Small Aquatic Organisms Research Articles

YOLO-GE: An Attention Fusion Enhanced Underwater Object Detection Algorithm

Underwater object detection is a challenging task with profound implications for fields such as aquaculture, marine ecological protection, and maritime rescue operations. The presence of numerous small aquatic organisms in the underwater environment often leads to issues of missed detections and false positives. Additionally, factors such as the water quality result in weak target features, which adversely affect the extraction of target feature information. Furthermore, the lack of illumination underwater causes image blur and low contrast, thereby increasing the difficulty of the detection task. To address these issues, we propose a novel underwater object detection algorithm called YOLO-GE (GCNet-EMA). First, we introduce an image enhancement module to mitigate the impact of underwater image quality issues on the detection task. Second, a high-resolution feature layer is added into the network to improve the problems of missed detections and false positives for small targets. Third, we propose GEBlock, an attention-based fusion module that captures long-range contextual information and suppresses noise from lower-level feature layers. Finally, we combine an adaptive spatial fusion module with the detection head to filter out conflicting feature information from different feature layers. Experiments on the UTDAC2020, DUO and RUOD datasets show that the proposed method achieves an optimal detection accuracy.

Metformin as an Emerging Pollutant in the Aquatic Environment: Occurrence, Analysis, and Toxicity.

The use of human and veterinary drugs has led to the accumulation of pharmaceuticals in various aquatic environments at progressively increasing levels, exhibiting strong ecological risks. Metformin is widely used as a first-line prescription drug for the treatment of type 2 diabetes mellitus as well as a livestock drug. Unlike other drugs, metformin is not metabolized in the body, and almost all of its intake is excreted and released into the aquatic environment via urine and feces, causing adverse effects on aquatic ecosystems. This review provides an overview of the occurrence and detection of metformin in the aquatic environment and its toxic effects on different aquatic organisms (fish, daphnia, rotifers, chlorella). Metformin has been documented in a variety of aqueous environments such as wastewater, surface water, and groundwater as well as drinking water. The wide distribution of metformin in the aqueous environment calls for the development of more accurate detection methods. This paper reviews detection methods for metformin in the aqueous environment and evaluates their advantages and disadvantages. Toxicity studies have shown that metformin can cause adverse reactions in fish, such as oxidative stress, genotoxicity, disruption of intestinal flora, and morphological alterations; it also affects the growth and reproduction of small aquatic organisms. Knowledge gaps in the field of metformin research were assessed, and future research priorities were identified.

Orthogonal-view microscope for the biomechanics investigations of aquatic organisms

Microscopes are essential for the biomechanical and hydrodynamical investigation of small aquatic organisms. We report a prototype of a do-it-yourself microscope that enables the visualization of organisms from two orthogonal imaging planes — top and side views. Compared to conventional imaging systems, this approach provides a comprehensive visualization strategy of organisms, which could have complex shapes and morphologies. The microscope was constructed by combining custom 3D-printed parts and off-the-shelf components. The system is designed for modularity and reconfigurability. Open-source design files and build instructions are provided in this report. Additionally, proof-of-use experiments (particularly with Hydra) and other organisms that combine the imaging with an analysis pipeline were demonstrated to highlight the system’s utility. Beyond the applications demonstrated, the system can be used or modified for various imaging applications.

Methods: A bioinformatic protocol for rapid analysis of zebrafish embryo photo-motory responses (PMR) in neurotoxicity testing

Chemobehavioural phenotyping presents unique opportunities for analyzing neurotoxicants and discovering behavior-modifying neuroceuticals in small aquatic model organisms such as zebrafish (Danio rerio). A recently popularized approach in this field involves the utilization of zebrafish embryos for a photo-motor response (PMR) bioassay. The PMR bioassay entails stimulating zebrafish embryos between 24 and 36 h post fertilization (hpf) with a high-intensity light stimulus, inducing a transient increase in the frequency of photo-induced embryo body flexions. These flexions can be computationally analyzed to derive behavioral signatures, enabling the categorization of neuromodulating chemicals.Despite the significant advantages of the PMR bioassay, its widespread implementation is hindered by lack of well described and straightforward high-throughput bioinformatic analysis of behavioral data. In this methods article, we present an easily implementable bioinformatics protocol specifically designed for rapid behavioral analysis of large cohorts of zebrafish specimens in PMR bioassays. We also address common pitfalls encountered during PMR analysis, discuss its limitations, and propose future directions for developing next-generation biometric analysis techniques in chemobehavioural assays utilizing zebrafish embryos.

