First Detection of Podocytes in the Circulatory System of Enigmatic Echiurids (Annelida: Thalassematidae).

The fine structure of echiurid blood vessels in the proboscis is known in detail, but the circulatory system of the trunk is still understood mainly at the level of general anatomy. The trunk circulatory system was studied in Bonellia viridis females, and specialized podocytes were found to form the walls of the ring vessel and the anterior part of the ventral vessel. Podocytes were for the first time described in the echiurid circulatory system. Podocytes of B. viridis displayed a typical cell architecture, which is known for other bilaterians. A podocyte consists of a cell body; primary processes; and pedicels, which extend from the primary processes and are interconnected via specialized slit diaphragms. The presence of podocytes indicates that the ventral and ring vessels act as ultrafiltration sites, where the plasma is filtered through the basal lamina into the body cavity.

Variations in cell plasticity and proliferation underlie distinct modes of regeneration along the antero-posterior axis in the annelid Platynereis.

The capacity to regenerate lost tissues varies significantly among animals. Some phyla, such as the annelids, display substantial regenerating abilities, although little is known about the cellular mechanisms underlying the process. To precisely determine the origin, plasticity and fate of the cells participating in blastema formation and posterior end regeneration after amputation in the annelid Platynereis dumerilii, we developed specific tools to track different cell populations. Using these tools, we find that regeneration is partly promoted by a population of proliferative gut cells whose regenerative potential varies as a function of their position along the antero-posterior axis of the worm. Gut progenitors from anterior differentiated tissues are lineage restricted, whereas gut progenitors from the less differentiated and more proliferative posterior tissues are much more plastic. However, they are unable to regenerate the stem cells responsible for the growth of the worms. Those stem cells are of local origin, deriving from the cells present in the segment abutting the amputation plane, as are most of the blastema cells. Our results favour a hybrid and flexible cellular model for posterior regeneration in Platynereis relying on different degrees of cell plasticity.

Evaluation of RNAlater as a Field-Compatible Preservation Method for Metaproteomic Analyses of Bacterium-Animal Symbioses.

Field studies are central to environmental microbiology and microbial ecology, because they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows investigators to study these communities "in situ," which requires protein preservation directly in the field because protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is nonhazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability under field conditions when targeted for metaproteomic analyses is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNAlater using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNAlater storage after 1 day, 1 week, and 4 weeks at room temperature (22°C to 23°C). We evaluated protein preservation using one-dimensional liquid chromatography-tandem mass spectrometry. We found that RNAlater and flash freezing preserved proteins equally well in terms of total numbers of identified proteins and relative abundances of individual proteins, and none of the test time points was altered, compared to time zero. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomic data and the logistical requirements for field deployment, we recommend RNAlater for protein preservation of field-collected samples targeted for metaproteomic analyses. IMPORTANCE Metaproteomics, the large-scale identification and quantification of proteins from microbial communities, provide direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory setups, samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations, due to their hazardous nature and transport restrictions. Our study shows that RNAlater can serve as a low-hazard, easy-to-transport alternative to flash freezing for field preservation of samples for metaproteomic analyses. We show that RNAlater preserves the metaproteome equally well, compared to flash freezing, and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature.

Comparative Aspects of Annelid Regeneration: Towards Understanding the Mechanisms of Regeneration.

The question of why animals vary in their ability to regenerate remains one of the most intriguing questions in biology. Annelids are a large and diverse phylum, many members of which are capable of extensive regeneration such as regrowth of a complete head or tail and whole-body regeneration, even from few segments. On the other hand, some representatives of both of the two major annelid clades show very limited tissue regeneration and are completely incapable of segmental regeneration. Here we review experimental and descriptive data on annelid regeneration, obtained at different levels of organization, from data on organs and tissues to intracellular and transcriptomic data. Understanding the variety of the cellular and molecular basis of regeneration in annelids can help one to address important questions about the role of stem/dedifferentiated cells and "molecular morphallaxis" in annelid regeneration as well as the evolution of regeneration in general.

