Stepwise emergence of the neuronal gene expression program in early animal evolution.

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.

The concept of intrinsic versus extrinsic apoptosis.

Regulated cell death is a vital and dynamic process in multicellular organisms that maintains tissue homeostasis and eliminates potentially dangerous cells. Apoptosis, one of the better-known forms of regulated cell death, is activated when cell-surface death receptors like Fas are engaged by their ligands (the extrinsic pathway) or when BCL-2-family pro-apoptotic proteins cause the permeabilization of the mitochondrial outer membrane (the intrinsic pathway). Both the intrinsic and extrinsic pathways of apoptosis lead to the activation of a family of proteases, the caspases, which are responsible for the final cell demise in the so-called execution phase of apoptosis. In this review, I will first discuss the most common types of regulated cell death on a morphological basis. I will then consider in detail the molecular pathways of intrinsic and extrinsic apoptosis, discussing how they are activated in response to specific stimuli and are sometimes overlapping. In-depth knowledge of the cellular mechanisms of apoptosis is becoming more and more important not only in the field of cellular and molecular biology but also for its translational potential in several pathologies, including neurodegeneration and cancer.

Marine Nemertean Worms for Immunoblotting Studies of Oocyte Aging.

Immunoblotting analyses employing phospho-specific antibodies can help elucidate potential roles played by protein kinases as oocytes age and lose their ability to undergo normal fertilization. This chapter updates a previously published protocol for conducting immunoblotting analyses of oocyte maturation in marine nemertean worms by adding general methods for obtaining adult worms and for handling their gametes in experiments assessing oocyte aging.

Optimization of a minimal sample preparation protocol for imaging mass spectrometry of unsectioned juvenile invertebrates.

Tissue sections have long been the subject matter for the application of imaging mass spectrometry, but recently the technique has been adapted for many other purposes including bacterial colonies and 3D cell culture. Here, we present a simple preparation method for unsectioned invertebrate tissue without the need for fixing, embedding, or slicing. The protocol was used to successfully prepare a Hawaiian bobtail squid hatchling for analysis, and the resulting data detected ions that correspond to compounds present in the host only during its symbiotic colonization by Vibrio fischeri.

Analysis of 13,312 benthic invertebrate samples from German streams reveals minor deviations in ecological status class between abundance and presence/absence data.

Benthic invertebrates are the most commonly used organisms used to assess ecological status as required by the EU Water Framework Directive (WFD). For WFD-compliant assessments, benthic invertebrate communities are sampled, identified and counted. Taxa × abundance matrices are used to calculate indices and the resulting scores are compared to reference values to determine the ecological status class. DNA-based tools, such as DNA metabarcoding, provide a new and precise method for species identification but cannot deliver robust abundance data. To evaluate the applicability of DNA-based tools to ecological status assessment, we evaluated whether the results derived from presence/absence data are comparable to those derived from abundance data. We analysed benthic invertebrate community data obtained from 13,312 WFD assessments of German streams. Broken down to 30 official stream types, we compared assessment results based on abundance and presence/absence data for the assessment modules "organic pollution" (i.e., the saprobic index) and "general degradation" (a multimetric index) as well as their underlying metrics. In 76.6% of cases, the ecological status class did not change after transforming abundance data to presence/absence data. In 12% of cases, the status class was reduced by one (e.g., from good to moderate), and in 11.2% of cases, the class increased by one. In only 0.2% of cases, the status shifted by two classes. Systematic stream type-specific deviations were found and differences between abundance and presence/absence data were most prominent for stream types where abundance information contributed directly to one or several metrics of the general degradation module. For a single stream type, these deviations led to a systematic shift in status from 'good' to 'moderate' (n = 201; with only n = 3 increasing). The systematic decrease in scores was observed, even when considering simulated confidence intervals for abundance data. Our analysis suggests that presence/absence data can yield similar assessment results to those for abundance-based data, despite type-specific deviations. For most metrics, it should be possible to intercalibrate the two data types without substantial efforts. Thus, benthic invertebrate taxon lists generated by standardised DNA-based methods should be further considered as a complementary approach.

BioCell2XML: a novel tool for converting cell lineage data from SIMI BioCell to MaMuT (Fiji).