Toward Real-Time Animal Tracking with Integrated Stimulus Control for Automated Conditioning in Aquatic Eco-Neurotoxicology.

Aquatic eco-neurotoxicology is an emerging field that requires new analytical systems to study the effects of pollutants on animal behaviors. This is especially true if we are to gain insights into one of the least studied aspects: the potential perturbations that neurotoxicants can have on cognitive behaviors. The paucity of experimental data is partly caused by a lack of low-cost technologies for the analysis of higher-level neurological functions (e.g., associative learning) in small aquatic organisms. Here, we present a proof-of-concept prototype that utilizes a new real-time animal tracking software for on-the-fly video analysis and closed-loop, external hardware communications to deliver stimuli based on specific behaviors in aquatic organisms, spanning three animal phyla: chordates (fish, frog), platyhelminthes (flatworm), and arthropods (crustacean). The system's open-source software features an intuitive graphical user interface and advanced adaptive threshold-based image segmentation for precise animal detection. We demonstrate the precision of animal tracking across multiple aquatic species with varying modes of locomotion. The presented technology interfaces easily with low-cost and open-source hardware such as the Arduino microcontroller family for closed-loop stimuli control. The new system has potential future applications in eco-neurotoxicology, where it could enable new opportunities for cognitive research in diverse small aquatic model organisms.

Numerical analysis of predatory potentiality of Toxorhynchites splendens against larval Aedes albopictus in laboratory and semi-field conditions

Larvae of the elephant mosquitoes, Toxorhynchites spp. (Diptera: Culicidae) are predacious on larvae of other mosquito species and some small aquatic organisms; this predatory behavior can be applied in (mosquito) vector control. The present study examined the feeding behavior of Toxorhynchites splendens on Aedes albopictus in relation to search area [volume of water (X1)] and prey density (X2), prey instars, predatory preference and larvae's functional response on variable prey densities. Experiments were conducted to determine changes in the feeding activity of T. splendens with different search areas and showed that rate of prey consumption was inversely proportional to the search area as evidenced by a negative value of X1 in the regression equation and positively related to prey density. The non-linear polynomial logistic regression estimated a significant linear parameter (P1 < 0) for the functional response analysis suggesting a Type II functional response. Differences in feeding response related to the different combinations of prey instars were statistically not significant (p > 0.05), expressing that all the instars of prey were equally susceptible to the predator. Toxorhynchites splendens preferred to consume Ae. albopictus larvae rather than Tubifex when supplied together as a food source.

Assessment of a new olfactometer for study of sensory ecology in small aquatic organisms

AbstractAll aquatic organisms rely on chemical stimuli at some point during their life cycles. These stimuli are typically associated with essential functions such as food‐, mate‐, or habitat‐finding, or predator avoidance. Historically, research on sensory ecology and behavior of aquatic organisms has been conducted using two‐channel choice flumes, which have many limitations when utilized on small organisms. Here, we describe functional features and results of pilot tests of an aquatic olfactometer, adapted from olfactometers used in studies on terrestrial arthropods, which eliminates these limitations. The initial implementation of this olfactometer is described. The structural integrity, hydrodynamics, and basic function of the olfactometer were tested and verified. The ability to clearly record the movements of a small organism within was also tested. Our experiments verify that the flow rate of water within the choice arena was accurate, water behaved as theorized in prior modeling, and chemical concentrations within each “zone” were distributed as expected based on computer simulations. We further show that video data could be obtained and analyzed using Ethovision XT to provide large amounts of raw data, including position, velocity, and acceleration, which can be used to investigate many potential questions associated with behavior. This olfactometer has the potential to become the new standard for research on chemotaxis and chemical ecology of small aquatic organisms.

