Small Organisms Research Articles

3D manipulation of small multicellular organisms in soft microtubes

Small multicellular organisms, such as C. elegans and zebrafish larvae, are essential models in biological research, but their 3D manipulation poses challenges due to their small size. Manual positioning is labor-intensive and imprecise. To address this, we developed a soft PDMS microtube that can be gently stretched and released, immobilizing organisms without anesthetization and damage. This tube enables easy rotation and adjustment of orientation, facilitating comprehensive imaging. Functionality testing showed effective immobilization as well as precise imaging and microinjection. Zebrafish larvae were successfully injected with fluorescein in the hindbrain without anesthesia. This technique offers a simple, efficient, and non-damaging method for 3D manipulation and imaging of small multicellular organisms and provides a versatile tool for biological research practices.

An optimized whole-body corticosteroid hormones quantification method by LC-MS/MS for assessing stress levels in European glass eels (Anguilla anguilla)

The European eel (Anguilla anguilla) juvenile stage exhibits facultative estuarine migration. The causes of this behavior are yet unknown but it may have an impact on the population's fate by altering the sex ratio of the population. Recent studies have highlighted potential stress-related issues in glass eels settling in estuaries but studying stress response in small organisms requires sensitive, accurate and precise analytical methods. The aims of the present study are (i) to develop a whole-body Liquid Chromatography-Mass Spectrometry (LC-MS/MS) method for the simultaneous determination of several stress hormones in low-body mass fish; (ii) to apply this method to glass eels to study their responses to acute stress (iii) to test the effect of anxiolytics (diazepam) on these responses. Our results showed that enhanced LC-MS/MS analysis reduced detection limits and improved accuracy and precision for the quantification at the individual level. Following an acute stress, cortisol concentration significantly increased in glass eels and a 15 h diazepam exposure significantly reduced cortisol levels highlighting a marked anxiolytic effect on this species.

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.

Disease models of Leigh syndrome: From yeast to organoids.

Leigh syndrome (LS) is a severe mitochondrial disease that results from mutations in the nuclear or mitochondrial DNA that impairs cellular respiration and ATP production. Mutations in more than 100 genes have been demonstrated to cause LS. The disease most commonly affects brain development and function, resulting in cognitive and motor impairment. The underlying pathogenesis is challenging to ascertain due to the diverse range of symptoms exhibited by affected individuals and the variability in prognosis. To understand the disease mechanisms of different LS-causing mutations and to find a suitable treatment, several different model systems have been developed over the last 30 years. This review summarizes the established disease models of LS and their key findings. Smaller organisms such as yeast have been used to study the biochemical properties of causative mutations. Drosophila melanogaster, Danio rerio, and Caenorhabditis elegans have been used to dissect the pathophysiology of the neurological and motor symptoms of LS. Mammalian models, including the widely used Ndufs4 knockout mouse model of complex I deficiency, have been used to study the developmental, cognitive, and motor functions associated with the disease. Finally, cellular models of LS range from immortalized cell lines and trans-mitochondrial cybrids to more recent model systems such as patient-derived induced pluripotent stem cells (iPSCs). In particular, iPSCs now allow studying the effects of LS mutations in specialized human cells, including neurons, cardiomyocytes, and even three-dimensional organoids. These latter models open the possibility of developing high-throughput drug screens and personalized treatments based on defined disease characteristics captured in the context of a defined cell type. By analyzing all these different model systems, this review aims to provide an overview of past and present means to elucidate the complex pathology of LS. We conclude that each approach is valid for answering specific research questions regarding LS, and that their complementary use could be instrumental in finding treatment solutions for this severe and currently untreatable disease.

