamphetamine at an Environmentally Relevant Concentration.

Methamphetamine (METH), a central nervous system stimulant used as a recreational drug, is frequently found in surface waters at potentially harmful concentrations. To determine effects of long-term exposure to environmentally relevant levels on nontarget organisms, we analysed cardiac and locomotor responses of signal crayfish Pacifastacus leniusculus to acute stress during a 21-day exposure to METH at 1 µg L-1 followed by 14 days depuration. Heart rate and locomotion were recorded over a period of 30 min before and 30 min after exposure to haemolymph of an injured conspecific four times during METH exposure and four times during the depuration phase. Methamphetamine-exposed crayfish showed a weaker cardiac response to stress than was observed in controls during both exposure and depuration phases. Similarly, methamphetamine-exposed crayfish, during METH exposure, showed lower locomotor reaction poststressor application in contrast to controls. Results indicate biological alterations in crayfish exposed to METH at low concentration level, potentially resulting in a shift in interactions among organisms in natural environment.

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities.

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities. In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model. This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

Synthetic progestin etonogestrel negatively affects mating behavior and reproduction in Endler's guppies (Poecilia wingei).

High rates of progestins consumption in the form of active ingredients in women's oral contraceptives and other hormonal preparations may lead to their increased concentrations in aquatic environments and subsequent harmful effect on fish reproduction. The objective of the present study was to assess the effect of etonogestrel, a third-generation synthetic progestin, on the reproductive behavior, fertility, gonads histology, and secondary sexual characteristics of male and female Endler's guppies (Poecilia wingei). Fish were subjected for 34 days to two concentrations of etonogestrel, including one possibly environmentally relevant (3.2 ng L-1) and one sublethal (320 ng L-1) concentration. A mating behavior study was subsequently conducted and revealed that the treatment with etonogestrel significantly reduced mating frequency in the exposed fish compared to controls. All the exposed females were unable to reproduce. In addition, female fish exposed to the highest level of etonogestrel were masculinized, as their anal fins and body coloration showed patterns similar to those of male fish. Etonogestrel-exposed females also had fewer developed oocytes. In conclusion, the low etonogestrel concentration (3.2 ng L-1) led to a reduction of mating activity in males without effect on their reproductive success, but it completely inhibited reproduction in females. Exposure to etonogestrel clearly has more severe consequences for females than males.

Localization of sound-producing fish in a water-filled tank.

In this paper, the authors introduce an algorithm for locating sound-producing fish in a small rectangular tank that can be used, e.g., in behavioral bioacoustical studies to determine which fish in a group is sound-producing. The technique consists of locating a single sound source in the tank using signals gathered by four hydrophones placed in the tank together with a group of fish under study. The localization algorithm used in this paper is based on a ratio of two spectra ratios: the spectra ratio between the sound pressure measured by hydrophones at two locations and the spectra ratio between the theoretical Green's functions at the same locations. The results are compared to a localization based on image processing technique and with video recordings acquired synchronously with the acoustic recordings.

Comparative Performance Analysis of Support Vector Machine, Random Forest, Logistic Regression and k-Nearest Neighbours in Rainbow Trout (Oncorhynchus Mykiss) Classification Using Image-Based Features.

The main aim of this study was to develop a new objective method for evaluating the impacts of different diets on the live fish skin using image-based features. In total, one-hundred and sixty rainbow trout (Oncorhynchus mykiss) were fed either a fish-meal based diet (80 fish) or a 100% plant-based diet (80 fish) and photographed using consumer-grade digital camera. Twenty-three colour features and four texture features were extracted. Four different classification methods were used to evaluate fish diets including Random forest (RF), Support vector machine (SVM), Logistic regression (LR) and k-Nearest neighbours (k-NN). The SVM with radial based kernel provided the best classifier with correct classification rate (CCR) of 82% and Kappa coefficient of 0.65. Although the both LR and RF methods were less accurate than SVM, they achieved good classification with CCR 75% and 70% respectively. The k-NN was the least accurate (40%) classification model. Overall, it can be concluded that consumer-grade digital cameras could be employed as the fast, accurate and non-invasive sensor for classifying rainbow trout based on their diets. Furthermore, these was a close association between image-based features and fish diet received during cultivation. These procedures can be used as non-invasive, accurate and precise approaches for monitoring fish status during the cultivation by evaluating diet's effects on fish skin.

