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Indirect development with an intermediate larva exists in all major animal lineages 1 , which makes larvae central to most scenarios of animal evolution 2-11 . Yet how larvae evolved remains disputed. Here we show that temporal shifts (that is, heterochronies) in trunk formation underpin the diversification of larvae and bilaterian life cycles. We performed chromosome-scale genome sequencing in the annelid Owenia fusiformis with transcriptomic and epigenomic profiling during the life cycles of this and two other annelids. We found that trunk development is deferred to pre-metamorphic stages in the feeding larva of O. fusiformis but starts after gastrulation in the non-feeding larva with gradual metamorphosis of Capitella teleta and the direct developing embryo of Dimorphilus gyrociliatus. Accordingly, the embryos of O. fusiformis develop first into an enlarged anterior domain that forms larval tissues and the adult head 12 . Notably, this also occurs in the so-called 'head larvae' of other bilaterians 13-17 , with which the O. fusiformis larva shows extensive transcriptomic similarities. Together, our findings suggest that the temporal decoupling of head and trunk formation, as maximally observed in head larvae, facilitated larval evolution in Bilateria. This diverges from prevailing scenarios that propose either co-option 9,10 or innovation 11 of gene regulatory programmes to explain larva and adult origins., (© 2023. The Author(s).)

The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling plays a key role in establishing the dorsal-ventral (D-V) axis and limiting the neuroectoderm to one side of that axis, leading to speculation about the conserved evolution of centralized nervous systems. Studies outside of insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (≈Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. To determine the function of BMP signaling during early development of the spiralian annelid Capitella teleta, we incubated embryos and larvae in BMP4 protein for different amounts of time. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, foregut, and ventral midline in a time-dependent manner. However, adding BMP did not block brain or VNC formation or majorly disrupt the D-V axis. We identified three key time windows of BMP activity. 1) BMP treatment around birth of the 3rd-quartet micromeres caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2) Treatment after the birth of micromere 4d induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3) Continuous BMP treatment from late cleavage (4d ​+ ​12 ​h) through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric ventral nerve cord, although the total amount of neural tissue was not greatly affected. Our results compared with those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue to one region of the D-V axis. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a non-axial organizing signal that was present in the last common ancestor of annelids and mollusks. Furthermore, in the last common ancestor of annelids, BMP signaling may have functioned in patterning ectodermal fates along the D-V axis in the trunk. Ultimately, studies on a wider range of spiralian taxa are needed to determine the role of BMP signaling during neural induction and neural patterning in the last common ancestor of this group. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pseudopotamilla occelata is a polychaete worm distributed widely in the northern part of the Pacific coast, having value as fishing bait as well as biological material for some basic research areas, including reproduction. Here we describe methods for handling the gametes and embryos of this worm, focusing on such topics as maintenance of adults, induction of oocyte maturation and fertilization, culture of embryos and larvae, microinjection into oocytes, and calcium (Ca 2+ ) imaging.

Platynereis dumerilii is a marine segmented worm (annelid) with externally fertilized embryos and it can be cultured for the full life cycle in the laboratory. The accessibility of embryos and larvae combined with the breadth of the established molecular and functional techniques has made P. dumerilii an attractive model for studying development, cell lineages, cell type evolution, reproduction, regeneration, the nervous system, and behavior. Traditionally, these worms have been kept in rooms dedicated for their culture. This allows for the regulation of temperature and light cycles, which is critical to synchronizing sexual maturation. However, regulating the conditions of a whole room has limitations, especially if experiments require being able to change culturing conditions. Here we present scalable and flexible culture methods that provide ability to control the environmental conditions, and have a multi-purpose culture space. We provide a closed setup shelving design with proper light conditions necessary for P. dumerilii to mature. We also implemented a standardized method of feeding P. dumerilii cultures with powdered spirulina which relieves the ambiguity associated with using frozen spinach, and helps standardize nutrition conditions across experiments and across different labs. By using these methods, we were able to raise mature P. dumerilii, capable of spawning and producing viable embryos for experimentation and replenishing culture populations. These methods will allow for the further accessibility of P. dumerilii as a model system, and they can be adapted for other aquatic organisms., Competing Interests: The authors have declared that no competing interests exist.

Chlorine-disinfected sewage effluents are typically dechlorinated by using NaHSO 3 , Na 2 SO 3 , or Na 2 S 2 O 3 , as chlorine residual could be harmful to aquatic organisms upon discharge of sewage effluents into receiving marine water. In this study, we systematically investigated the effects of dechlorination-related factors on the developmental toxicity of a chlorinated saline primary sewage effluent, via direct exposure of the embryos of a marine polychaete to the effluent. The results showed that dechlorination ratio (i.e., the ratio of the dosed amount to the requisite stoichiometric amount of a dechlorination agent) and mixing condition were critical factors affecting the toxicity of the effluent. The toxicity of the effluent under insufficient dechlorination conditions was mainly caused by residual chlorine, especially monochloramine. Although the three dechlorination agents generally performed similarly, dechlorination with Na 2 S 2 O 3 required a more vigorous mixing condition than that with NaHSO 3 or Na 2 SO 3 , as the relatively high density of Na 2 S 2 O 3 might affect the mixing efficiency. Under insufficient mixing conditions, a prolonged dechlorination time was beneficial to achieving complete dechlorination and thus lowered the toxicity of the effluent. Moreover, because disinfection byproducts (DBPs) may have chronic effects on aquatic organisms, the developmental toxicity of the DBP mixtures in the chlorinated effluent in different dechlorination scenarios was also evaluated. The results indicated that increasing the dechlorination ratio reduced the developmental toxicity of the DBP mixture in the chlorinated saline sewage effluent, which might be ascribed to the decrease of the levels of overall brominated and iodinated DBPs; the dechlorination agent (NaHSO 3 or Na 2 S 2 O 3 ) might act as a nucleophile in the nucleophilic substitution and cause the substitution of bromine or iodine atoms in brominated and iodinated DBPs. The results from this study might aid in the design and operation of dechlorination facilities in sewage treatment plants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: During early development, patterns of cell division-embryonic cleavage-accompany the gradual restriction of blastomeres to specific cell fates. In Spiralia, which include annelids, mollusks, and flatworms, "spiral cleavage" produces a highly stereotypic, spiral-like arrangement of blastomeres and swimming trochophore-type larvae with rotational (spiral) symmetry. However, starting at larval stages, spiralian larvae acquire elements of bilateral symmetry, before they metamorphose into fully bilateral juveniles. How this spiral-to-bilateral transition occurs is not known and is especially puzzling for the early differentiating brain and head sensory organs, which emerge directly from the spiral cleavage pattern. Here we present the developmental cell lineage of the Platynereis larval episphere., Results: Live-imaging recordings from the zygote to the mid-trochophore stage (~ 30 hpf) of the larval episphere of the marine annelid Platynereis dumerilii reveal highly stereotypical development and an invariant cell lineage of early differentiating cell types. The larval brain and head sensory organs develop from 11 pairs of bilateral founders, each giving rise to identical clones on the right and left body sides. Relating the origin of each bilateral founder pair back to the spiral cleavage pattern, we uncover highly divergent origins: while some founder pairs originate from corresponding cells in the spiralian lineage on each body side, others originate from non-corresponding cells, and yet others derive from a single cell within one quadrant. Integrating lineage and gene expression data for several embryonic and larval stages, we find that the conserved head patterning genes otx and six3 are expressed in bilateral founders representing divergent lineage histories and giving rise to early differentiating cholinergic neurons and head sensory organs, respectively., Conclusions: We present the complete developmental cell lineage of the Platynereis larval episphere, and thus the first comprehensive account of the spiral-to-bilateral transition in a developing spiralian. The bilateral symmetry of the head emerges from pairs of bilateral founders, similar to the trunk; however, the head founders are more numerous and show striking left-right asymmetries in lineage behavior that we relate to differential gene expression.