A concentric 3D-printed centrifuge rotor to study the effects of hypergravity on small organisms at three different but concurrent radial accelerations

For life to reach space, it must first escape Earth's gravity—hence exposure to hypergravity during launch, and interplanetary acceleration and deceleration. Long-term colonisation of space requires the establishment of sustainable, in situ, food production systems. Earthworms may be involved as a regolith/soil ameliorant, a source of protein, and for organic waste conversion. Earthworms, however, have hydrostatic skeletons that may be susceptible to hypergravity exposure. We investigated whether earthworms (Eisenia andrei) would survive prolonged centrifugal hypergravity using a table-top centrifuge. Our intention was to concurrently test three different hypergravity treatments (plus a 1 G control). Our concentric rotor concept, with appropriate design adaptations, allows concurrent testing of three different gravity treatments on small- and microorganisms, cell cultures, plants, microcosm, and chemical/biochemical reactions for extended periods of hours to months. Our concentric concept therefore is easily adaptable and may facilitate other terrestrial-based research on chemical, molecular, and organismal levels.•We designed a 3D-printed centrifuge rotor with three concentric rings, each ring with four separate 125 mL compartments.•This design was tested with an 8-day pilot run with four young worms per container at 1.7 G, 2.5 G, and 3.1 G, with a 1 G control.•All worms at all treatments gained body mass, the mean increases of which were almost indistinguishable between treatments, illustrating the utility of the design.

Degradation of Polymer Materials in the Environment and Its Impact on the Health of Experimental Animals: A Review

The extensive use of polymeric materials has resulted in significant environmental pollution, prompting the need for a deeper understanding of their degradation processes and impacts. This review provides a comprehensive analysis of the degradation of polymeric materials in the environment and their impact on the health of experimental animals. It identifies common polymers, delineates their degradation pathways, and describes the resulting products under different environmental conditions. The review covers physical, chemical, and biological degradation mechanisms, highlighting the complex interplay of factors influencing these processes. Furthermore, it examines the health implications of degradation products, using experimental animals as proxies for assessing potential risks to human health. By synthesizing current research, the review focuses on studies related to small organisms (primarily rodents and invertebrates, supplemented by fish and mollusks) to explore the effects of polymer materials on living organisms and underscores the urgency of developing and implementing effective polymer waste management strategies. These strategies are crucial for mitigating the adverse environmental and health impacts of polymer degradation, thus promoting a more sustainable interaction between human activities and the natural environment.

Visceral leishmaniasis complicated with hemophagocytic lymphohistiocytosis and resistant to amphotericin B: a case report

IntroductionHemophagocytic lymphohistiocytosis characterized by hemophagocytosis leading to uncontrolled inflammation; the most common etiology in secondary cases of hemophagocytic lymphohistiocytosis is viral infections, especially Epstein–Barr virus. Visceral leishmaniasis is a vectorborne protozoal disease caused by Leishmania donovani complex. It is common in tropical and subtropical regions, with 50,000–90,000 new cases annually.Case presentationA 15-month-old Arab female was admitted to our hospital with 15 days of fever and decreased weight. On clinical examination, she had a markedly enlarged liver and spleen that were palpable 4 cm and 6 cm below the costal margin, respectively. The peripheral blood smear showed hypochromic microcytic anemia, poikilocytosis, reactive lymphocytosis, and mild thrombocytopenia. Bone marrow aspiration did not show malignancy or any other pathological findings. The patient was put on antibiotic therapy without improvement. Repeated bone marrow aspiration showed erythrophagocytosis; intracellular small round organisms looked like the amastigote form of Leishmania (Donovan bodies) with no evidence of malignancies. Her lab values showed ferritin greater than 500 ug/L, pancytopenia, and hypertriglyceridemia. The patient was diagnosed with hemophagocytic lymphohistiocytosis secondary to visceral leishmaniasis.ConclusionHemophagocytic lymphohistiocytosis secondary to visceral leishmaniasis is an extensively rare phenomenon in the medical literature that causes challenges in diagnosis and management. Steroids should be used wisely to not cover the symptoms of infections or malignancy, and amphotericin B resistance should be kept in mind in unresponsive Leishmania cases.

Frontiers in artificial intelligence-directed light-sheet microscopy for uncovering biological phenomena and multi-organ imaging.