Crayfish can distinguish between natural and chemical stimuli as assessed by cardiac and locomotor reactions.

In this study, cardiac and locomotor activities of signal crayfish Pacifastacus leniusculus were investigated under exposure to a range of natural (i.e., odors of conspecific crayfish, predatory fish, food, and injured conspecific) and one chemical (i.e., disinfectant chloramine-T) stimuli. Crayfish locomotion was simultaneously initiated with an increase in heart rate only when affected by chloramine-T, while locomotor response was delayed in all cases (or was not manifested at all by some specimens) when disturbed by the natural stressors. The heart rate differences measured before and during the stimulation were arranged as follows: odor of conspecific crayfish (9.2 ± 7.1%) < predator (18.4 ± 13%) < food (33.5 ± 15.7%) < chloramine-T (41.1 ± 14.7%) < injured conspecific (51.8 ± 28.4%). Analysis of the peculiarities of crayfish heartbeat under exposure to the tested stimuli revealed complex cardiac responses as was previously observed by an electrocardiography approach, that is, a slowed heart rate followed by a delayed increase. Evaluation of the intrinsic parameters of crayfish bioindicators remains essential due to the possibility of detection of the substantial ethological responses even in motionless animals. The role and appropriateness of signal crayfish as a bioindicator of water quality is discussed; they seem to be an applicable species for this task due to their sufficient sensitivity and broad availability. In addition to providing a better understanding of stereotypic crayfish behaviors induced by common and chemical stressors, the results of this study may serve as reference data for the evaluation of crayfish suitability for water quality tests.

BioWes-from design of experiment, through protocol to repository, control, standardization and back-tracking.

BACKGROUND: One of the main challenges in modern science is the amount of data produced by the experimental work; it is difficult to store, organize and share the scientific data and to extract the wealth of knowledge. Experimental method descriptions in scientific publications are often incomplete, which complicates experimental reproducibility. The proposed system was created in order to address these issues. It provides a solution for management of the experimental data and metadata to support the reproducibility. IMPLEMENTATION: The system is implemented as a repository for experiment descriptions and experimental data. It has three main entry points: desktop application for protocol design and data processing, web interface dedicated for protocol and data management, and web-based interface for mobile devices suitable for the field experiments. The functionality of desktop client can be extended using the custom plug-ins for data extraction and data processing. The system provides several methods to support experimental reproducibility: standardized terminology support, data and metadata at a single location, standardized protocol design or protocol evolution. RESULTS AND DISCUSSION: The system was tested in the framework of international infrastructure project AQUAEXCEL with five pilot installations at different institutes. The general testing in Tissue culture certified laboratory, Institute of complex systems and IFREMER verified the usability under different research infrastructures. The specific testing focused on the data processing modules and plug-ins demonstrated the modularity of the system for the specific conditions. The BioWes system represents experimental data as black box and therefore can handle any data type so as to provide broad usability for a variety of experiments and provide the data management infrastructure to improve the reproducibility and data sharing. CONCLUSIONS: The proposed system provides the tools for standard data management operations and extends the support by the standardization possibilities, protocol evolution with visualization features and modularity based on the data processing modules and device communication plug-ins. The software can be used at different organization levels: from a single researcher (to improve data organization) to research consortium through the central protocols management repository. Support from the protocol design until being shared with the standardization features helps to improve the reproducibility of research work. The platform provides support from experimental protocol design to cooperation using simple sharing.

Segmentation Method of Time-Lapse Microscopy Images with the Focus on Biocompatibility Assessment.