Evaluating Dermal Bone as a Novel Source of Endocrine Information in Ninespine and Threespine Stickleback Fish.

Monitoring the physiology of small aquatic and marine teleost fish presents challenges. Blood samples, often the first choice for endocrinologists, can be difficult or even impossible to obtain and alternative matrices currently used for hormone analyses do not occur in fishes (e.g., hair, feathers etc.) or are not easily collected from small aquatic organisms (e.g., urine and feces). Some teleosts, however, have enlarged bony dermal elements that possibly accumulate and store steroid hormones in physiological relevant concentrations. Both threespine stickleback (Gasterosteus aculeatus) and ninespine stickleback (Pungitius pungitius) have a series of external, lateral bony plates, dorsal spines, and a pair of pelvic spines attached to the pelvic girdle. We investigated if cortisol, the primary circulating glucocorticoid in teleosts, could be extracted from stickleback dermal bone and quantified using a commercially available enzyme immunoassay (EIA). We successfully validated a cortisol EIA for dermal bone extracts, determined that cortisol was detectable in both species, and found that dermal bone cortisol levels significantly correlated with cortisol levels in whole body homogenate. Ninespine stickleback had significantly higher dermal bone cortisol concentrations than threespine stickleback and female threespine stickleback tended to have over twice the mean dermal bone cortisol concentration than males. Because both stickleback species are widely used for ecotoxicological studies, using dermal bone as a source of endocrine information, while leaving the body for contaminant, genomic, histological, and stable isotope analyses, could be a powerful and parsimonious tool. Further investigation and physiological validations are necessary to fully understand the utility of this new sample matrix.

In situ size and motility measurement of aquatic invertebrates with an underwater stereoscopic camera system using tilted lenses

Abstract In situ observation of traits of aquatic organisms, including size and motility, requires three‐dimensional measurements that are commonly done with a stereoscopic imaging system. However, to observe traits of small aquatic invertebrates, the imaging system requires relatively high magnification, which results in a small overlapping volume between the two cameras of a conventional stereoscopic system. The provision of a larger shared volume would therefore be of great advantage, especially, when the organism abundance is low. We implement a stereoscopic system that utilizes a tilted lens approach, known as the Scheimpflug principle, to increase the common imaging volume of two cameras. The system was calibrated and tested in the laboratory and then deployed in a saltmarsh to observe water boatmen Trichocorixa californica. Processing of the image data from the field deployments resulted in the simultaneous estimation of the traits of body length and swimming speed of the aquatic insects. Our stereo setup with tilted lenses increased the sampling volume by 3.1 times compared to a traditional stereo setup with the same optical parameters. The in situ data and subsequent processing reveal that the instrument can capture stereoscopic images that resolve both body length and swimming speed of the aquatic insects. Results indicate that the relationship between the body length and the swimming speed of the water boatmen is linear in the log–log space with an exponent of . Furthermore, the insects experience Reynold's number in the range of –. Our results demonstrated that the system can be used to observe key traits of small aquatic organisms in an ecologically relevant context. This work expands the capability of underwater imaging systems to measure important traits of an individual aquatic invertebrate in its natural environment and aids in providing a trait‐based approach to zooplankton ecology.

Impact of test chamber design on spontaneous behavioral responses of model crustacean zooplankton Artemia franciscana.

The use of small aquatic model organisms to investigate the behavioral effects of chemical exposure is becoming an integral component of aquatic ecotoxicology research and neuroactive drug discovery. Despite the increasing use of invertebrates for behavioral phenotyping in toxicological studies and chemical risk assessments, little is known regarding the potential for environmental factors-such as geometry, size, opacity and depth of test chambers-to modulate common behavioral responses. In this work, we demonstrate that test chamber geometry, size, opacity and depth can affect spontaneous, unstimulated behavioral responses of euryhaline crustacean Artemia franciscana first instar larval stages. We found that in the absence of any obvious directional cues, A. franciscana exhibited a strong innate wall preference behavior. Using different test chamber sizes and geometries, we found both increased wall preference and lowered overall distance traveled by the test shrimp in a smaller chamber with sharper-angled vertices. It was also determined through quantifiable changes in the chambers' color that the A. franciscana early larval stages can perceive, differentiate and react to differences in color or perhaps rather to light transmittance of the test chambers. The interaction between innate edge preference and positive phototaxis could be consistently altered with a novel photic stimulus system. We also observed a strong initial preference for depth in A. franciscana first instar larval stages, which diminished through the acclimatization. We postulate that the impact of test chamber designs on neurobehavioral baseline responses warrants further investigation, in particular considering the increased interest in behavioral eco-neurotoxicology applications.