It has been proposed that animals have a pattern of developmental evolution resembling an hourglass because the most conserved development stage-often called the phylotypic stage-is always in midembryonic development. Although the topic has been debated for decades, recent studies using molecular data such as RNA-seq gene expression data sets have largely supported the existence of periods of relative evolutionary conservation in middevelopment, consistent with the phylotypic stage and the hourglass concepts. However, so far this approach has only been applied to a limited number of taxa across the tree of life. Here, using established phylotranscriptomic approaches, we found a surprising reverse hourglass pattern in two molluscs and a polychaete annelid, representatives of the Spiralia, an understudied group that contains a large fraction of metazoan body plan diversity. These results suggest that spiralians have a divergent midembryonic stage, with more conserved early and late development, which is the inverse of the pattern seen in almost all other organisms where these phylotranscriptomic approaches have been reported. We discuss our findings in light of proposed reasons for the phylotypic stage and hourglass model in other systems., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Spiral cleavage is a mode of embryonic cell division found in species from several Phyla, including molluscs, annelids and flatworms. It reflects a tilting in the direction of spindle orientation and cell division at the 4 to 8-cell stage, which may be dextral or sinistral, and propagates into later organismal asymmetry. Genetic analysis in a small number of gastropod molluscs shows the direction of spiral cleavage is determined by maternal genotype, though whether this is also the case more generally for spiralians, and whether spiral cleavage at the 4-8 cell stage is preceded by earlier internal chirality in any spiralian species, is unknown. Here we study the early cleavage stages of two equal-cleaving spiralians, the dextral annelid Spirobranchus lamarcki and the sinistral mollusc Biomphalaria glabrata, using light sheet microscopy to image subcellular vesicles in live embryos and asking if chirality of movement is identifiable. We observe variability in the early cleavage of S. lamarcki , including a viable 3-cell stage. Image data are analysed by both particle tracking and particle image velocimetry. Neither finds evidence for chiral movement in 1-, 2-, 3-, or 4-cell embryos, nor do we detect consistent differences between the embryos of the dextral and sinistrai species. The methodological and evolutionary implications of this are discussed., (© 2019 Zoological Society of Japan.)

Background: The marine polychaete annelid Platynereis dumerilii has recently emerged as a prominent organism for the study of development, evolution, stem cells, regeneration, marine ecology, chronobiology and neurobiology within metazoans. Its phylogenetic position within the spiralian/ lophotrochozoan clade, the comparatively high conservation of ancestral features in the Platynereis genome, and experimental access to any stage within its life cycle, make Platynereis an important model for elucidating the complex regulatory and functional molecular mechanisms governing early development, later organogenesis, and various features of its larval and adult life. High resolution RNA-seq gene expression data obtained from specific developmental stages can be used to dissect early developmental mechanisms. However, the potential for discovery of these mechanisms relies on tools to search, retrieve, and compare genome-wide information within Platynereis, and across other metazoan taxa., Results: To facilitate exploration and discovery by the broader scientific community, we have developed a web-based, searchable online research tool, PdumBase, featuring the first comprehensive transcriptome database for Platynereis dumerilii during early stages of development (2 h ~ 14 h). Our database also includes additional stages over the P. dumerilii life cycle and provides access to the expression data of 17,213 genes (31,806 transcripts) along with annotation information sourced from Swiss-Prot, Gene Ontology, KEGG pathways, Pfam domains, TmHMM, SingleP, and EggNOG orthology. Expression data for each gene includes the stage, the normalized FPKM, the raw read counts, and information that can be leveraged for statistical analyses of differential gene expression and the construction of genome-wide co-expression networks. In addition, PdumBase offers early stage transcriptome expression data from five further species as a valuable resource for investigators interested in comparing early development in different organisms. To understand conservation of Platynereis gene models and to validate gene annotation, most Platynereis gene models include a comprehensive phylogenetic analysis across 18 species representing diverse metazoan taxa., Conclusions: PdumBase represents the first online resource for the early developmental transcriptome of Platynereis dumerilii. It serves as a research platform for discovery and exploration of gene expression during early stages, throughout the Platynereis life cycle, and enables comparison to other model organisms. PdumBase is freely available at http://pdumbase.gdcb.iastate.edu .

Embryonic organizers are signaling centers that coordinate developmental events within an embryo. Localized to either an individual cell or group of cells, embryonic organizing activity induces the specification of other cells in the embryo and can influence formation of body axes. In the spiralian Capitella teleta, previous cell deletion studies have shown that organizing activity is localized to a single cell, 2d, and this cell induces the formation of the dorsal-ventral axis and bilateral symmetry. In this study, we attempt to identify the signaling pathway responsible for the organizing activity of 2d. Embryos at stages when organizing activity is occurring were exposed to various small molecule inhibitors that selectively inhibited either the Activin/Nodal or the BMP branch of the TGF-β signaling pathway. Embryos were then raised to larval stages, and scored for axial anomalies analogous to 2d ablated phenotypes. Our results show that interference with the Activin/Nodal pathway through a short three hour exposure to the inhibitor SB431542 results in larvae that lack bilateral symmetry and a detectable dorsal-ventral axis. However, interference with the BMP signaling pathway through exposure to the inhibitors DMH1 and dorsomorphin dihydrochloride does not appear to play a role in specification by 2d of the dorsal-ventral axis or bilateral symmetry. Our findings highlight species differences in how the molecular architecture of the conserved TGF-β superfamily signaling pathway components was utilized to mediate the organizing activity signal during early spiralian development., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification.

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos.

Spiralians, including molluscs, annelids and platyhelminths, share a unique development process that includes the typical geometry of early cleavage and early segregation of cell fate in blastomeres along the animal-vegetal axis. However, the molecular mechanisms underlying this early cell fate segregation are largely unknown. Here, we report spiralian-specific expansion of the three-amino-acid loop extension (TALE) class of homeobox genes. During early development, some of these TALE genes are expressed in staggered domains along the animal-vegetal axis in the limpet Nipponacmea fuscoviridis and the polychaete Spirobranchus kraussii. Inhibition or overexpression of these genes alters the developmental fate of blastomeres, as predicted by the gene expression patterns. These results suggest that the expansion of novel TALE genes plays a critical role in the establishment of a novel cell fate segregation mechanism in spiralians.