Computer-assisted 4D manual cell tracking has been a valuable method for understanding spatial-temporal dynamics of embryogenesis (e.g., Stach & Anselmi BMC Biol, 13(113), 1-11 2015; Vellutini et al. BMC Biol, 15(33), 1-28 2017; Wolff et al. eLife, 7, e34410 2018) since the method was introduced in the late 1990s. Since two decades SIMI® BioCell (Schnabel et al. Dev Biol, 184, 234-265 1997), a software which initially was developed for analyzing data coming from the, at that time new technique of 4D microscopy, is in use. Many laboratories around the world use SIMI BioCell for the manual tracing of cells in embryonic development of various species to reconstruct cell genealogies with high precision. However, the software has several disadvantages: limits in handling very large data sets, the virtually no maintenance over the last 10 years (bound to older Windows versions), the difficulty to access the created cell lineage data for analyses outside SIMI BioCell, and the high cost of the program. Recently, bioinformatics, in close collaboration with biologists, developed new lineaging tools that are freely available through the open source image processing platform Fiji. Here we introduce a software tool that allows conversion of SIMI BioCell lineage data to a format that is compatible with the Fiji plugin MaMuT (Wolff et al. eLife, 7, e34410 2018). Hereby we intend to maintain the usability of SIMI BioCell created cell lineage data for the future and, for investigators who wish to do so, facilitate the transition from this software to a more convenient program.

Circuit architecture for somatotopic action selection in invertebrates.

Invertebrate species have significantly contributed to neuroscience owing to the accessibility they provide to cellular- and molecular-level understanding of brain functions. Somatotopic action selection is one of the key features of animal behavior, and studying this process in invertebrates is potentially a sweet spot in understanding the general relationship between neuronal morphology, circuit structure, and animal behavior. In this review, we introduce circuit architectures that realize somatotopic action selection, from simple reflexes to patterned motor outputs, in different invertebrate species. We then discuss future directions towards understanding the general principles underlying the development and evolution of the circuit architecture that enables sensorimotor transformation and action selection in the animal kingdom.

Pseudocnidae of archinemerteans (Nemertea, Palaeonemertea) and their implications for nemertean systematics.

The structure of pseudocnidae of 16 species of Palaeonemertea clade Archinemertea (= Cephalotrichida s.l.) was investigated with confocal laser, scanning, and transmission electron microscopy (TEM). All species of the genus Cephalothrix possess two kinds of pseudocnidae, large and small. Only one type of pseudocnida is present in Balionemertes and Cephalotrichella. TEM revealed variation in the ultrastructure of large and small pseudocnidae of four species of Cephalothrix. Pseudocnidae of Balionemertes, Cephalotrichella, and Cephalothrix differ in substructure: in Balionemertes and Cephalotrichella the medulla is located in the basal half of the pseudocnidae with а precore layer situated in the apical half, whereas in Cephalothrix spp. and other palaeonemerteans the medulla surrounds a precore layer. Our results confirm the division of archinemerteans into Cephalotrichidae (with genus Cephalothrix) and Cephalotrichellidae (with genera Cephalotrichella and Balionemertes). The synapomorphy of Cephalotrichidae is pseudocnida dimorphism and the synapomorphies of Cephalotrichellidae are the position of the pseudocnidae on epithelial ridges and the distinct organization of pseudocnida layers, specifically the relative position of the medulla and precore layers. The pseudocnida lateral process, one or more of which is present in most species observed, is a probable synapomorphy of the clade Archinemertea. This is the first application of pseudocnida features to distinguish super-generic nemertean taxa and the results suggest that pseudocnidae provide a useful source of characters for nemertean systematics.

Marine Nemertean Worms for Studies of Oocyte Maturation and Aging.

Many marine invertebrates are capable of providing an abundant supply of oocytes that are fertilized external to the female body, thereby making these specimens well suited for studies of development. Along with intensively analyzed model systems belonging to such groups as echinoderms, tunicates, mollusks, and annelids, various lesser-studied taxa can undergo an external mode of fertilization. For example, nemertean worms constitute a relatively small phylum of marine protostome worms whose optically clear oocytes are easily collected and fertilized in the laboratory. Thus, to help promote the use of nemertean oocytes as a potential model in embryological analyses, this chapter begins by describing general methods for obtaining adults and for handling their gametes. After presenting such protocols, this chapter concludes with some representative results obtained with these specimens by summarizing the roles played by adenosine monophosphate-activated kinase (AMPK) during oocyte maturation and by c-Jun N-terminal kinase (JNK) during oocyte aging and death.