Light-sheet fluorescence microscopy (LSFM) introduces fast scanning of biological phenomena with deep photon penetration and minimal phototoxicity. This advancement represents a significant shift in 3-D imaging of large-scale biological tissues and 4-D (space + time) imaging of small live animals. The large data associated with LSFM requires efficient imaging acquisition and analysis with the use of artificial intelligence (AI)/machine learning (ML) algorithms. To this end, AI/ML-directed LSFM is an emerging area for multi-organ imaging and tumor diagnostics. This review will present the development of LSFM and highlight various LSFM configurations and designs for multi-scale imaging. Optical clearance techniques will be compared for effective reduction in light scattering and optimal deep-tissue imaging. This review will further depict a diverse range of research and translational applications, from small live organisms to multi-organ imaging to tumor diagnosis. In addition, this review will address AI/ML-directed imaging reconstruction, including the application of convolutional neural networks (CNNs) and generative adversarial networks (GANs). In summary, the advancements of LSFM have enabled effective and efficient post-imaging reconstruction and data analyses, underscoring LSFM's contribution to advancing fundamental and translational research.

Deciphering macrobenthic biological traits in response to long-term eutrophication in Xiangshan Bay, China

As an emerging global issue in coastal marine ecosystems, eutrophication may lead to profound ecological consequences or disasters. Six locations in Xiangshan Bay were sampled during 2012–2022 along the eutrophication gradient from the innermost bay with the most eutrophication to the outer bay with the least eutrophication. A trait-based method was adopted to explore the ecological effects of eutrophication on macrobenthic communities. The results showed that the community composition is mostly characterized by deposit feeders and predators with small (1–3 cm) and large (> 10 cm) body sizes, classified as indifferent and tolerant species (AMBI ecological groups), deposit feeders and predators (feeding mode), and a preference for a free living lifestyle. The RLQ and fourth-corner analyses further confirmed that there was a negative correlation between the abundance of small macrobenthic organisms (< 1 cm) and nitrate concentration. Phosphorus was a crucial influencing factor for macrobenthic spatial patterns and was strongly affected by the activities of deposit feeders and the decomposition of macrobenthos. Due to mass organic deposition resulting from increased primary production, long-term eutrophication had led to an increase in the proportion of detritus feeders. In addition, the significant negative correlation between the concentration of dissolved oxygen and first-order opportunistic species represented by the polychaete Capitella capitata indicated tolerance to hypoxia. The macrobenthic community in Xiangshan Bay had been negatively affected but maintains considerable stability in functional diversity and functional redundancy under the influence of long-term eutrophication.

Digital Microfluidics for Sample Preparation in Low-Input Proteomics.

Low-input proteomics, also referred to as micro- or nanoproteomics, has become increasingly popular as it allows one to elucidate molecular processes in rare biological materials. A major prerequisite for the analytics of minute protein amounts, e.g., derived from low cell numbers, down to single cells, is the availability of efficient sample preparation methods. Digital microfluidics (DMF), a technology allowing the handling and manipulation of low liquid volumes, has recently been shown to be a powerful and versatile tool to address the challenges in low-input proteomics. Here, an overview is provided on recent advances in proteomics sample preparation using DMF. In particular, the capability of DMF to isolate proteomes from cells and small model organisms, and to perform all necessary chemical sample preparation steps, such as protein denaturation and proteolytic digestion on-chip, are highlighted. Additionally, major prerequisites to making these steps compatible with follow-up analytical methods such as liquid chromatography-mass spectrometry will be discussed.