Structural and Functional Characterization of the FGF Signaling Pathway in Regeneration of the Polychaete Worm Alitta virens (Annelida, Errantia).

Epimorphic regeneration of lost body segments is a widespread phenomenon across annelids. However, the molecular inducers of the cell sources for this reparative morphogenesis have not been identified. In this study, we focused on the role of fibroblast growth factor (FGF) signaling in the posterior regeneration of Alitta virens. For the first time, we showed an early activation of FGF ligands and receptor expression in an annelid regenerating after amputation. The expression patterns indicate that the entire regenerative bud is competent to FGFs, whose activity precedes the initiation of cell proliferation. The critical requirement of FGF signaling, especially at early stages, is also supported by inhibitor treatments followed by proliferation assay, demonstrating that induction of blastemal cells depends on FGFs. Our results show that FGF signaling pathway is a key player in regenerative response, while the FGF-positive wound epithelium, ventral nerve cord and some mesodermal cells around the gut could be the inducing tissues. This mechanism resembles reparative regeneration of vertebrate appendages suggesting such a response to the injury may be ancestral for all bilaterians.

Collective dynamics in entangled worm and robot blobs.

Living systems at all scales aggregate in large numbers for a variety of functions including mating, predation, and survival. The majority of such systems consist of unconnected individuals that collectively flock, school, or swarm. However, some aggregations involve physically entangled individuals, which can confer emergent mechanofunctional material properties to the collective. Here, we study in laboratory experiments and rationalize in theoretical and robophysical models the dynamics of physically entangled and motile self-assemblies of 1-cm-long California blackworms (Lumbriculus variegatus, Annelida: Clitellata: Lumbriculidae). Thousands of individual worms form braids with their long, slender, and flexible bodies to make a three-dimensional, soft, and shape-shifting "blob." The blob behaves as a living material capable of mitigating damage and assault from environmental stresses through dynamic shape transformations, including minimizing surface area for survival against desiccation and enabling transport (negative thermotaxis) from hazardous environments (like heat). We specifically focus on the locomotion of the blob to understand how an amorphous entangled ball of worms can break symmetry to move across a substrate. We hypothesize that the collective blob displays rudimentary differentiation of function across itself, which when combined with entanglement dynamics facilitates directed persistent blob locomotion. To test this, we develop a robophysical model of the worm blobs, which displays emergent locomotion in the collective without sophisticated control or programming of any individual robot. The emergent dynamics of the living functional blob and robophysical model can inform the design of additional classes of adaptive mechanofunctional living materials and emergent robotics.

Quantifying Cell Proliferation During Regeneration of Aquatic Worms.

Many species of aquatic worms, including members of the phyla Nemertea, Annelida, Platyhelminthes, and Xenacoelomorpha, can regenerate large parts of their body after amputation. In most species, cell proliferation plays key roles in the reconstruction of lost tissues. For example, in annelids and flatworms, inhibition of cell proliferation by irradiation or chemicals prevents regeneration. Cell proliferation also plays crucial roles in growth, body patterning (e.g., segmentation) and asexual reproduction in many groups of aquatic worms. Cell proliferation dynamics in these organisms can be studied using immunohistochemical detection of proteins expressed during proliferation-associated processes or by incorporation and labeling of thymidine analogues during DNA replication. In this chapter, we present protocols for labeling and quantifying cell proliferation by (a) antibody-based detection of either phosphorylated histone H3 during mitosis or proliferating cell nuclear antigen (PCNA) during S-phase, and (b) incorporation of two thymidine analogues, 5'-bromo-2'-deoxyuridine (BrdU) and 5'-ethynyl-2'-deoxyuridine (EdU), detected by immunohistochemistry or inorganic "click" chemistry, respectively. Although these protocols have been developed for whole mounts of small (<2 cm) marine and freshwater worms, they can also be adapted for use in larger specimens or tissue sections.

Genes with spiralian-specific protein motifs are expressed in spiralian ciliary bands.