Comprehensive Multiphase NMR Probehead with Reduced Radiofrequency Heating Improves the Analysis of Living Organisms and Heat-Sensitive Samples

Comprehensive multiphase (CMP) NMR, first described in 2012, combines all of the hardware components necessary to analyze all phases (solid, gel, and solution) in samples in their natural state. In combination with spectral editing experiments, it can fully differentiate phases and study the transfer of chemical species across and between phases, providing unprecedented molecular-level information in unaltered natural systems. However, many natural samples, such as swollen soils, plants, and small organisms, contain water, salts, and ionic compounds, making them electrically lossy and susceptible to RF heating, especially when using high-strength RF fields required to select the solid domains. While dedicated reduced-heating probes have been developed for solid-state NMR, to date, all CMP-NMR probes have been based on solenoid designs, which can lead to problematic sample heating. Here, a new prototype CMP probe was developed, incorporating a loop gap resonator (LGR) for decoupling. Temperature increases are monitored in salt solutions analogous to those in small aquatic organisms and then tested in vivo on Hyalella azteca (freshwater shrimp). In the standard CMP probe (solenoid), 80% of organisms died within 4 h under high-power decoupling, while in the LGR design, all organisms survived the entire test period of 12 h. The LGR design reduced heating by a factor of ∼3, which allowed 100 kHz decoupling to be applied to salty samples with generally ≤10 °C sample heating. In addition to expanding the potential for in vivo research, the ability to apply uncompromised high-power decoupling could be beneficial for multiphase samples containing true crystalline solids that require the strongest possible decoupling fields for optimal detection.

A new approach to assessing the space use behavior of macroinvertebrates by automated video tracking.

Individual space and resource use are central issues in ecology and conservation. Recent technological advances such as automated tracking techniques are boosting ecological research in this field. However, the development of a robust method to track space and resource use is still challenging for at least one important ecosystem component: motile aquatic macroinvertebrates. The challenges are mostly related to the small body size and rapid movement of many macroinvertebrate species and to light scattering and wave signal interference in aquatic habitats.We developed a video tracking method designed to reliably assess space use behavior among individual aquatic macroinvertebrates under laboratory (microcosm) conditions. The approach involves the use of experimental apparatus integrating a near infrared backlight source, a Plexiglas multi‐patch maze, multiple infrared cameras, and automated video analysis. It allows detection of the position of fast‐moving (~ 3 cm/s) and translucent individuals of small size (~ 5 mm in length, ~1 mg in dry weight) on simulated resource patches distributed over an experimental microcosm (0.08 m2).To illustrate the adequacy of the proposed method, we present a case study regarding the size dependency of space use behavior in the model organism Gammarus insensibilis, focusing on individual patch selection, giving‐up times, and cumulative space used.In the case study, primary data were collected on individual body size and individual locomotory behavior, for example, mean speed, acceleration, and step length. Individual entrance and departure times were recorded for each simulated resource patch in the experimental maze. Individual giving‐up times were found to be characterized by negative size dependency, with patch departure occurring sooner in larger individuals than smaller ones, and individual cumulative space used (treated as the overall surface area of resource patches that individuals visited) was found to scale positively with body size.This approach to studying space use behavior can deepen our understanding of species coexistence, yielding insights into mechanistic models on larger spatial scales, for example, home range, with implications for ecological and evolutionary processes, as well as for the management and conservation of populations and ecosystems. Despite being specifically developed for aquatic macroinvertebrates, this method can also be applied to other small aquatic organisms such as juvenile fish and amphibians.