Customized digestion protocols for copepods, euphausiids, chaetognaths and fish larvae facilitate the isolation of ingested microplastics

Degradation of oceanic plastic waste leads to the formation of microplastics that are ingested by a wide range of animals. Yet, the amounts that are taken up, especially by small zooplankton, are largely unknown. This is mostly due to the complex methodology that is required for isolating ingested microplastics from organisms. We developed customised, effective and benign digestion protocols for four important zooplankton taxa (copepods, euphausiids, chaetognaths and fish larvae), and assessed their digestion efficacy and their potential to cause particle loss or to alter microplastics using six polymers (HDPE, LDPE, PS, PET, PVC, PMMA). All protocols are based on an incubation of the organic matrix with 10% KOH at 38 °C, which is optionally combined with digestive enzymes (chitinase, proteinase K). This yielded digestion efficacies of > 98.2%, recovery rates of > 91.8%, < 2.4% change in microplastics’ size, while no visual alteration of the microplastics and no changes in their spectra were observed when analysing them with a hyperspectral imaging camera. The proposed protocols are inexpensive (< 2.15 € per sample), but require several days when enzymatic digestion is included. They will facilitate research on microplastic ingestion by small marine organisms and thus enable well-founded conclusions about the threat that microplastics pose to these animals as well as about the role of biota in determining the vertical distribution of microplastics in oceanic environments.

Freshwater Ecosystems: Carbon Sequestration Champions

Nature helps us fight climate change by removing carbon from the atmosphere and storing it underground—a process called carbon sequestration. Some freshwater ecosystems, like lakes, are great at carbon sequestration. Surprisingly, small organisms play a big role here! When tiny algae and bacteria in the water photosynthesize, they take carbon dioxide from the atmosphere and convert it to organic carbon (the carbon that forms all living organisms). The organic carbon moves through the ecosystem as it is eaten by other organisms or as it sinks to the bottom as either poop or the remains of dead organisms. When organic carbon reaches the bottom, it can remain buried for a long time. Aquatic plants and material from land that washes into freshwater can also sink and end up as sequestered carbon. It seems like freshwater ecosystems sequester carbon more efficiently than other environments, but scientists still have more work to do. Because of the huge diversity of water bodies across the world, scientists need to know which are the real carbon sequestration champions.

Machine learning-based bee recognition and tracking for advancing insect behavior research

The study of insect behavior, particularly that of honey bees, has a broad scope and significance. Tracking bee flying patterns grants much helpful information about bee behavior. However, tracking a small yet fast-moving object, such as a bee, is difficult. Hence, we present artificial intelligence, machine-learning-based bee recognition, and tracking systems to assist the researcher in studying the bee’s behavior. To develop a machine learning system, a labeled database is required for model training. To address this, we implemented an automated system for analyzing and labeling bee videos. This labeled database served as the foundation for two distinct bee-tracking solutions. The first solution (planar bee tracking system) tracked individual bees in closed mazes using a neural network. The second solution (spatial bee tracking system) utilized a neural network and a tracking algorithm to identify and track flying bees in open environments. Both systems tackle the challenge of tracking small-bodied creatures with rapid and diverse movement patterns. Although we applied these systems to entomological cognition research in this paper, their relevance extends to general insect research and developing tracking solutions for small organisms with swift movements. We present the complete architecture and detailed methodologies to facilitate the utilization of these models in future research endeavors. Our approach is a simple and inexpensive method that contributes to the growing number of image-analysis tools used for tracking animal movement, with future potential applications under less sterile field conditions. The tools presented in this paper could assist the study of movement ecology, specifically in insects, by providing accurate movement specifications. Following the movement of pollinators or natural enemies, for example, greatly contributes to the study of pollination or biological control, respectively, in natural and agro-ecosystems.

Ecological succession shapes size–density scaling relationships of trees and soil invertebrates

Abstract The widely observed negative scaling relationship between organism size and abundance is predicted to have a universal −0.75 scaling exponent across all life forms. However, factors influencing frequently observed deviations from this exponent, such as ecosystem succession and organism traits, remain poorly understood. We explore the dependence of size–density scaling on ecosystem succession and organism traits by analysing size–density relationships in trees and soil invertebrates across 183 temperate forest plots comprising urban secondary forests, urban old‐growth forests and non‐urban natural forests. Exponents of scaling relationships in urban tree and invertebrate communities progressively steepened with increasing restored (planted) forest stand age as small organisms increased in abundance. In contrast, non‐urban tree scaling relationships flattened during succession with exponents veering away from −0.75, whereas urban tree and invertebrate communities converged towards this prediction in later successional stages. Our results shed light on how the body size structure of tree and soil invertebrate communities spanning multiple trophic levels shift over successional time as the relative abundances of large versus small‐bodied organisms increase. This study emphasises the fundamental influence of organismal traits and ecosystem succession on scaling relationships of organism body size and abundance. Read the free Plain Language Summary for this article on the Journal blog.