Spiralia is a large, ancient and diverse clade of animals, with a conserved early developmental program but diverse larval and adult morphologies. One trait shared by many spiralians is the presence of ciliary bands used for locomotion and feeding. To learn more about spiralian-specific traits we have examined the expression of 20 genes with protein motifs that are strongly conserved within the Spiralia, but not detectable outside of it. Here, we show that two of these are specifically expressed in the main ciliary band of the mollusc Tritia (also known as Ilyanassa). Their expression patterns in representative species from five more spiralian phyla-the annelids, nemerteans, phoronids, brachiopods and rotifers-show that at least one of these, lophotrochin, has a conserved and specific role in particular ciliated structures, most consistently in ciliary bands. These results highlight the potential importance of lineage-specific genes or protein motifs for understanding traits shared across ancient lineages.

Development of an annelid-like peristaltic crawling soft robot using dielectric elastomer actuators.

The annelid, which consists of several identical segments, exploits its soft structures to move effectively in complex natural environments. Elongation and shortening of different segments produce a reverse peristaltic wave while retractable setae generate a variable friction, enabling bidirectional crawling locomotion. Although several designs have applied soft technologies towards the construction of annelid-like robots, these robots do not exhibit the homonymous segmentation, reverse peristaltic wave and variable friction. This paper reports the development of an annelid-like soft robot based on an improved dielectric elastomer (DE) minimum energy structure actuator to have these annelidan features. Each biomimetic segment of the robot is supported by a polyethylene terephthalate (PET) frame adhered to the DE actuator. The DE actuator induces segment elongation or shortening, which causes silica gel pads attached to the PET frame to contact or separate from the ground, producing a variable friction. The designed robot, whose identical segments conform to the homonymous segmentation, achieves forward or backward movement via the cooperative efforts of all the biomimetic segments. This cooperative movement, which produces the reverse peristaltic wave, strongly resembles that of natural annelidan locomotion. In addition, the kinematic analysis of the robot is investigated. Experimental results confirm that the designed robot is capable of bidirectional and rapid locomotion. The robot can achieve a maximum velocity of 11.5 mm s-1 and a maximum velocity/mass ratio of 86.25 mm (min-1 g-1). Compared to other existing annelid-like soft robots, this designed robot exhibits a superior average velocity, velocity/length ratio, body length/cycle, and velocity/mass ratio, and its performance affords the best approximation to that of the natural annelid.

First Discovery of Pogonophora (Annelida, Siboglinidae) in the Kara Sea Coincide with the Area of High Methane Concentration.

Here we report the first finding of a frenulate pogonophoran (Annelida, Siboglinidae) in the southern part of the Kara Sea. This finding was made in the Yenisei Gulf in the region of the highest methane concentrations, resulting from the degradation of permafrost under the influence of river flow. It has been suggested that pogonophorans are indicators of hydrocarbon manifestations of various genesis.

On the genus Sabidius Strelzov, 1973 (Annelida: Paraonidae), with a redescription of the type species and the description of a new species.

Two species of Sabidius Strelzov, 1973 were collected during a benthic survey, from 10-3,000 m deep, in Espírito Santo Basin, off southeastern Brazilian coast. Those species are S. cornatus (Hartman, 1965) and S. antennatus sp. nov. The genus Sabidius was monotypic until the present study, in which a new species is proposed, together with a redescription of the type species, with expansion of the geographic distribution of the genus and of the type species. The diagnostic feature of the genus is the morphology of prostomium, with crested anterior margin. The main feature that differentiates the two species within the genus is the presence/absence of a median antenna, which is present in the new species described in the present study and absent in Sabidius cornatus.

Parapodial glandular organs in Owenia borealis (Annelida: Oweniidae) and their possible relationship with nephridia.