Biocompatibility testing of new materials is often performed in vitro by measuring the growth rate of mammalian cancer cells in time-lapse images acquired by phase contrast microscopes. The growth rate is measured by tracking cell coverage, which requires an accurate automatic segmentation method. However, cancer cells have irregular shapes that change over time, the mottled background pattern is partially visible through the cells and the images contain artifacts such as halos. We developed a novel algorithm for cell segmentation that copes with the mentioned challenges. It is based on temporal differences of consecutive images and a combination of thresholding, blurring, and morphological operations. We tested the algorithm on images of four cell types acquired by two different microscopes, evaluated the precision of segmentation against manual segmentation performed by a human operator, and finally provided comparison with other freely available methods. We propose a new, fully automated method for measuring the cell growth rate based on fitting a coverage curve with the Verhulst population model. The algorithm is fast and shows accuracy comparable with manual segmentation. Most notably it can correctly separate live from dead cells.

The significance of droughts for hyporheic dwellers: evidence from freshwater crayfish.

Freshwater biodiversity is globally threatened by various factors while severe weather events like long-term droughts may be substantially devastating. In order to remain in contact with the water or stay in a sufficiently humid environment at drying localities, the ability to withstand desiccation by dwelling in the hyporheic zone, particularly through vertical burrowing is crucial. We assessed the ability of three European native and five non-native crayfish as models to survive and construct vertical burrows in a humid sandy-clayey substrate under a simulated one-week drought. Three native species (Astacus astacus, A. leptodactylus, and Austropotamobius torrentium) suffered extensive mortalities. Survival of non-native species was substantially higher while all specimens of Cherax destructor and Procambarus clarkii survived. The native species and Pacifastacus leniusculus exhibited no ability to construct vertical burrows. Procambarus fallax f. virginalis and P. clarkii constructed bigger and deeper burrows than C. destructor and Orconectes limosus. In the context of predicted weather fluctuations, the ability to withstand desiccation through constructing vertical burrows into the hyporheic zone under drought conditions might play a significant role in the success of particular crayfish species, as well as a wide range of further hyporheic-dwelling aquatic organisms in general.

The cell monolayer trajectory from the system state point of view.

Time-lapse microscopic movies are being increasingly utilized for understanding the derivation of cell states and predicting cell future. Often, fluorescence and other types of labeling are not available or desirable, and cell state-definitions based on observable structures must be used. We present the methodology for cell behavior recognition and prediction based on the short term cell recurrent behavior analysis. This approach has theoretical justification in non-linear dynamics theory. The methodology is based on the general stochastic systems theory which allows us to define the cell states, trajectory and the system itself. We introduce the usage of a novel image content descriptor based on information contribution (gain) by each image point for the cell state characterization as the first step. The linkage between the method and the general system theory is presented as a general frame for cell behavior interpretation. We also discuss extended cell description, system theory and methodology for future development. This methodology may be used for many practical purposes, ranging from advanced, medically relevant, precise cell culture diagnostics to very utilitarian cell recognition in a noisy or uneven image background. In addition, the results are theoretically justified.

Analysis of biological time-lapse microscopic experiment from the point of view of the information theory.

We report objective analysis of information in the microscopic image of the cell monolayer. The process of transfer of information about the cell by the microscope is analyzed in terms of the classical Shannon information transfer scheme. The information source is the biological object, the information transfer channel is the whole microscope including the camera chip. The destination is the model of biological system. The information contribution is analyzed as information carried by a point to overall information in the image. Subsequently we obtain information reflection of the biological object. This is transformed in the biological model which, in information terminology, is the destination. This, we propose, should be constructed as state transitions in individual cells modulated by information bonds between the cells. We show examples of detected cell states in multidimensional state space. This space is reflected as colour channel intensity phenomenological state space. We have also observed information bonds and show examples of them.

Analysis of biological time-lapse microscopic experiment from the point of view of the information theory.

We report objective analysis of information in the microscopic image of the cell monolayer. The process of transfer of information about the cell by the microscope is analyzed in terms of the classical Shannon information transfer scheme. The information source is the biological object, the information transfer channel is the whole microscope including the camera chip. The destination is the model of biological system. The information contribution is analyzed as information carried by a point to overall information in the image. Subsequently we obtain information reflection of the biological object. This is transformed in the biological model which, in information terminology, is the destination. This, we propose, should be constructed as state transitions in individual cells modulated by information bonds between the cells. We show examples of detected cell states in multidimensional state space reflected in space an colour channel intensity phenomenological state space. We have also observed information bonds and show examples of them.