In the deuterostomes and ecdysozoans that have been studied (e.g. chordates and insects), neural fate specification relies on signaling from surrounding cells. However, very little is known about mechanisms of neural specification in the third major bilaterian clade, spiralians. Using blastomere isolation in the annelid Capitella teleta, a spiralian, we studied to what extent extrinsic versus intrinsic signals are involved in early neural specification of the brain and ventral nerve cord. For the first time in any bilaterian, we found that brain neural ectoderm is autonomously specified. This occurs in the daughters of first-quartet micromeres, which also generate anterior neural ectoderm in other spiralians. In contrast, isolation of the animal cap, including the 2d micromere, which makes the trunk ectoderm and ventral nerve cord, blocked ventral nerve cord formation. When the animal cap was isolated with the 2D macromere, the resulting partial larvae had a ventral nerve cord. These data suggest that extrinsic signals from second-quartet macromeres or their daughters, which form mesoderm and endoderm, are required for nerve cord specification in C. teleta and that the 2D macromere or its daughters are sufficient to provide the inductive signal. We propose that autonomous specification of anterior neural ectoderm evolved in spiralians in order to enable them to quickly respond to environmental cues encountered by swimming larvae in the water column. In contrast, a variety of signaling pathways could have been co-opted to conditionally specify the nerve cord. This flexibility of nerve cord development may be linked to the large diversity of trunk nervous systems present in Spiralia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chlorination is extensively applied for disinfecting sewage effluents, but it unintentionally generates disinfection byproducts (DBPs). Using seawater for toilet flushing introduces a high level of bromide into domestic sewage. Chlorination of sewage effluent rich in bromide causes the formation of brominated DBPs. The objectives of achieving a disinfection goal, reducing disinfectant consumption and operational costs, as well as diminishing adverse effects to aquatic organisms in receiving water body remain a challenge in sewage treatment. In this study, we have demonstrated that, with the same total chlorine dosage, a three-step chlorination (dosing chlorine by splitting it into three equal portions with a 5-min time interval for each portion) was significantly more efficient in disinfecting a primary saline sewage effluent than a one-step chlorination (dosing chlorine at one time). Compared to one-step chlorination, three-step chlorination enhanced the disinfection efficiency by up to 0.73-log reduction of Escherichia coli. The overall DBP formation resulting from one-step and three-step chlorination was quantified by total organic halogen measurement. Compared to one-step chlorination, the DBP formation in three-step chlorination was decreased by up to 23.4%. The comparative toxicity of one-step and three-step chlorination was evaluated in terms of the development of embryo-larva of a marine polychaete Platynereis dumerilii. The results revealed that the primary sewage effluent with three-step chlorination was less toxic than that with one-step chlorination, indicating that three-step chlorination could reduce the potential adverse effects of disinfected sewage effluents to aquatic organisms in the receiving marine water., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The expression of transcription factors with endodermal and mesodermal roles in bilaterians is characterized during the development of Hydroides elegans, a serpulid polychaete with planktotrophic trochophore. GATA 4/5/6 is expressed in endodermal and mesodermal precursors during embryogenesis and in the midgut of trochophore larvae. HeGATA1/2/3a is expressed in animal hemisphere blastomeres 1d121 and 1d122, in dorsal ectoderm and in 4d endomesodermal derivatives that maintain their expression in trochophore larvae. HeGATA1/2/3b is not expressed during embryogenesis, but in several regions of the larva during postembryonic development. During very early gastrulation, Brn1/2/4 is first expressed in cells associated with the prospective oral/foregut side of the blastopore, and during larval development in 4d blastomere descendants. Comparison with orthologs in other metazoans suggests ancestral expression of GATA4/5/6 in the midgut of the last common ancestor of protostomes and deuterostomes. The conserved expression of Brn1/2/4 in the foregut precursors of Hydroides and sea urchins suggests an ancestral role in patterning the tripartite gut of planktotrophic larvae. Broader analysis of these and other regulatory genes reveals variability of developmental gene expression among polychaetes with lecithotrophic larvae, suggesting that they are evolutionarily derived from polychaetes with planktotrophic larvae., (© 2016 Wiley Periodicals, Inc.)

The stereotypic cleavage pattern shared by spiralian embryos provides unique opportunities to compare mechanisms of cell fate specification of homologous blastomeres, and can give insights into how changes in fate may have influenced the evolution of novel structures and morphological diversity. The potential of cells to undergo regulation and the timing of cell fate specification were investigated during early development in the polychaete annelid, Capitella teleta. Targeted laser deletions of the first quartet micromeres were performed, with a focus on the eye-forming cells 1a and 1c. Most of the larvae resulting from deletion of the 1a or 1c micromeres lack both the pigment cell and sensory cell of the eye as predicted by the C. teleta fate map. In a minority of cases, however, both left and right larval eye spots develop, suggesting that other blastomeres within the embryo regulate for loss of these cells. Deletion of the 1a and 1c derivatives, 1a(1) or 1c(1), also largely result in larvae with one pigment spot, although there are larvae with two eye spots, suggesting that the ability to regulate for loss of an eye-generating cell persists for an additional cell cycle. Cell deletion in conjunction with intracellular labeling indicates that all four quadrants retain the ability to generate eyes, including those that normally do not. Deletion of all four first quartet micromeres provides evidence that only the first quartet micromeres have eye-forming potential. Additionally, in contrast to the right side of the head where larval and adult eye sensory cells are derived from the same cell (1c), on the left side, the larval and adult eye sensory cells are generated by different embryonic lineages. We hypothesize that cell-cell interactions and cell position are important for regulative ability in Capitella. To our knowledge, this is one of the first detailed deletion studies of the first quartet micromeres and the first convincing example of regulation in polychaetes, which are often thought to be non-regulative in nature., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Chlorine/chloramine residuals are maintained in drinking water distribution systems to prevent microbial contamination and microorganism regrowth. During household cooking processes (e.g., soup making), the residual chlorine/chloramines in tap water may react with the iodide in iodized table salt to form hypoiodous acid, which could react with remaining natural organic matter in tap water and organic matter in food to generate iodinated disinfection byproducts (I-DBPs). However, I-DBPs formed during cooking with chloraminated/chlorinated tap water are almost completely new to researchers. In this work, by adopting precursor ion scan of m/z 127 using ultra performance liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry, many new polar I-DBPs formed during cooking with chloraminated/chlorinated tap water were detected and proposed with structures, of which 3-iodo-4-hydroxybenzaldehyde, 3-iodo-4-hydroxybenzoic acid, 3-iodo-4-hydroxy-5-methylbenzoic acid, diiodoacetic acid, 3,5-diiodo-4-hydroxybenzaldehyde, 3,5-diiodo-4-hydroxybenzoic acid, 2,6-diiodo-4-nitrophenol, 2,4-diiodo-6-nitrophenol, and 2,4,6-triiodophenol were confirmed with standard compounds. With the aid of ultra fast liquid chromatography/ion trap-time of flight-mass spectrometry, molecular formula identification of five new I-DBPs (C8H5O4I, C7H4NO4I, C8H5O5I, C7H4NO5I, and C8H6O3I2) was achieved. A developmental toxicity with a recently developed sensitive bioassay was conducted for the newly identified I-DBPs, suggesting that phenolic I-DBPs (except for iodinated carboxyphenols) were about 50-200 times more developmentally toxic than aliphatic I-DBPs. The major I-DBPs in a baseline simulated cooking water sample were determined to be from 0.72 to 7.63 μg/L. Polar I-DBPs formed under various disinfection and cooking conditions were compared, and suggestions for controlling their formation were provided., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Matured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation.