Oweniidae is a basal group of recent annelids and nowadays it attracts the attention of researchers of many biological fields. Surprisingly, details of their anatomy, like the adult excretory system, remain obscure. Researchers recently suggested that the paired organs of tubeworms in the family Oweniidae are related to nephridia. In the current study of Owenia borealis adults, we determined that these structures are parapodial glandular organs (PGOs) and are located in the first two segments of adults. The PGOs are complex subepidermal multicellular glands that contain secretory cells, that is, goblet cells, which are differentiated by the type of the producing tube matter. The goblet cells are surrounded by muscles that are used to extrude material stored in the PGO's lumen into the external environment. The anterior pair of PGOs have very well-developed rough endoplasmatic reticulum in the proximal cells, spacious Golgi complexes, numerous nail-shaped microvilli, and apocrine secretory processes in the goblet cells of the distal parts. The posterior pair of PGOs only consists of cells, which probably produce proteinaceous fibrils. We discuss the homology of goblet cells with specific nail-shaped microvilli that produce ß-chitin within annelids. We also discuss the possibility that PGOs and nephridia have a common origin. This study provides new information on the ultrastructure of cells that secrete the organic material used to form the tubes inhabited by tube-dwelling annelids.

Description of ovary organization and oogenesis in a phreodrilid clitellate.

Phreodrilidae is a small family uniting about 50 species of minute freshwater clitellate annelids inhabiting mainly the Southern hemisphere. Other than the male and spermathecal genitalia, their internal organization is poorly known. Here, we present results of our study of the ovaries and oogenesis in Insulodrilus bifidus, a phreodrilid from Western Australia using light and electron microscopy. The ovaries are paired and located in segment XII. They are inconspicuous and composed of several (10-12) spherical germ-line cysts loosely interconnected by flattened somatic cells. The cysts usually comprise 32 germ cells and each cell is connected via a cytoplasmic bridge (ring canal) to the central cytoplasmic mass (the cytophore). In ovaries, germ cells in a given cyst develop in full synchrony. However, there is no synchrony among cysts, so there is a developmental gradient of cysts (from oogonial to early meiotic) along the longitudinal ovary axis. Within the cysts that are located in the distal end of the ovary the synchrony is finally lost and interconnected cells diversify into two morphologically distinct categories: an oocyte and 31 nurse cells. Such cysts detach from the ovaries and further development occurs within the body cavity. The oocyte gathers nutrients, mainly in form of yolk spheres, whereas nurse cells grow slightly and do not gather yolk. Organelles such as ribosomes, mitochondria and endoplasmic reticulum pass freely through the ring canals and are present within the cytophore, which suggests cytoplasmic transfer towards the oocyte. The formation of female germ-line cysts equipped with cytophore and cells differentiated into oocyte and nurse cells matches the general pattern of oogenesis found in clitellates. In details, the ovary organization and oogenesis found in I. bifidus resembles the situation described in some representatives of Naidinae and Enchytraeidae.

Temperature sensitivity patterns of carbon and nitrogen processes in decomposition of boreal organic soils - Quantification in different compounds and molecule sizes based on a multifactorial experiment.

Climate warming and organic matter decomposition are connected in a recursive manner; this recursion can be described by temperature sensitivity. We conducted a multifactorial laboratory experiment to quantify the temperature sensitivity of organic carbon (C) and nitrogen (N) decomposition processes of common boreal organic soils. We incubated 36 mor and 36 slightly decomposed Carex-Sphagnum peat samples in a constant moisture and ambient temperature for 6 months. The experiment included three temperature and two moisture levels and two food web manipulations (samples with and without fungivore enchytraeid worms). We determined the release of carbon dioxide (CO2) and dissolved organic carbon (DOC) in seven molecular size classes together with ammonium N and dissolved organic N in low molecular weight and high molecular weight fractions. The temperature sensitivity function Q10 was fit to the data. The C and N release rate was almost an order of magnitude higher in mor than in peat. Soil fauna increased the temperature sensitivity of C release. Soil fauna played a key role in N release; when fauna was absent in peat, the N release was ceased. The wide range of the studied C and N compounds and treatments (68 Q10 datasets) allowed us to recognize five different temperature sensitivity patterns. The most common pattern (37 out of 68) was a positive upwards temperature response, which was observed for CO2 and DOC release. A negative downward pattern was observed for extractable organic nitrogen and microbial C. Sixteen temperature sensitivity patterns represented a mixed type, where the Q10function was not applicable, as this does not allow changing the sign storage change rate with increasing or decreasing temperature. The mixed pattern was typically connected to intermediate decomposition products, where input and output fluxes with different temperature sensitivities may simultaneously change the storage. Mixed type was typical for N processes. Our results provide useful parameterization for ecosystem models that describe the feedback loop between climate warming, organic matter decomposition, and productivity of N-limited vegetation.