Combinations of bacterial probiotics and yeast postbiotics influence fat deposition and growth in the nematode C. elegans

BackgroundProbiotics are live microorganisms with intended benefits on human health including obesity. As a small and fast-growing whole organism model, Caenorhabditis elegans has been used to assess the health effects of probiotics where mechanisms can be assessed through available genetic tools. Results from C. elegans can provide data on the effect of specific probiotic strains and combinations with prebiotics and postbiotics on health-related physiology to inform selections of interventions for further study. We hypothesized that specific combinations with prebiotics and postbiotics could both speed up worm development and reduce fat deposition, suggesting they allow for more effective nutrient utilization. MethodsHere we expose C. elegans to the ABB S20 strain of Lactiplantibacillus plantarum in combination with different prebiotics and postbiotics. We then measure how these affect growth and development speed as well as fat deposition by measuring the time until the appearance of progeny and measuring Oil Red O staining respectively. ResultsOur results show that the combination of probiotic L. plantarum ABB S20 plus the postbiotic inactive yeasts K. marxianus ABB S8 and S. boulardii ABB S3 resulted in fast growth and reduced fat deposition compared to L. plantarum ABB S20 alone. ConclusionThese results demonstrate the usefulness of C. elegans as a model to efficiently screen though combinations of probiotics, prebiotics and postbiotics to find those that are candidates to help with effective nutrition use and therefore weight management.

Experience-dependent modulation of collective behavior in larval zebrafish.

Complex group behavior can emerge from simple inter-individual interactions. Commonly, these interactions are considered static and hardwired and little is known about how experience and learning affect collective group behavior. Young larvae use well described visuomotor transformations to guide interindividual interactions and collective group structure. Here, we use naturalistic and virtual-reality (VR) experiments to impose persistent changes in population density and measure their effects on future visually evoked turning behavior and the resulting changes in group structure. We find that neighbor distances decrease after exposure to higher population densities, and increase after the experience of lower densities. These adaptations develop slowly and gradually, over tens of minutes and remain stable over many hours. Mechanistically, we find that larvae estimate their current group density by tracking the frequency of neighbor-evoked looming events on the retina and couple the strength of their future interactions to that estimate. A time-varying state-space model that modulates agents' social interactions based on their previous visual-social experiences, accurately describes our behavioral observations and predicts novel aspects of behavior. These findings provide concrete evidence that inter-individual interactions are not static, but rather continuously evolve based on past experience and current environmental demands. The underlying neurobiological mechanisms of experience dependent modulation can now be explored in this small and transparent model organism.

Impacts on Aquatic Life for Indiscriminate Exploitation of Baby Shrimp (P. monodon) in the Coastal Area, South-Western Region of Bangladesh

Fishing for baby shrimp (Penaeus monodon post-larvae) is a major source of income for most fishermen in the coastal regions of the southwestern part of Bangladesh during during July, 2022 to June, 2023. These post-larvae collectors lack financial security and are socially regressive. Other aquatic species are destroyed by the indiscriminate exploitation of baby shrimp for aquaculture. According to the present study, about 98-99% of other larvae are destroyed to catch nearly 1-2% of indiscriminate exploitation baby shrimp in our study area where averagely 0.35% Penaeus indicus, 0.06% Penaeus merguiensis, 0.037% Metapenaeus Monoceros, 3.58% Other shrimp, 6.59% Fin-fish larvae, 5.95% Crab larvae, and 82.42% Small organisms. Actually, the government of Bangladesh already has declared a ban period and illegal baby shrimp catching. But to maintain the poor fisherman's livelihood, they are doing that continuously. Furthermore, because wild baby shrimp are said to have a far higher survival rate than baby shrimp raised in hatcheries, farmers prefer wild baby shrimp. For thousands of Bangladeshi coastal landless and jobless poor people, wild prawn baby shrimp fishing has created employment opportunities. This study describes the impact of wild baby shrimp fishing in coastal areas of Bangladesh.