Revealing and Revelling in the Floods on Country: Memory Poles within Toonooba

Revealing and Revelling in the Floods on Country: Memory Poles within Toonooba

Assessing urban microplastic pollution in a benthic habitat of Patagonia Argentina

Plastic pollution in the oceans has become a global problem, but its documentation is disparate around the world. We assess the abundance and type of microplastics in three benthic matrices: mussels, small fishes, and bottom water; in three sites nearby Puerto Madryn city (Patagonia, Argentina). Microplastics were present in the three matrices for all sites sampled. The average amounts of items observed were 1.6 and 0.3 per total wet weight in fishes and mussels, respectively, and 10.5 per liter in bottom water. Mussels and fishes presented a difference of microplastics size comparing with the surrounding bottoms waters; fishes also presented color discrimination, suggesting the necessity of more than one bioindicator to perform microplastic pollution monitoring. Moreover, small fishes had more MPs in their gastrointestinal tracts than bigger ones. The present study is the first one about the interaction between MPs and small aquatic organisms in coastal marine environments from Patagonia.

Invasion syndromes: a systematic approach for predicting biological invasions and facilitating effective management

Our ability to predict invasions has been hindered by the seemingly idiosyncratic context-dependency of individual invasions. However, we argue that robust and useful generalisations in invasion science can be made by considering “invasion syndromes” which we define as “a combination of pathways, alien species traits, and characteristics of the recipient ecosystem which collectively result in predictable dynamics and impacts, and that can be managed effectively using specific policy and management actions”. We describe this approach and outline examples that highlight its utility, including: cacti with clonal fragmentation in arid ecosystems; small aquatic organisms introduced through ballast water in harbours; large ranid frogs with frequent secondary transfers; piscivorous freshwater fishes in connected aquatic ecosystems; plant invasions in high-elevation areas; tall-statured grasses; and tree-feeding insects in forests with suitable hosts. We propose a systematic method for identifying and delimiting invasion syndromes. We argue that invasion syndromes can account for the context-dependency of biological invasions while incorporating insights from comparative studies. Adopting this approach will help to structure thinking, identify transferrable risk assessment and management lessons, and highlight similarities among events that were previously considered disparate invasion phenomena.

Impact of digital video analytics on accuracy of chemobehavioural phenotyping in aquatic toxicology.

Chemobehavioural phenotypic analysis using small aquatic model organisms is becoming an important toolbox in aquatic ecotoxicology and neuroactive drug discovery. The analysis of the organisms’ behavior is usually performed by combining digital video recording with animal tracking software. This software detects the organisms in the video frames, and reconstructs their movement trajectory using image processing algorithms. In this work we investigated the impact of video file characteristics, video optimization techniques and differences in animal tracking algorithms on the accuracy of quantitative neurobehavioural endpoints. We employed larval stages of a free-swimming euryhaline crustacean Artemia franciscana,commonly used for marine ecotoxicity testing, as a proxy modelto assess the effects of video analytics on quantitative behavioural parameters. We evaluated parameters such as data processing speed, tracking precision, capability to perform high-throughput batch processing of video files. Using a model toxicant the software algorithms were also finally benchmarked against one another. Our data indicates that variability in video file parameters; such as resolution, frame rate, file containers types, codecs and compression levels, can be a source of experimental biases in behavioural analysis. Similarly, the variability in data outputs between different tracking algorithms should be taken into account when designing standardized behavioral experiments and conducting chemobehavioural phenotyping.

Flow-based in vivo NMR spectroscopy of small aquatic organisms.

NMR applied to living organisms is arguably the ultimate tool for understanding environmental stress responses and can provide desperately needed information on toxic mechanisms, synergistic effects, sublethal impacts, recovery, and biotransformation of xenobiotics. To perform in vivo NMR spectroscopy, a flow cell system is required to deliver oxygen and food to the organisms while maintaining optimal line shape for NMR spectroscopy. In this tutorial, two such flow cell systems and their constructions are discussed: (a) a single pump high-volume flow cell design is simple to build and ideal for organisms that do not require feeding (i.e., eggs) and (b) a more advanced low-volume double pump flow cell design that permits feeding, maintains optimal water height for water suppression, improves locking and shimming, and uses only a small recirculating volume, thus reducing the amount of xenobiotic required for testing. In addition, key experimental aspects including isotopic enrichment, water suppression, and 2D experiments for both 13 C enriched and natural abundance organisms are discussed.