Oogenesis in the viviparous phoronid, Phoronis embryolabi.

The study of gametogenesis is useful for phylogenetic analysis and can also provide insight into the physiology and biology of species. This report describes oogenesis in the Phoronis embryolabi, a newly described species, which has an unusual type of development, that is, a viviparity of larvae. Phoronid oogonia are described here for the first time. Yolk formation is autoheterosynthetic. Heterosynthesis occurs in the peripheral cytoplasm via fusion of endocytosic vesicles. Simultaneously, the yolk is formed autosynthetically by rough endoplasmic reticulum in the central cytoplasm. Each developing oocyte is surrounded by the follicle of vasoperitoneal cells, whose cytoplasm is filled with glycogen particles and various inclusions. Cytoplasmic bridges connect developing oocytes and vasoperitoneal cells. These bridges and the presence of the numerous glycogen particles in the vasoperitoneal cells suggest that nutrients are transported from the follicle to oocytes. Phoronis embryolabi is just the second phoronid species in which the ultrastructure of oogenesis has been studied, and I discuss the data obtained comparing them with those in Phoronopsis harmeri. Finally, I discuss the distribution of reproductive patterns across both, molecular and morphological phylogenetic trees in Phoronida proving that parental care has evolved independently several times in this phylum.

EvoRegen in animals: Time to uncover deep conservation or convergence of adult stem cell evolution and regenerative processes.

How do animals regenerate specialised tissues or their entire body after a traumatic injury, how has this ability evolved and what are the genetic and cellular components underpinning this remarkable feat? While some progress has been made in understanding mechanisms, relatively little is known about the evolution of regenerative ability. Which elements of regeneration are due to lineage specific evolutionary novelties or have deeply conserved roots within the Metazoa remains an open question. The renaissance in regeneration research, fuelled by the development of modern functional and comparative genomics, now enable us to gain a detailed understanding of both the mechanisms and evolutionary forces underpinning regeneration in diverse animal phyla. Here we review existing and emerging model systems, with the focus on invertebrates, for studying regeneration. We summarize findings across these taxa that tell us something about the evolution of adult stem cell types that fuel regeneration and the growing evidence that many highly regenerative animals harbor adult stem cells with a gene expression profile that overlaps with germline stem cells. We propose a framework in which regenerative ability broadly evolves through changes in the extent to which stem cells generated through embryogenesis are maintained into the adult life history.

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.

Acoel regeneration mechanisms indicate an ancient role for muscle in regenerative patterning.

Positional information is required for animal regeneration, yet how it is harbored in adult tissues is poorly understood. In planarians, positional control genes (PCGs) control regeneration outcomes and are regionally expressed predominately in the musculature. Acoels are early diverging bilaterally symmetric animals, having separated from other bilaterians > 550 million years ago. Here, we find that PCGs in the acoel Hofstenia miamia are expressed together and specifically in a primary differentiated cell type: muscle. The vast majority of Hofstenia muscle cells in regions tested express PCGs, suggesting positional information is a major feature of muscle. PCG expression domains are dynamic in muscle after injury, consistent with known PCG roles in guiding regeneration. These data demonstrate an instructive positional role for Hofstenia muscle and this similarity with planarians suggests mesodermal muscle originated at the base of the Bilateria not only for contraction, but also as the source of positional information guiding regeneration.

Manufacturing Clinical Grade Recombinant Adeno-Associated Virus Using Invertebrate Cell Lines.