The serpulid tubeworm, Hydroides elegans, is an ecologically and economically important species whose biology has been fairly well studied, especially in the context of larval development and settlement on man-made objects (biofouling). Nevertheless, ontogenetic changes associated with calcareous tube composition and structures have not yet been studied. Here, the ultrastructure and composition of the calcareous tubes built by H. elegans was examined in the three early calcifying juvenile stages and in the adult using XRD, FTIR, ICP-OES, SEM and Raman spectroscopy. Ontogenetic shifts in carbonate mineralogy were observed, for example, juvenile tubes contained more amorphous calcium carbonate and were predominantly aragonitic whereas adult tubes were bimineralic with considerably more calcite. The mineral composition gradually shifted during the tube development as shown by a decrease in Sr/Ca and an increase of Mg/Ca ratios with the tubeworm's age. The inner tube layer contained calcite, whereas the outer layer contained aragonite. Similarly, the tube complexity in terms of ultrastructure was associated with development. The sequential appearance of unoriented ultrastructures followed by oriented ultrastructures may reflect the evolutionary history of serpulid tube biominerals. As aragonitic structures are more susceptible to dissolution under ocean acidification (OA) conditions but are more difficult to be removed by anti-fouling treatments, the early developmental stages of the tubeworms may be vulnerable to OA but act as the important target for biofouling control., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Segmented body organizations are widely represented in the animal kingdom. Whether the last common bilaterian ancestor was already segmented is intensely debated. Annelids display broad morphological diversity but many species are among the most homonomous metameric animals. The front end (prostomium) and tail piece (pygidium) of annelids are classically described as non-segmental. However, the pygidium structure and development remain poorly studied., Results: Using different methods of microscopy, immunolabelling and a number of molecular markers, we describe the neural and mesodermal structures of the pygidium of Platynereis dumerilii. We establish that the pygidium possesses a complicated nervous system with a nerve ring and a pair of sensory ganglia, a complex intrinsic musculature, a large terminal circular blood sinus and an unusual unpaired torus-shaped coelomic cavity. We also describe some earlier steps of pygidial development and pygidial structure of mature animals after epitokous transformation., Conclusions: We describe a much more complex organization of the pygidium of P. dumerilii than previously suggested. Many of the characteristics are strikingly similar to those found in the trunk segments, opening the debate on whether the pygidium and trunk segments derive from the same ancestral metameric unit. We analyze these scenarios in the context of two classical theories on the origin of segmentation: the cyclomeric/archicoelomate concept and the colonial theory. Both theories provide possible explanations for the partial or complete homology of trunk segments and pygidium.

The notochord is considered an evolutionary novelty and one of the defining characters of chordates. A new study of an annelid challenges this view and proposes an earlier evolutionary origin in the most recent common ancestor of chordates and annelids., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Marine sediments can contain B vitamins, presumably incorporated from settled, decaying phytoplankton and microorganisms associated with decomposition. Because B vitamins may be advantageous for the energetically intensive processes of metamorphosis, post-metamorphic growth, and reproduction, we tested several B vitamins to determine if they would stimulate larvae of the deposit-feeding polychaete Capitella teleta to settle and metamorphose. Nicotinamide and riboflavin individually stimulated larvae of C. teleta to settle and metamorphose, generally within 1-2 hours at nicotinamide concentrations as low as 3 µM and riboflavin concentrations as low as 50 µM. More than 80% of the larvae metamorphosed within 30 minutes at a nicotinamide concentration of 7 µM. The pyridine channel agonist pyrazinecarboxamide also stimulated metamorphosis at very low concentrations. In contrast, neither lumichrome, thiamine HCl, pyridoxine HCl, nor vitamin B12 stimulated larvae of C. teleta to metamorphose at concentrations as high as 500 µM. Larvae also did not metamorphose in response to either nicotinamide or pyrazinecarboxamide in calcium-free seawater or with the addition of 4-acetylpyridine, a competitive inhibitor of the pyridine receptor. Together, these results suggest that larvae of C. teleta are responding to nicotinamide and riboflavin via a chemosensory pyridine receptor similar to that previously reported to be present on crayfish chela and involved with food recognition. Our data are the first to implicate B vitamins as possible natural chemical settlement cues for marine invertebrate larvae.

The origin of chordates has been debated for more than a century, with one key issue being the emergence of the notochord. In vertebrates, the notochord develops by convergence and extension of the chordamesoderm, a population of midline cells of unique molecular identity. We identify a population of mesodermal cells in a developing invertebrate, the marine annelid Platynereis dumerilii, that converges and extends toward the midline and expresses a notochord-specific combination of genes. These cells differentiate into a longitudinal muscle, the axochord, that is positioned between central nervous system and axial blood vessel and secretes a strong collagenous extracellular matrix. Ancestral state reconstruction suggests that contractile mesodermal midline cells existed in bilaterian ancestors. We propose that these cells, via vacuolization and stiffening, gave rise to the chordate notochord., (Copyright © 2014, American Association for the Advancement of Science.)