Selective Amino Acid-Only in Vivo NMR: A Powerful Tool To Follow Stress Processes.

In vivo NMR of small 13C-enriched aquatic organisms is developing as a powerful tool to detect and explain toxic stress at the biochemical level. Amino acids are a very important category of metabolites for stress detection as they are involved in the vast majority of stress response pathways. As such, they are a useful proxy for stress detection in general, which could then be a trigger for more in-depth analysis of the metabolome. 1H–13C heteronuclear single quantum coherence (HSQC) is commonly used to provide additional spectral dispersion in vivo and permit metabolite assignment. While some amino acids can be assigned from HSQC, spectral overlap makes monitoring them in vivo challenging. Here, an experiment typically used to study protein structures is adapted for the selective detection of amino acids inside living Daphnia magna (water fleas). All 20 common amino acids can be selectively detected in both extracts and in vivo. By monitoring bisphenol-A exposure, the in vivo amino acid-only approach identified larger fluxes in a greater number of amino acids when compared to published works using extracts from whole organism homogenates. This suggests that amino acid-only NMR of living organisms may be a very sensitive tool in the detection of stress in vivo and is highly complementary to more traditional metabolomics-based methods. The ability of selective NMR experiments to help researchers to “look inside” living organisms and only detect specific molecules of interest is quite profound and paves the way for the future development of additional targeted experiments for in vivo research and monitoring.

A review of fish identification methods applied on small fish

AbstractMost of the research on marking of aquatic organisms has been done using large organisms since they tend to have better retention rates, their swimming behaviour is less affected by the size of the tag, they have less trauma due to injectable tags or from the injector itself and they are easier to handle and able to withstand the stress of tagging and recapture. The development of small tags has grown from a necessity to study smaller fish species, earlier life stages of fish and to study other small aquatic organisms. As an endangered species, delta smelt (Hypomesus transpacificus) are being intensively studied in the upper San Francisco Estuary (CA, USA). A crucial part of many of these studies is tracking delta smelt, which requires a distinctive mark on each individual or group of fish. Unfortunately, many of the experimental fish suffer inadvertent mortality, as delta smelt are very sensitive to environmental and handling stress. The purpose of this review was to synthesize the different tagging and marking studies on small fish and aquatic organisms and to evaluate the advantages and disadvantages of each method for applications on sensitive species such as delta smelt. This review suggests the most effective methods for tracking delta smelt are visible implant alphanumeric (VIA) tagging and adipose fin clips for subadult to adult fish and calcein marks for young life stages. Among these methods, only VIA tagging currently provides individual organism identification.

Quantification of Cutaneous Ionocytes in Small Aquatic Organisms.

Aquatic organisms have specialized cells called ionocytes that regulate the ionic composition, osmolarity, and acid/base status of internal fluids. In small aquatic organisms such as fishes in their early life stages, ionocytes are typically found on the cutaneous surface and their abundance can change to help cope with various metabolic and environmental factors. Ionocytes profusely express ATPase enzymes, most notably Na+/K+ ATPase, which can be identified by immunohistochemistry. However, quantification of cutaneous ionocytes is not trivial due to the limited camera's focal plane and the microscope's field-of-view. This protocol describes a technique to consistently and reliably identify, image, and measure the relative surface area covered by cutaneous ionocytes through software-mediated focus-stacking and photo-stitching-thereby allowing the quantification of cutaneous ionocyte area as a proxy for ion transporting capacity across the skin. Because ionocytes are essential for regulating ionic composition, osmolarity, and acid/base status of internal fluids, this technique is useful for studying physiological mechanisms used by fish larvae and other small aquatic organisms during development and in response to environmental stress.