Recombinant adeno-associated virus (rAAV) vectors are proving to be a reliable gene transfer system for several clinical applications, with an increasing body of evidence supporting safety and efficacy. Realizing the clinical and commercial potential of rAAV depends on a reliable source of high-quality, well-characterized rAAV lots. This requirement has been very challenging to achieve due to limits of manufacturing platforms, lot-to-lot variability, or differences in the rigor applied to quality-control assays. In addition to reliable, high-quality vectors, limited quantities of rAAV have hampered clinical development and discouraged investigations into applications that require large therapeutic doses or quantities needed to treat large patient populations. A minimal number of vector production runs should be sufficient to support all phases of clinical development, including non-clinical, pharmacological, and toxicological studies, as well as clinical studies and commercial supply. The production platform using the Sf9 invertebrate cell line has emerged as a scalable and economical source of rAAV. Access to larger quantities of rAAV has now enabled evaluation of gene therapeutics for diseases that require large doses per patient or diseases with large patient populations. The only licensed rAAV product, Glybera, was produced in Sf9 cells, and other rAAV products are in clinical trials in the United States and Europe. The development of the Sf9 rAAV genetics, processes, and overview of the current system are described.

Biomonitoring of water quality of the Osumi, Devolli, and Shkumbini rivers through benthic macroinvertebrates and chemical parameters.

Environmental monitoring of river water quality in Albania, using biological and chemical parameters, is a fast and effective way to assess the quality of water bodies.The aim of this study was to investigate Ephemeroptera, Plecoptera and Trichoptera (EPT), Biotic index-Richness using macroinvertebrates to assess the water quality, with special reference to nutrient (phosphorus and nitrogen) levels in the Devolli, Shkumbini and Osumi rivers. Our objective was to investigate the relationships between the measures of benthic macroinvertebrate communities and nutrient concentrations to assess water quality. The rivers' benthic macroinvertebrates were collected during different seasons in 2012. The biological and chemical parameters used in the current study identified them as quick indicators of water quality assessment. The total number of macroinvertebrate individuals (n = 15,006) (Osumi river: n = 5,546 organisms; Devolli river: n = 3,469 organisms; and Shkumbini river: n = 5,991 organisms), together with the EPT group (Ephemeroptera, Plecoptera, and Trichoptera), showed that the water quality at the river stations during the above-mentioned period belonged to Classes II and III (fair water quality and good water quality, respectively). The classification of the water quality was also based on the nitrogen and total phosphorus contents. The pollution tolerance levels of macroinvertebrate taxa varied from the non-tolerating forms encountered in environments with low pollution levels to the tolerating forms that are typical of environments with considerable pollution levels.

Circulating phagocytes: the ancient and conserved interface between immune and neuroendocrine function.

Immune and neuroendocrine functions display significant overlap in highly divergent and evolutionarily distant models such as molluscs, crustaceans, insects and mammals. Fundamental players in this crosstalk are professional phagocytes: macrophages in vertebrates and immunocytes in invertebrates. Although they have different developmental origins, macrophages and immunocytes possess comparable functions and differentiate under the control of evolutionarily conserved transcription factors. Macrophages and immunocytes share their pools of receptors, signalling molecules and pathways with neural cells and the neuro-endocrine system. In crustaceans, adult transdifferentiation of circulating haemocytes into neural cells has been documented recently. In light of developmental, molecular and functional evidence, we propose that the immune-neuroendocrine role of circulating phagocytes pre-dates the split of protostomian and deuterostomian superphyla and has been conserved during the evolution of the main groups of metazoans.

Two new species of dicyemid mesozoans (Dicyemida: Dicyemidae) from Octopus maya Voss & Solis-Ramirez (Octopodidae) off Yucatan, Mexico.

Two new dicyemid species are described from the endemic cephalopod Octopus maya Voss & Solis-Ramirez collected off Yucatan, Mexico. The renal sacs of 40 juvenile and adult octopuses from four localities were examined. Dicyema hochbergi n. sp. is a medium-sized species that reaches 2,245 µm in length. The vermiform stages consist of 18-24 peripheral cells, a conical calotte and the extension of the axial cell between the base and middle of the metapolar cells. Infusoriform embryos consist of 39 cells with urn cell containing one germinal cell, two nuclei and solid refringent bodies. Dicyema mexcayae n. sp. is a relatively small species that reaches 1,114 µm in length. The vermiform stages are constituted by 14-16 peripheral cells, an elongate calotte and the axial cell extending forward to the middle of the metapolar cells. The infusoriform embryos consist of 37 cells, two solid refringent bodies and urn cells with two nuclei each. The present study represents the first description of a dicyemid species from O. maya and increases the number of described species from Mexican waters to 11.