The complexity of porphyrin-like pigments in a marine annelid sheds new light on haem metabolism in aquatic invertebrates.

True green pigments in the animal kingdom are scarce and are almost invariably porphyrinoids. Endogenous porphyrins resulting from the breakdown of haem are usually known as "bile pigments". The pigmentation of intertidal Polychaeta has long gained attention due to its variety and vivid patterning that often seems incompatible with camouflage, as it occurs with Eulalia viridis, one of the few truly green Polychaeta. The present study combined UV and bright-field microscopy with HPLC to address the presence and distribution of pigments in several organs. The results showed two major types of porphyrin-like pigments, yellowish and greenish in colour, that are chiefly stored as intraplasmatic granules. Whereas the proboscis holds yellow pigments, the skin harbours both types in highly specialised cells. In their turn, oocytes and intestine have mostly green pigments. Despite some inter-individual variation, the pigments tend to be stable after prolonged storage at -20 °C, which has important implications for future studies. The results show that, in a foraging predator of the intertidal where melanins are circumscribed to lining the nervous system, porphyrinoid pigments have a key role in protection against UV light, in sensing and even as chemical defence against foulants and predators, which represents a remarkable adaptive feature.

Siboglinids (Annelida, Siboglinidae) as Possible Hydrocarbon Indicators as Exemplified by the Sea of Okhotsk.

The geographic and bathymetric distribution of siboglinids in the Sea of Okhotsk was studied. At least 75% of all siboglinid findings were at a depth up to 400 m. Most of them were concentrated in the northwestern part of the shelf. Comparison of the data on siboglinid distribution in the Sea of Okhotsk and the aggregate geological data on hydrocarbon distribution showed that, in the Sea of Okhotsk, siboglinids were mostly in the regions of hydrocarbon manifestations, but they were absent in the central areas with the minimum methane and hydrocarbon concentrations in both benthic sediments and the uppermost water layers.

A Behavioral Assay to Study Effects of Retinoid Pharmacology on Nervous System Development in a Marine Annelid.

Autonomous animal locomotion, such as swimming, is modulated by neuronal networks acting on cilia or muscles. Understanding how these networks are formed and coordinated is a complex scientific problem, which requires various technical approaches. Among others, behavioral studies of developing animals treated with exogenous substances have proven to be a successful approach for studying the functions of neuronal networks. One such substance crucial for the proper development of the nervous system is the vitamin A-derived morphogen retinoic acid (RA). In the larva of the marine annelid Platynereis dumerilii , for example, RA is involved in the specification and differentiation of individual neurons and responsible for orchestrating the swimming behavior of the developing larva. Here, we report a workflow to analyze the effects of RA on the locomotion of the P. dumerilii larva. We provide a protocol for both the treatment with RA and the recording of larval swimming behavior. Additionally, we present a pipeline for the analysis of the obtained data in terms of swimming speed and movement trajectory. This chapter thus summarizes the methodology for analyzing the effects of a specific drug treatment on larval swimming behavior. We expect this approach to be readily adaptable to a wide variety of pharmacological compounds and aquatic species.

CRISPR/CAS9 mutagenesis of a single r-opsin gene blocks phototaxis in a marine larva.

