Small Multicellular 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.

Microswimmers learning chemotaxis with genetic algorithms

Various microorganisms and some mammalian cells are able to swim in viscous fluids by performing nonreciprocal body deformations, such as rotating attached flagella or by distorting their entire body. In order to perform chemotaxis (i.e., to move toward and to stay at high concentrations of nutrients), they adapt their swimming gaits in a nontrivial manner. Here, we propose a computational model, which features autonomous shape adaptation of microswimmers moving in one dimension toward high field concentrations. As an internal decision-making machinery, we use artificial neural networks, which control the motion of the microswimmer. We present two methods to measure chemical gradients, spatial and temporal sensing, as known for swimming mammalian cells and bacteria, respectively. Using the genetic algorithm NeuroEvolution of Augmenting Topologies, surprisingly simple neural networks evolve. These networks control the shape deformations of the microswimmers and allow them to navigate in static and complex time-dependent chemical environments. By introducing noisy signal transmission in the neural network, the well-known biased run-and-tumble motion emerges. Our work demonstrates that the evolution of a simple and interpretable internal decision-making machinery coupled to the environment allows navigation in diverse chemical landscapes. These findings are of relevance for intracellular biochemical sensing mechanisms of single cells or for the simple nervous system of small multicellular organisms such as Caenorhabditis elegans.

Contingent interactions among biofilm‐forming bacteria determine preservation or decay in the first steps toward fossilization of marine embryos

Fossils of soft tissues provide important records of early animals and embryos, and there is substantial evidence for a role for microbes in soft tissue fossilization. We are investigating the initial events in interactions of bacteria with freshly dead tissue, using marine embryos as a model system. We previously found that microbial invasion can stabilize embryo tissue that would otherwise disintegrate in hours or days by generating a bacterial pseudomorph, a three dimensional biofilm that both replaces the tissue and replicates its morphology. In this study, we sampled seawater at different times and places near Sydney, Australia, and determined the range and frequency of different taphonomic outcomes. Although destruction was most common, bacteria in 35% of seawater samples yielded morphology‐preserving biofilms. We could replicate the taphonomic pathways seen with seawater bacterial communities using single cultured strains of marine gammaproteobacteria. Each given species reproducibly generated a consistent taphonomic outcome and we identified species that yielded each of the distinct pathways produced by seawater bacterial communities. Once formed,bacterial pseudomorphs are stable for over a year and resist attack by other bacteria and destruction by proteases and other lytic enzymes. Competition studies showed that the initial action of a pseudomorphing strain can be blocked by a strain that destroys tissues. Thus embryo preservation in nature may depend on contingent interactions among bacterial species that determine if pseudomorphing occurs.We used Artemia nauplius larvae to show that bacterial biofilm replacement of tissue is not restricted to embryos, but is relevant for preservation of small multicellular organisms. We present a model for bacterial self‐assembly of large‐scale three‐dimensional tissue pseudomorphs, based on smallscaleinteractions among individual bacterial cells to form local biofilms at structural boundaries within the tissue. Localbiofilms then conjoin to generate the pseudomorph.

Wormometry‐on‐a‐chip: Innovative technologies for in situ analysis of small multicellular organisms

Small multicellular organisms such as nematodes, fruit flies, clawed frogs, and zebrafish are emerging models for an increasing number of biomedical and environmental studies. They offer substantial advantages over cell lines and isolated tissues, providing analysis under normal physiological milieu of the whole organism. Many bioassays performed on these alternative animal models mirror with a high level of accuracy those performed on inherently low-throughput, costly, and ethically controversial mammalian models of human disease. Analysis of small model organisms in a high-throughput and high-content manner is, however, still a challenging task not easily susceptible to laboratory automation. In this context, recent advances in photonics, electronics, as well as material sciences have facilitated the emergence of miniaturized bioanalytical systems collectively known as Lab-on-a-Chip (LOC). These technologies combine micro- and nanoscale sciences, allowing the application of laminar fluid flow at ultralow volumes in spatially confined chip-based circuitry. LOC technologies are particularly advantageous for the development of a wide array of automated functionalities. The present work outlines the development of innovative miniaturized chip-based devices for the in situ analysis of small model organisms. We also introduce a new term "wormometry" to collectively distinguish these up-and-coming chip-based technologies that go far beyond the conventional meaning of the term "cytometry."

A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

BackgroundMotile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. Although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. Here we present evidence for p38 mitogen-activated protein kinase (p38 MAPK) playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite Schistosoma mansoni.ResultsFluorescence confocal microscopy revealed that non-motile miracidia trapped within eggs prior to hatching displayed phosphorylated (activated) p38 MAPK associated with their ciliated surface. In contrast, freshly-hatched, rapidly swimming, miracidia lacked phosphorylated p38 MAPK. Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument. Freshly-hatched miracidia possessed swim velocities between 2.17 - 2.38 mm/s. Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min. In contrast, SB 203580, a p38 MAPK inhibitor, increased swim velocity by up to 15% over this duration. Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.ConclusionsThis study supports a role for p38 MAPK in the regulation of ciliary-beat. Given the evolutionary conservation of signalling processes and cilia structure, we hypothesize that p38 MAPK may regulate ciliary beat and beat-frequency in a variety of eukaryotes.

Read-out of soft x-ray contact microscopy microradiographs by focused ion beam/scanning electron microscope

A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.