Development of a Simple Cost Effective Oxygenation System for In Vivo Solution State NMR in 10 mm NMR Tubes.

In vivo NMR is evolving into an important tool to understand biological processes and environmental responses. Current approaches use flow systems to sustain the organisms with oxygenated water and food (e.g., algae) inside the NMR. However, such systems have the potential to leak and clog (potentially damaging costly hardware), require large volumes of media, and multiple expensive HPLC pumps. The proposed "oxygenation system", uses a simple "double slit" adapter and a single air/oxygen flow line into the NMR. The design is especially suited to larger diameter probes given that standard flow systems would require higher flow rates thus amplifying the potential and impact of leaks/clogs. Traditionally, in vivo NMR of small organisms (e.g., Daphnia) have required 2D NMR in combination with 13C enrichment to overcome susceptibility distortions and provide information rich metabolic profiles. Here Daphnia magna, Eisenia fetida and Artemia franciscana are used to demonstrate the potential of the oxygenation system. Survivability tests and 1H time-resolved monitoring were first performed on D. magna, while E. fetida contained enough biomass to permit 1H-13C HSQC, 13C-1H HETCOR and 31P NMR without isotopic enrichment. Finally, STOCSY of 1D 13C NMR was used to follow the growth of A. franciscana (without 13C enrichment) for 48 h after birth, which helps visualize trends across a series of 1D in vivo data. In summary, application of the oxygenation system toward larger diameter probes allows the collection of NMR data without enrichment, offering a promising solution to better understand processes in vivo.

Low-input PacBio sequencing generates high-quality individual fly genomes and characterizes mutational processes

Long-read sequencing, exemplified by PacBio, revolutionizes genomics, overcoming challenges like repetitive sequences. However, the high DNA requirement ( > 1 µg) is prohibitive for small organisms. We develop a low-input (100 ng), low-cost, and amplification-free library-generation method for PacBio sequencing (LILAP) using Tn5-based tagmentation and DNA circularization within one tube. We test LILAP with two Drosophila melanogaster individuals, and generate near-complete genomes, surpassing preexisting single-fly genomes. By analyzing variations in these two genomes, we characterize mutational processes: complex transpositions (transposon insertions together with extra duplications and/or deletions) prefer regions characterized by non-B DNA structures, and gene conversion of transposons occurs on both DNA and RNA levels. Concurrently, we generate two complete assemblies for the endosymbiotic bacterium Wolbachia in these flies and similarly detect transposon conversion. Thus, LILAP promises a broad PacBio sequencing adoption for not only mutational studies of flies and their symbionts but also explorations of other small organisms or precious samples.

Fly Me to the Micron: Microtechnologies for Drosophila Research.

Multicellular model organisms, such as Drosophila melanogaster (fruit fly), are frequently used in a myriad of biological research studies due to their biological significance and global standardization. However, traditional tools used in these studies generally require manual handling, subjective phenotyping, and bulk treatment of the organisms, resulting in laborious experimental protocols with limited accuracy. Advancements in microtechnology over the course of the last two decades have allowed researchers to develop automated, high-throughput, and multifunctional experimental tools that enable novel experimental paradigms that would not be possible otherwise. We discuss recent advances in microtechnological systems developed for small model organisms using D. melanogaster as an example. We critically analyze the state of the field by comparing the systems produced for different applications. Additionally, we suggest design guidelines, operational tips, and new research directions based on the technical and knowledge gaps in the literature. This review aims to foster interdisciplinary work by helping engineers to familiarize themselves with model organisms while presenting the most recent advances in microengineering strategies to biologists.