Hypoxia likely compromises the reproductive success of those marine organisms carrying out external fertilization because their gametes and embryos are inevitably exposed to the external environment. Hydroides elegans, a dominant serpulid polychaete in Hong Kong waters, can spawn throughout the year but the number of recruits drops during summer when hypoxia commonly occurs. This study attempted to explain such observation by investigating the gamete quality, including sperm motility, egg size, complexity and viability, after 1-h hypoxic exposure (1 mg O2 l(-1)). In addition, how gamete quality affects fertilization success and embryonic development was examined. After 1-h hypoxic exposure, sperm motility was significantly reduced, compromising fertilization success. Although the eggs remained viable, more malformed embryos and retarded embryonic development were observed. We interpreted that the harmful effect of hypoxia on embryonic development was attributed to the teratogenicity and induced oxidative stress, ultimately causing the reduction in recruitment during summer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, we aimed to identify molecular mechanisms involved in the specification of the 4d (mesentoblast) lineage in Platynereis dumerilii. We employ RT-PCR and in situ hybridization against the Platynereis dumerilii twist homolog (Pdu-twist) to reveal mesodermal specification within this lineage. We show that Pdu-twist mRNA is already maternally distributed. After fertilization, ooplasmatic segregation leads to relocation of Pdu-twist transcripts into the somatoblast (2d) lineage and 4d, indicating that the maternal component of Pdu-twist might be an important prerequisite for further mesoderm specification but does not represent a defining characteristic of the mesentoblast. However, after the primordial germ cells have separated from the 4d lineage, zygotic transcription of Pdu-twist is exclusively observed in the myogenic progenitors, suggesting that mesodermal specification occurs after the 4d stage. Previous studies on spiral cleaving embryos revealed a spatio-temporal correlation between the 4d lineage and the activity of an embryonic organizer that is capable to induce the developmental fates of certain micromeres. This has raised the question if specification of the 4d lineage could be connected to the organizer activity. Therefore, we aimed to reveal the existence of such a proposed conserved organizer in Platynereis employing antibody staining against dpERK. In contrast to former observations in other spiralian embryos, activation of MAPK signaling during 2d and 4d formation cannot be detected which questions the existence of a conserved connection between organizer function and specification of the 4d lineage. However, our experiments unveil robust MAPK activation in the prospective nephroblasts as well as in the macromeres and some micromeres at the blastopore in gastrulating embryos. Inhibition of MAPK activation leads to larvae with a shortened body axis, defects in trunk muscle spreading and improper nervous system condensation, indicating a critical function for MAPK signaling for the reorganization of embryonic tissues during the gastrulation process.

Elucidating the origin of germ cells in embryos and larvae is often obscured by the fact that the typical germ cell markers vasa, nanos and piwi are not exclusively expressed in primordial germ cells (PGCs), but are also commonly found in undifferentiated somatic tissues and stem cells as part of an evolutionary conserved 'germline multipotency program' (Juliano et al., 2010). Hidden in the crowd of undifferentiated cells, the PGCs have occasionally been overlooked and their formation during early embryogenesis was only revealed recently by new methodological approaches (e.g. Wu et al., 2011). Spiralians are excellent model organisms to deepen our understanding of PGC formation, given the highly stereotypical cleavage that occurs during embryogenesis. In these species, detailed cell lineage studies enable the tracing of single cells up to gastrulation stages. Here, I review our knowledge of the origin of PGCs in these invertebrates. Similarities in PGC formation among spiralian phyla as well as peculiarities of the highly derived clitellates are discussed with respect to developmental mode and evolution. Furthermore, the issue of gonad regeneration in platyhelminths and the asexually reproducing oligochaete Enchytraeus japonensis is addressed. An alternative strategy of compensating for caudal regeneration is presented for the polychaete Platynereis dumerilli. Finally, the molecular bases of PGC specification and the question of germplasm are discussed.

The highly conserved wnt gene family has roles in developmental processes ranging from axis formation to cell fate determination. The polychaete Platynereis dumerilii has retained 12 of the 13 ancient wnt subfamilies and is a good model system to study the roles of the wnt ligands in spiralian development. While it has been shown that Platynereis uses a global beta-catenin-mediated binary cell fate specification module in development, the early roles of the 12 wnt genes present in Platynereis are unknown. Transcriptional profiling by RNA-Seq during early development and whole-mount in situ hybridization of embryo and larval stages were used to determine the temporal and spatial regulation of the wnt complement in Platynereis. None of the 12 wnt transcripts were maternally provided at significant levels. In pregastrula embryos, zygotic wntA, wnt4, and wnt5 transcripts exhibited distinctive patterns of differential gene expression. In contrast, in trochophore larvae, all 12 wnt ligands were expressed and each had a distinct expression pattern. While three wnt ligands were expressed in early development, none were expressed in the right place for a widespread role in beta-catenin-mediated binary specification in early Platynereis development. However, the expression patterns of the wnt ligands suggest the presence of numerous wnt signaling centers, with the most prominent being a bias for staggered posterior wnt expression in trochophore larvae. The similarity to wnt expression domains in cnidarians around the blastopore and the tail organizer in chordates supports a hypothesis of a common evolutionary origin of posterior organizing centers.

Hydroides elegans is an indirectly developing polychaete with equal spiral cleavage, gastrulation by invagination, and a feeding trochophore. Expression of several transcription factors and differentiation genes has been characterized. Comparative analysis reveals evolutionarily conserved roles. For example, the synexpression of transcription factors FoxA and Brachyury suggests homology of primary and secondary gut openings in protostomes and deuterostomes, and the expression of Sall suggests similar regulatory controls in the posterior growth zone of bilaterians. Differences in gene expression suggest regulatory differences control gastrulation by invagination in polychaetes with a feeding trochophore and gastrulation by epiboly in polychaetes without a feeding trochophore. Association of histone variant H2A.Z with transcriptional potency and its expression suggest a developmental role during both embryogenesis and the larva-to-adult transformation. Methods are being developed for experimental exploration of the gene regulatory networks involved in trochophore development in Hydroides. It is unknown if polychaete feeding trochophores evolved from a larval stage already present in the life cycle of the last common ancestor of protostomes and deuterostomes. Previous evolutionary scenarios about larval origins overemphasize the discontinuity between larval and adult development and require the early evolution of undifferentiated and transcriptionally potent "set aside" cells. Indirect development may proceed by developmental remodeling of differentiated cells and could have evolved after gradual transformation of juveniles into larvae; undifferentiated and transcriptionally potent cells would have evolved secondarily. Comprehensive characterization of gene regulatory networks for feeding trochophore development may help resolve these major evolutionary questions.

The marine polychaete Streblospio benedicti exhibits two distinct larval types, making it a model for the study of developmental evolution. Females produce either large eggs or small ones, which develop into distinct lecithotrophic or planktotrophic larvae with concomitant morphological and life-history differences. Here, we investigate the inheritance of key morphological traits that distinguish the larval types. We used genetic crosses to establish the influence of maternal and zygotic differences on larval phenotypes. We found a large maternal effect on larval size and the number of larval chaetae, while the number and length of these chaetae were also strongly influenced by zygotic genotype. Interestingly, the distribution of larval phenotypes produced by these crosses suggests traits intermediate to the two parental types should not be uncommon. Yet, despite gene flow between the types in natural populations, such intermediates are rarely found in nature, suggesting that selection may be maintaining distinct larval modes.

Spiralian development is characterized by the conservation of spindle orientation and cell geometry during early cleavage stages, as well as features of the ultimate fates of identified cells. This complex set of characters is shared by a number of animal lineages including nemerteans, polyclad platyhelminthes, annelids and mollusks. How a similar, highly stereotypical cleavage program can give rise to such diversity of larval and adult forms has intrigued researchers for many years. This review summarizes recent data from polychaete annelids, and highlights both conservation and variation in the cellular and molecular mechanisms that guide the spiral cleavage developmental program. There is a specific focus on comparisons of fate maps, patterns of cleavage, mechanisms of cell fate specification, organizing activity, and differences in molecular patterning. Some of the differences in early development represent intra-clade variation within annelids, and others hint at differences between annelids and other taxa. Because much of the classic work on spiralians has focused on mollusks, these new data from annelids have expanded our knowledge about the evolutionary flexibility in spiralian development and potentially its role in body plan evolution.