Species differentiation of insects and other multicellular organisms using matrix‐assisted laser desorption/ionization time of flight mass spectrometry protein profiling

Abstract. Protein profiling by ‘whole cell MALDI’ is a well developed tool for species identification in bacteria but this is the first report that this inexpensive and simple technique can also be used for the identification of small multicellular organisms such as insects. A proof‐of‐concept experiment demonstrates that the method can distinguish between sibling species from the Drosophila melanogaster subgroup. This suggests that the technique may be used as a rapid screen for cryptic species and to identify known species.

PERSPECTIVE: THE SIZE-COMPLEXITY RULE

It is widely accepted that bigger entities have a greater division of labor than smaller ones and this is reflected in the fact that larger multicellular organisms have a corresponding increase in the number of their cell types. This rule is examined in some detail from very small organisms to large animals, and plants, and societies. Compared to other size-related rules, the size-complexity rule is relatively rough and approximate, yet clearly it holds throughout the whole range of living organisms, as well as for societies. The relationship between size and complexity is analyzed by examining the effects of size increase and decrease: size increase requires an increase in complexity, whereas size decrease permits, and sometimes requires, a decrease in complexity. Conversely, an increase or decrease in complexity permits, but does not require changes in size. An especially compelling argument for the close relation between size and complexity can be found in size quorum sensing in very small multicellular organisms.

PERSPECTIVE: THE SIZE-COMPLEXITY RULE

It is widely accepted that bigger entities have a greater division of labor than smaller ones and this is reflected in the fact that larger multicellular organisms have a corresponding increase in the number of their cell types. This rule is examined in some detail from very small organisms to large animals, and plants, and societies. Compared to other size-related rules, the size-complexity rule is relatively rough and approximate, yet clearly it holds throughout the whole range of living organisms, as well as for societies. The relationship between size and complexity is analyzed by examining the effects of size increase and decrease: size increase requires an increase in complexity, whereas size decrease permits, and sometimes requires, a decrease in complexity. Conversely, an increase or decrease in complexity permits, but does not require changes in size. An especially compelling argument for the close relation between size and complexity can be found in size quorum sensing in very small multicellular organisms.

Trophic structure of ecosystems and ecotoxicology of soil organisms

Studies on the ecotoxicology of soil organisms have led to the revision of views on the trophic struc- ture of ecosystems. It was found that the microbial link is obligatory and controls the migration of toxicants and their effects in the food chain. Differences in effects are accounted for by both the physioMgical stability of organisms and their affiliation with relatively independent and biogeochemically closed ecosystems differing in their spatiotemporal scales. The latter form a hierarchical three-level structure: ecosystems of unicellular organisms---ecosystems of small multicellular organisms--the ecosystem of large multicellular organisms, or a biogeocenosis. Trophic networks within the structure are united by ecosystemophagy as the type of feeding of large multiceUular organisms, and this accounts for the importance of the latter as indicators of long-term changes in a biogeocenosis.

Use and misuse of biomarker tests in “environmental conditions”

Use and misuse of biomarker tests in “environmental conditions”

Diffusible signal molecules controlling cell differentiation and patterning in Dictyostelium

Abstract Slime moulds, such as Dictyostelium discoideum, have biochemical, physiological and probably developmental features in common with both plants and animals. During development separate Dictyostelium amoebae first aggregate into collecting centers to form small multicellular organisms which, in their slug form, can migrate over the substratum toward light. Eventually a slug culminates to form a fruiting body consisting of a cellular stalk supporting a mass of spores. Development is highly regulative, indicating that it is controlled by signalling between the cells. A number of diffusible signal molecules have been discovered, including cyclic AMP, the chemoattractant in aggregation, and DIF-1, a novel chlorinated phenyl alkanone, which acts as a specific inducer of stalk cell differentiation. The migrating slug contains three types of precursor cell: prespore, prestalk A and prestalk B cells. Differentiation of these cells from uncommitted amoebae can be brought about in ceil cultures by cyclic AMP and DIF-1 acting in combination: cyclic AMP alone favours prespore, DIF-1 alone favours prestalk B, cyclic AMP and DIF-1 together favour prestalk A cell differentiation. There is evidence suggesting that these signals act in the same way in the intact aggregate. A cytoplasmic DIF-1 binding protein has been discovered, whose level increases as cells become sensitive to DIF-1 and which binds DIF-1 with an affinity and specificity suggestive of a receptor. At the same time, cells are able to inactivate DIF-1 by a metabolic pathway involving at least 12 metabolites. Metabolism may also serve to produce gradients of DIF-1 in the aggregate or other signal molecules from DIF-1. Manipulation of the DIF-1 signalling system will be an important means of further elucidating its role in development.

Interruption of Synthesis as a Cost of Sex in Small Organisms

Three processes associated with sex-meiosis, gametic union, and nuclear fusion-have a mechanical component, i.e., they require movements of supramolecular bodies. Times required for these processes are surprisingly high, probably because of the mechanical component, thus insuring that production of cells by sexual processes will require much longer than production of an equal number of cells by asexual processes. This is especially evident in direct comparisons of meiosis and mitosis. Meiosis requires much longer than two mitotic divisions. For unicellular organisms, and for small multicellular organisms in which the egg-to-egg time is very short, the additional time required by sexual processes at the cellular level lowers the feasibility of sex by slowing the reproduction rate. This is called the "cellular-mechanical" cost of sex. The cellular-mechanical cost of sex is at least twofold and will often exceed 10-fold by comparison with asexual reproduction in unicellular eucaryotes. The cellular mechanical cost of sex thus is a strong selective force acting against sex in unicellular and small organisms and, because it does not apply when the organism stops growing, tends to restrict sexuality to the end of the growth period.