[Evolution and phylogeny of B lymphocytes]. / Evolución y filogenia de los linfocitos B.

B lymphocytes are one of the most important cell types involved in the immune response of mammals. The origin and evolution of this cellular type is unknown, but the B lymphocyte bona fide appeared first in fish. In this review we analize the principal components of the immune response of invertebrates, their phylogenetic distribution and the permancence of some properties that allowed the emergence of the B lymphocyte. We started from the idea that many of the components that characterize the B lymphocyte are found distributed among the invertebrates, however, it is in the B lymphocyte, where all these components that give this type of cell its identity, converged. The actual knowledge we have in regards of the lymphocytes comes, in the most part, from physiological studies in mammals, being the mice the more representative. The origin of the B lymphocyte, its alternative mechanisms for generating receptor diversity, its immune effector response, and the generation of memory, require an evolutionary and multidisiplinary approach for its study.

Los linfocitos B son unos de los tipos celulares más importantes de la respuesta inmunitaria de los mamíferos. El origen y evolución de este tipo celular se desconocen, pero el linfocito B bona fide aparece en peces. En esta revisión se analizan los principales componentes de la respuesta inmunitaria en invertebrados, su distribución filogenética y la permanencia de algunas propiedades que permitieron el surgimiento del linfocito B. Se parte de la idea de que muchos de los componentes que caracterizan a los linfocitos B están distribuidos desde los invertebrados; sin embargo, es en el linfocito donde se integran todos estos componentes que le dan identidad a este tipo celular. El conocimiento actual de los linfocitos proviene, en su mayor parte, del estudio de la fisiología en mamíferos y como mayor representante el ratón. El origen del linfocito B, sus mecanismos alternativos de generación de diversidad de receptores, su respuesta inmunitaria efectora y la generación de memoria, requieren para su estudio de un abordaje multidisciplinario y con enfoque evolutivo.

The genetic program for cartilage development has deep homology within Bilateria.

The evolution of novel cell types led to the emergence of new tissues and organs during the diversification of animals. The origin of the chondrocyte, the cell type that synthesizes cartilage matrix, was central to the evolution of the vertebrate endoskeleton. Cartilage-like tissues also exist outside the vertebrates, although their relationship to vertebrate cartilage is enigmatic. Here we show that protostome and deuterostome cartilage share structural and chemical properties, and that the mechanisms of cartilage development are extensively conserved--from induction of chondroprogenitor cells by Hedgehog and ß-catenin signalling, to chondrocyte differentiation and matrix synthesis by SoxE and SoxD regulation of clade A fibrillar collagen (ColA) genes--suggesting that the chondrogenic gene regulatory network evolved in the common ancestor of Bilateria. These results reveal deep homology of the genetic program for cartilage development in Bilateria and suggest that activation of this ancient core chondrogenic network underlies the parallel evolution of cartilage tissues in Ecdysozoa, Lophotrochozoa and Deuterostomia.

Oocyte aging in a marine protostome worm: The roles of maturation-promoting factor and extracellular signal regulated kinase form of mitogen-activated protein kinase.

The roles of maturation-promoting factor (MPF) and an extracellular signal regulated kinase form of mitogen-activated protein kinase (ERK MAPK) are analyzed during oocyte aging in the marine protostome worm Cerebratulus. About a day after removal from the ovary, unfertilized metaphase-I-arrested oocytes of Cerebratulus begin to flatten and swell before eventually lysing, thereby exhibiting characteristics of a necroptotic mode of regulated cell death. Based on immunoblots probed with phospho-specific antibodies, MPF and ERK are initially active in freshly mature specimens. However, as oocytes age, both kinase activities decline, with ERK deactivation occurring well before MPF downregulation. Experiments using pharmacological modulators indicate that oocyte degradation is promoted by the maturation-initiated activation of ERK as well as by the deactivation of MPF that occurs in extensively aged specimens. The potential significance of these findings is discussed relative to previously published results for apoptotic eggs and oocytes of echinoderm and vertebrate deuterostomes.