The nereidid annelid Platynereis dumerilii emerged as a well-understood model organism. P. dumerilii and P. massiliensis are sister taxa, which are morphologically indistinguishable as adults. Interestingly, they exhibit highly contrasting life-histories: while P. dumerilii is a gonochorostic species with planktonic feeding larvae, P. massiliensis is a protandric hermaphrodite with lecitotrophic semi-direct -development in brood tubes. Using light microscopy and immunohistochemical methods coupled with confocal laser scanning microscopy, we describe the development of P. massiliensis. Musculature was stained with phalloidin-rhodamine. FMRFamide, acetylated α-tubulin, and serotonin were targeted by antibodies for the staining of neuronal structures. Additionally, eye development was investigated with the specific 22C10-antibody. The development of P. massiliensis is characterized by the absence of a free-swimming stage, a late development of food uptake, and the presence of a large amount of yolk even in late juvenile stages. Most notably, early juvenile stages already exhibit an organization of several organ systems that resembles those of adults. Larval characters present in the free-swimming feeding larvae of P. dumerilii, as e.g. the apical organ and larval eyes, are absent and regarded to be lost in developing stages of P. massiliensis. Many of the differences found in the development of these two species can be described in the context of heterochronic changes. We strongly advocate expanding evolutionary developmental studies from the well-established model annelid P. dumerilii to the closely related P. massiliensis to study the evolutionary conservation and divergence of genetic pathways involved in developmental processes.

Sipuncula is an ancient clade of unsegmented marine worms that develop through a conserved pattern of unequal quartet spiral cleavage. They exhibit putative character modifications, including conspicuously large first-quartet micromeres and prototroch cells, postoral metatroch with exclusive locomotory function, paired retractor muscles and terminal organ system, and a U-shaped digestive architecture with left-right asymmetric development. Four developmental life history patterns are recognized, and they have evolved a unique metazoan larval type, the pelagosphera. When compared with other quartet spiral-cleaving models, sipunculan development is understudied, challenging and typically absent from evolutionary interpretations of spiralian larval and adult body plan diversity. If spiral cleavage is appropriately viewed as a flexible character complex, then understudied clades and characters should be investigated. We are pursuing sipunculan models for modern molecular, genetic and cellular research on evolution of spiralian development. Protocols for whole mount gene expression studies are established in four species. Molecular labeling and confocal imaging techniques are operative from embryogenesis through larval development. Next-generation sequencing of developmental transcriptomes has been completed for two species with highly contrasting life history patterns, Phascolion cryptum (direct development) and Nephasoma pellucidum (indirect planktotrophy). Looking forward, we will attempt intracellular lineage tracing and fate-mapping studies in a proposed model sipunculan, Themiste lageniformis. Importantly, with the unsegmented Sipuncula now repositioned within the segmented Annelida, sipunculan worms have become timely and appropriate models for investigating the potential for flexibility in spiralian development, including segmentation. We briefly review previous studies, and discuss new observations on the spiralian character complex within Sipuncula.

Sperm production and motility, fecundity, and egg size, complexity and viability of serpulid polychaetes Hydroides elegans and Hydroides diramphus after 2-week treatment to hypoxia (2 mg O2 l(-1)) was compared with those under normoxia (6 mg O2 l(-1)). Despite reduced fecundity, the effect of parental hypoxic exposure on gamete quality was not discernible for both species. However, regardless of their subsequent dissolved oxygen environment, eggs spawned by H. elegans after hypoxic exposure were found to have lower fertilization success, slower embryonic development and a significantly higher yield of malformed embryos than those with a parental normoxic treatment. In contrast, neither fertilization success nor rate of embryonic development was affected for H. diramphus. The results implied that hypoxia was a potential stress reducing the recruitment of H. elegans through non-adaptive epigenetic effect, whereas H. diramphus was a more tolerant species to survive hypoxic events., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The basic helix-loop-helix transcription factor twist plays a key role during mesoderm development in Bilateria. In this study, we identified a twist ortholog in the polychaete annelid Platynereis dumerilii and analyze its expression during larval development, postlarval growth up to the adult stage, and caudal regeneration after amputation of posterior segments. At late larval stages, Pdu-twist is expressed in the mesodermal anlagen and in developing muscles. During adulthood and caudal regeneration, Pdu-twist is expressed in the posterior growth zone, in mesodermal cells within the newly forming segments and budding parapodia. Our results indicate that Pdu-twist is involved in mesoderm formation during larval development, posterior growth, and caudal regeneration.

Many lophotrochozoans (i.e., molluscs, annelids, nemerteans, and polyclad flatworms) display a well-conserved early developmental program called spiral cleavage that contrasts with the high diversity of adult body forms present in this group. Due to this stereotypical development, each cell can be uniquely identified and its lineage history known following intracellular injection of lineage tracers. Cell deletion experiments performed mainly in molluscs have demonstrated that one or two cells associated with the endomesodermal lineage represent an embryonic organizer of subsequent development and are causally involved in cell fate and body patterning. Utilizing the published fate map of the spiral-cleaving annelid Capitella teleta, we used infrared laser cell deletions to dissect the role of individual cells on the patterning of the larval body. Thirteen uniquely identifiable individual blastomeres and two double cell combination deletions were studied to assess larval phenotypes by scoring multiple morphological structures and cell type-specific molecular markers differentially expressed along the antero-posterior and dorso-ventral axes. Surprisingly, our results show that in C. teleta, the cellular identity of the "organizing cell" and the timing of the organizing activity are different from that of other spiralians. retain-->In C. teleta, the ectodermal primary somatoblast, 2d, is the key cell responsible for organizing activity during early embryonic development, and is necessary for bilateral symmetry and dorso-ventral axis organization of the head as well as neural, foregut and mesoderm tissue formation. Furthermore, we show that the ERK/MAPK signaling pathway does not appear to be involved in organizing activity in retain-->C. teleta. This contrasts with data from molluscs and the molecular mechanism suggested for another polychaete, Hydroides elegans, highlighting likely molecular level variation among spiralian embryos. These results reinforce the idea that an embryonic organizing activity is present across spiralians. Our data also emphasize the developmental variation within lophotrochozoans, and may ultimately provide insight into the role of developmental processes in the evolution of diverse body forms in metazoans., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Expression of the transcription factor genes brachyury, Tbx2/3 and sall is characterized in detail for the first time in an indirectly developing spiralian with a feeding trochophore. In Hydroides elegans, gut formation proceeds by invagination during embryogenesis and is followed by feeding-dependent posterior growth during larval stages. Posterior growth gives rise to the reproductive and segmented portion of the adult and derives primarily from multipotent dorsal blastomeres. Dorsal fate becomes morphologically evident at the 60-cell stage during spiral cleavage, although the timing of dorsal specification remains uncertain. Expression of brachyury anticipates the morphogenetic events associated with both gastrulation by invagination in the endoderm and ventral midline convergent extension in the ectoderm. The absence of brachyury expression in endoderm precursors previously reported in annelids that do not have feeding larvae suggests evolutionarily conserved roles associated with morphogenesis rather than endoderm specification. Synexpression of brachyury and FoxA in the blastopore of eumetazoans as well as in the secondarily formed anus of some protostomes and the mouth of deuterostomes suggests shared regulatory circuits during the formation of both oral and anal openings in protostomes and deuterostomes. Expression of sall during gastrulation, in the protonephridium, and in posterior growth zone precursors, also suggests evolutionarily conserved roles. The dorsal sides of the Hydroides and sea urchin embryos express Tbx2/3 in all three germ layer precursors, suggesting evolutionarily conserved dorsal regionalization functions. The results suggest specific gene usage during tubular gut formation, endoderm specification, dorsoventral specification and anteroposterior body elongation in the context of development by feeding larva.