Many marine animals depend upon a larval phase of their life cycle to locate suitable habitat, and larvae use light detection to influence swimming behaviour and dispersal. Light detection is mediated by the opsin genes, which encode light-sensitive transmembrane proteins. Previous studies suggest that r-opsins in the eyes mediate locomotory behaviour in marine protostomes, but few have provided direct evidence through gene mutagenesis. Larvae of the marine annelid Capitella teleta have simple eyespots and are positively phototactic, although the molecular components that mediate this behaviour are unknown. Here, we characterize the spatio-temporal expression of the rhabdomeric opsin genes in C. teleta and show that a single rhabdomeric opsin gene, Ct-r-opsin1, is expressed in the larval photoreceptor cells. To investigate its function, Ct-r-opsin1 was disrupted using CRISPR/CAS9 mutagenesis. Polymerase chain reaction amplification and DNA sequencing demonstrated efficient editing of the Ct-r-opsin1 locus. In addition, the pattern of Ct-r-opsin1 expression in photoreceptor cells was altered. Notably, there was a significant decrease in larval phototaxis, although the eyespot photoreceptor cell and associated pigment cell formed normally and persisted in Ct-r-opsin1-mutant animals. The loss of phototaxis owing to mutations in Ct-r-opsin1 is similar to that observed when the entire photoreceptor and pigment cell are deleted, demonstrating that a single r-opsin gene is sufficient to mediate phototaxis in C. teleta. These results establish the feasibility of gene editing in animals like C. teleta, and extend previous work on the development, evolution and function of the C. teleta visual system . Our study represents one example of disruption of animal behaviour by gene editing through CRISPR/CAS9 mutagenesis, and has broad implications for performing genome editing studies in a wide variety of other understudied animals.

Exploring the Use of Species Sensitivity Distributions to Define Protective Limits for the Use of Organic Wastes as Soil Amendments.

The use of organic wastes as soil amendments can be an important measure to improve soil quality and reduce waste accumulation and landfilling. However, the potential contaminant loads of such wastes, can be a source of environmental concern. Consequently, legislation has been developed to regulate the use of these wastes in agricultural soils. However, the regulations only consider chemical parameters, which are insufficient to establish the level of environmental risk. A possible solution is the use of species sensitivity distributions (SSDs), employing ecotoxicological data from test batteries that could be incorporated into legislation. In the present study, 2 different hazardous concentrations affecting 5 and 50% of the soil community (HC5 and HC50, respectively) were determined using ecotoxicological data (effect concentrations, 10 and 50% [EC10 and EC50, respectively]) for 5 different wastes. The results demonstrate that, as expected, current legislative thresholds do not translate to environmental risk/protection and that SSDs may be an important tool allowing the simple inclusion and interpretation of ecotoxicological data from test batteries in legislation. On the other hand, SSDs must be used with caution because there are still doubts about their actual value in risk prediction and about which estimates provide adequate protection. For instance, the use of HC50EC10 values is not recommended; these values overlap with the more conservative HC5EC50 data, highlighting the fact that the use of lower effect concentrations may not always provide the most protective approach. Also, hazardous concentrations need to be calibrated at the field or semifield level, to verify environmental protection in different soils/environments and the adequacy of standard test organisms. Environ Toxicol Chem 2019;38:1569-1576. © 2019 SETAC.

Twenty years of the 'Preparation for Oxidative Stress' (POS) theory: Ecophysiological advantages and molecular strategies.

Freezing, dehydration, salinity variations, hypoxia or anoxia are some of the environmental constraints that many organisms must frequently endure. Organisms adapted to these stressors often reduce their metabolic rates to maximize their chances of survival. However, upon recovery of environmental conditions and basal metabolic rates, cells are affected by an oxidative burst that, if uncontrolled, leads to (oxidative) cell damage and eventually death. Thus, a number of adapted organisms are able to increase their antioxidant defenses during an environmental/functional hypoxic transgression; a strategy that was interpreted in the 1990s as a "preparation for oxidative stress" (POS). Since that time, POS mechanisms have been identified in at least 83 animal species representing different phyla including Cnidaria, Nematoda, Annelida, Tardigrada, Echinodermata, Arthropoda, Mollusca and Chordata. Coinciding with the 20th anniversary of the postulation of the POS hypothesis, we compiled this review where we analyze a selection of examples of species showing POS-mechanisms and review the most recent advances in understanding the underlying molecular mechanisms behind those strategies that allow animals to survive in harsh environments.