Cilia-based locomotion is the major form of locomotion for microscopic planktonic organisms in the ocean. Given their negative buoyancy, these organisms must control ciliary activity to maintain an appropriate depth. The neuronal bases of depth regulation in ciliary swimmers are unknown. To gain insights into depth regulation we studied ciliary locomotor control in the planktonic larva of the marine annelid, Platynereis. We found several neuropeptides expressed in distinct sensory neurons that innervate locomotor cilia. Neuropeptides altered ciliary beat frequency and the rate of calcium-evoked ciliary arrests. These changes influenced larval orientation, vertical swimming, and sinking, resulting in upward or downward shifts in the steady-state vertical distribution of larvae. Our findings indicate that Platynereis larvae have depth-regulating peptidergic neurons that directly translate sensory inputs into locomotor output on effector cilia. We propose that the simple circuitry found in these ciliated larvae represents an ancestral state in nervous system evolution.

The ability to produce more than one kind of offspring, or poecilogony, is a striking example of reproductive variability. Traditionally, larval nutrition has been classified as a dichotomy: if offspring obtain nutrition from their mothers (lecithotrophy), there is lower fecundity and greater chance of offspring survival than when they get their nutrition from plankton (planktotrophy). The polychaete Boccardia proboscidea (Spionidae) produces both types of embryos using three different reproductive strategies. In this study, we examined the roles of genetic history and phenotypic plasticity on explaining natural variation in B. proboscidea along the Pacific coast of the United States using two genetic mitochondrial markers, 16S rDNA and Cyt b, and common garden experiments. These data show a single North American West Coast network that is structured, geographically, by the well-documented biogeographic break near Point Conception, California. The southern group within this network covers a smaller range, but has larger haplotype diversity, than the northern group. Some individuals differing in reproductive type had the same haplotype, indicating independence of these features; however, differences between laboratory and field data suggest additional geographic variation within one of the reproductive types. Females from higher latitudes provide offspring with larger supplies of extra embryonic nutrition than females from southern latitudes. Results herein suggest that both genetic history and developmental plasticity are playing a role in the maintenance of this reproductive polymorphism., (© 2011 Wiley Periodicals, Inc.)

For the first time, under laboratory conditions, development of the polychaete Namanereis littoralis (Grube, 1876) is investigated. Under conditions of the Sea of Japan, its reproduction occurs in July and is confined to the season of monsoon rains. Fertilization is external. Spawning manifests no epitocous transformations. Fecundity is low, ovicells are rich in yolk, and development is nonpelagic, lecithotrophic, embryonized, characterized by a high rate--5-8 days--and occurs in mucous clutches up to hatching of benthic juveniles. Temperature and salinity optima of development are 22-27 degrees C and 16-21 per thousand, respectively, characterizing the species as subtropical brackish-water by its origin. Archaic and specialized traits are noted in the early ontogenesis of N. littoralis.

SUMMARY Annelids and arthropods, despite their distinct classification as Lophotrochozoa and Ecdysozoa, present a morphologically similar, segmented body plan. To elucidate the evolution of segmentation and, ultimately, to align segments across remote phyla, we undertook a refined expression analysis to precisely register the expression of conserved regionalization genes with morphological boundaries and segmental units in the marine annelid Platynereis dumerilii. We find that Pdu-otx defines a brain region anterior to the first discernable segmental entity that is delineated by a stripe of engrailed-expressing cells. The first segment is a "cryptic" segment that lacks chaetae and parapodia. This and the subsequent three chaetigerous larval segments harbor the anterior expression boundary of gbx, hox1, hox4, and lox5 genes, respectively. This molecular segmental topography matches the segmental pattern of otx, gbx, and Hox gene expression in arthropods. Our data thus support the view that an ancestral ground pattern of segmental identities existed in the trunk of the last common protostome ancestor that was lost or modified in protostomes lacking overt segmentation., (© 2011 Wiley Periodicals, Inc.)

The evolution of the highest-order human brain center, the "pallium" or "cortex," remains enigmatic. To elucidate its origins, we set out to identify related brain parts in phylogenetically distant animals, to then unravel common aspects in cellular composition and molecular architecture. Here, we compare vertebrate pallium development to that of the mushroom bodies, sensory-associative brain centers, in an annelid. Using a newly developed protocol for cellular profiling by image registration (PrImR), we obtain a high-resolution gene expression map for the developing annelid brain. Comparison to the vertebrate pallium reveals that the annelid mushroom bodies develop from similar molecular coordinates within a conserved overall molecular brain topology and that their development involves conserved patterning mechanisms and produces conserved neuron types that existed already in the protostome-deuterostome ancestors. These data indicate deep homology of pallium and mushroom bodies and date back the origin of higher brain centers to prebilaterian times., (Copyright 2010 Elsevier Inc. All rights reserved.)

Immunochemical labeling of neuronal elements and laser confocal microscopy have considerably expanded the capacity of comparative morphology and allowed us to monitor the neurogenesis of various trochophore animals at the level of individual identified neurons and their projections. It has been demonstrated that many generally accepted concepts of the larval nervous system and the phylogenetic theories constructed on this basis are incorrect. Comparative analysis has demonstrated that the orthogonal brain is absent at all developmental stages in the representative Lophotrochozoa members. Fundamental differences in the structure and development of the nervous system have been found in the trochophores belonging to different taxonomic groups within Lophotrochozoa; these differences demonstrate that the trochophore larva in these groups are not homologous, while their similarity is most likely a result of convergence. Our results challenge the concept of trochophore as the ancestral form common for all trochophore animals. It is necessary to exclude from phylogenetic discussions the orthogon as a basic plan for the structure of the nervous system and the trochophore as an ancestral form for all Lophtrochozoa.

Molecular data for nephridial development in polychaetes are not available yet. The scope of our work was to establish a reference system for future investigations using two markers for nephridial development: beta-tubulin as marker for cilia and alkaline phosphatase (AP) activity for secretory epithelia. The markers identified, unexpectedly, three consecutively forming generations of nephridia: (1) a transitory unciliated, but AP-positive head kidney, (2) a transitory larval nephridium, which undergoes a morphological transition from a protonephridium to a funnelled nephridium concomitant with the development of the coelomic cavity and finally, (3) the serially arranged metanephridia. The spatial arrangement of larval and definitive nephridia, revealed an up to now unknown developmental boundary between the synchronously forming larval and the serially proliferating definitive segments. Development of three consecutive sets of nephridia with different morphology and biochemical properties was unexpected and reveals an interesting multistep process in the development of excretory structures in Platynereis., ((c) 2010 Wiley-Liss, Inc.)

Background: The internal phylogenetic relationships of Annelida, one of the key lophotrochozoan lineages, are still heavily debated. Recent molecular analyses suggest that morphologically distinct groups, such as the polychaetes, are paraphyletic assemblages, thus questioning the homology of a number of polychaete morphological characters. Serpulid polychaetes are typically recognized by having fused anterior ends bearing a tentacular crown and an operculum. The latter is commonly viewed as a modified tentacle (= radiole) and is often used as an important diagnostic character in serpulid systematics., Results: By reconstructing the developmental neuroanatomy of the serpulid polychaete Spirorbis cf. spirorbis (Spirorbinae), we found striking differences in the overall neural architecture, the innervation pattern, and the ontogenetic establishment of the nervous supply of the operculum and the radioles in this species. Accordingly, the spirorbin operculum might not be homologous to the radioles or to the opercula of other serpulid taxa such as Serpula and Pomatoceros and is thus probably not a part of the tentacular crown., Conclusion: We demonstrate that common morphological traits such as the prostomial appendages may have evolved independently in respective serpulid sublineages and therefore require reassessment before being used in phylogenetic analyses. Our findings corroborate recent molecular studies that argue for a revision of serpulid systematics. In addition, our data on Spirorbis neurogenesis provide a novel set of characters that highlight the developmental plasticity of the segmented annelid nervous system.

Intertaxonomic comparisons are important for understanding neurogenesis and evolution of nervous systems, but high-resolution, cellular studies of early central nervous system development and the molecular mechanisms controlling this process in lophotrochozoans are still lacking. We provide a detailed cellular and molecular description of early brain neurogenesis in a lophotrochozoan annelid, Capitella sp. I. We utilized different approaches including DiI lineage tracing, immunohistochemistry, BrdU labeling, and gene expression analyses to characterize neural precursor cells in Capitella sp. I. Brain neurogenesis proceeds by the ingression of single cells from the anterior ectoderm to generate a stratified epithelial layer. Most cell divisions are restricted to apically localized cells with mitotic spindles oriented parallel to the epithelial layer. Prior to and during this process, an achaete-scute complex homolog, CapI-ash1, is expressed in clusters of surface cells in the anterior ectoderm, consistent with a proneural function for CapI-ash1. In contrast, a homolog of the neural differentiation marker elav, CapI-elav1, is restricted to basally localized cells within the forming brain. Unlike insects, Capitella sp. I does not have morphologically obvious enlarged neuroblasts, although Capitella sp. I brain neurogenesis displays several similarities with non-insect arthropod and vertebrate neurogenesis, providing a useful lophotrochozoan model for comparison.

The expression of Blimp and T-brain was characterized during embryogenesis of the indirectly developing polychaete Hydroides elegans. The expression of both genes in the vegetal blastomeres of this lophotrochozoan is restricted to the lateral and oral regions of the blastopore. Both transcription factors also have expression patterns consistent with ancestral neural functions. HeBlimp is expressed in a couple of animal cap cells and exhibits a marked left bias that correlates with earlier development of the left eye in H. elegans. HeT-brain is expressed in animal cap blastomeres and eventually becomes restricted to apical tuft cells of early trochophore stages. The expression of Blimp and T-brain among directly and indirectly developing bilaterians suggests ancestral sensory cell-type specification, gastrulation by invagination, endoderm specification, mesoderm specification, and/or tripartite gut subdivision. These characterizations add to the long-term goal of understanding the regulatory evolution functions that underlies complex life cycles in metazoans., (Copyright 2008 Wiley-Liss, Inc.)

Members of the vasa and nanos gene families are involved in germ line development in a number of diverse animals. As a polychaete annelid model for studies of the germ line, Capitella sp. I has several advantages including the presence of dedicated gonads, individuals that reproduce multiple times, and the presence of males, females, and hermaphrodites. Germ line development has not been characterized in Capitella sp. I, nor is the mechanism of germ line specification generally well understood in annelids. We have cloned vasa and nanos orthologues from Capitella sp. I and found that both CapI-vasa and CapI-nanos transcripts are expressed in developing gametes of sexually mature adults. Characterization of both these genes during embryonic, larval, and juveniles stages reveals expression in multiple somatic tissues for CapI-vasa and CapI-nanos with largely overlapping but not identical expression patterns. In early cleavage stages, both transcripts are broadly expressed; following gastrulation, expression is observed in the presumptive brain, mesodermal bands, and developing foregut. Using CapI-nanos and CapI-vasa as markers, we have identified putative primordial germ cells (PGCs) in larvae, which are initially present as small bilateral clusters in segment 4 and as a single cluster at late larval stages. In adults, a single large cluster of putative PGCs is present in segments 5 and 6. In addition to highlighting differences in expression profiles for these two genes among lophotrochozoans, we present a hypothesis concerning the origin and development of PGCs in Capitella sp. I.

Notch signaling is involved in a large range of developmental processes, and has been functionally implicated in body plan segmentation in two of the three diverse segmented taxa, the vertebrates and arthropods. Here we investigate expression of Notch, Delta, and hes gene homologues during larval and juvenile development in the polychaete annelid Capitella sp. I., a member of the third group of segmented animals. During larval stages, CapI-Notch, CapI-Delta, CapI-hes2, and CapI-hes3 transcripts are initially detected in broad ectodermal domains in future segments as well as in the brain and foregut; later, CapI-Notch, CapI-Delta, and CapI-hes2 transcripts are detected in the presumptive chaetal sacs. In contrast, CapI-hes1 has a segmentally reiterated pattern in a restricted region of the mesoderm in each presumptive segment. CapI-Notch, CapI-Delta, CapI-hes2, and CapI-hes3 and CapI-hes1 are all expressed in the terminal growth zone that generates post-metamorphic segments, however, CapI-hes1 has a non-overlapping complementary expression pattern to that of CapI-Notch and CapI-Delta. CapI-Delta and CapI-Notch transcripts are localized to already formed segments, with posterior boundaries that correlate with the posterior boundary of the nascent segment, while CapI-hes1 lies posterior to CapI-Notch and CapI-Delta. The localization of CapI-Notch, CapI-Delta, and CapI-hes transcripts correlate with areas of rapid cell proliferation in Capitella, which include the brain, foregut, and terminal growth zone.