Morphological Study and 3D Reconstruction of the Larva of the Ascidian Halocynthia roretzi

The swimming larva represents the dispersal phase of ascidians, marine invertebrates belonging to tunicates. Due to its adhesive papillae, the larva searches the substrate, adheres to it, and undergoes metamorphosis, thereby becoming a sessile filter feeding animal. The larva anatomy has been described in detail in a few species, revealing a different degree of adult structure differentiation, called adultation. In the solitary ascidian Halocynthia roretzi, a species reared for commercial purposes, embryogenesis has been described in detail, but information on the larval anatomy is still lacking. Here, we describe it using a comparative approach, utilizing 3D reconstruction, as well as histological/TEM observations, with attention to its papillae. The larva is comparable to those of other solitary ascidians, such as Ciona intestinalis. However, it displays a higher level of adultation for the presence of the atrium, opened outside by means of the atrial siphon, and the peribranchial chambers. It does not reach the level of complexity of the larva of Botryllus schlosseri, a phylogenetically close colonial ascidian. Our study reveals that the papillae of H. roretzi, previously described as simple and conform, exhibit dynamic changes during settlement. This opens up new considerations on papillae morphology and evolution and deserves to be further investigated.

The ontology of the anatomy and development of the solitary ascidian Ciona: the swimming larva and its metamorphosis

Ciona robusta (Ciona intestinalis type A), a model organism for biological studies, belongs to ascidians, the main class of tunicates, which are the closest relatives of vertebrates. In Ciona, a project on the ontology of both development and anatomy is ongoing for several years. Its goal is to standardize a resource relating each anatomical structure to developmental stages. Today, the ontology is codified until the hatching larva stage. Here, we present its extension throughout the swimming larva stages, the metamorphosis, until the juvenile stages. For standardizing the developmental ontology, we acquired different time-lapse movies, confocal microscope images and histological serial section images for each developmental event from the hatching larva stage (17.5 h post fertilization) to the juvenile stage (7 days post fertilization). Combining these data, we defined 12 new distinct developmental stages (from Stage 26 to Stage 37), in addition to the previously defined 26 stages, referred to embryonic development. The new stages were grouped into four Periods named: Adhesion, Tail Absorption, Body Axis Rotation, and Juvenile. To build the anatomical ontology, 203 anatomical entities were identified, defined according to the literature, and annotated, taking advantage from the high resolution and the complementary information obtained from confocal microscopy and histology. The ontology describes the anatomical entities in hierarchical levels, from the cell level (cell lineage) to the tissue/organ level. Comparing the number of entities during development, we found two rounds on entity increase: in addition to the one occurring after fertilization, there is a second one during the Body Axis Rotation Period, when juvenile structures appear. Vice versa, one-third of anatomical entities associated with the embryo/larval life were significantly reduced at the beginning of metamorphosis. Data was finally integrated within the web-based resource "TunicAnatO", which includes a number of anatomical images and a dictionary with synonyms. This ontology will allow the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

Effect of Polycyclic Aromatic Hydrocarbons on Development of the Ascidian Ciona intestinalis Type A

Polycyclic aromatic hydrocarbons (PAHs) are pollutants that exert harmful effects on marine invertebrates; however, the molecular mechanism underlying PAH action remains unclear. We investigated the effect of PAHs on the ascidian Ciona intestinalis type A (Ciona robusta). First, the influence of PAHs on early Ciona development was evaluated. PAHs such as dibenzothiophene, fluorene, and phenanthrene resulted in formation of abnormal larvae. PAH treatment of swimming larva induced malformation in the form of tail regression. Additionally, we observed the Ciona aryl hydrocarbon receptor (Ci-AhR) mRNA expression in swimming larva, mid body axis rotation, and early juvenile stages. The time correlation between PAH action and AhR mRNA expression suggested that Ci-AhR could be associated with PAH metabolism. Lastly, we analyzed Ci-AhR mRNA localization in Ciona juveniles. Ci-AhR mRNA was localized in the digestive tract, dorsal tubercle, ganglion, and papillae of the branchial sac, suggesting that Ci-AhR is a candidate for an environmental pollutant sensor and performs a neural function. Our results provide basic knowledge on the biological function of Ci-AhR and PAH activity in marine invertebrates.

A cis-regulatory change underlying the motor neuron-specific loss of terminal selector gene expression in immotile tunicate larvae

The evolutionary history of animal body plans cannot be fully reconstructed without considering the roles of both novelties and losses. Some of the more remarkable examples of massively parallel evolutionary losses in animals comes from many species in the tunicate genusMolgulathat have independently lost the swimming larva and instead develop as tail-less, immotile larvae that bypass the period of swimming and dispersal observed in other tunicates, marine invertebrate chordates that alternate between motile larval and sessile adult life cycle stages. The larvae ofMolgula occultaand other tail-less species do not fully develop structures that are essential for swimming behavior, including notochord, tail muscles, and otolith, and loss-of-function mutations have been identified in various genes required for the differentiation of these tissues. However, little is known about the extent of development of the larval nervous system inM. occulta. While differentiated neurons might in principle be entirely dispensable to the non-swimming larva, the adult has a fully functional nervous system like any other tunicate. To further investigate this conundrum, we studied the specification and patterning of theM. occultaMotor Ganglion, which is the key central nervous system compartment that drives the motor movements of swimming tunicate larvae. We found that the expression patterns of important regulators of MG neuron subtype specification are highly conserved during the development of the non-swimming larvae ofM. occulta, suggesting that the gene networks regulating their expression are largely intact in this species, despite the loss of swimming ability. However, we identified aM. occulta-specific reduction in expression of the important motor neuron terminal selector geneEbf (Collier/Olf/EBF or COE)in the Motor Ganglion. AlthoughM. occulta Ebfis predicted to encode a fully functional protein, its expression was reduced in developing motor neurons when compared to species with swimming larvae, which was corroborated by measuring allele-specific expression ofEbfin interspecific hybrid embryos produced by crossingM. occultawith the closely related swimming speciesM. oculata. Comparative reporter construct experiments also revealed a specificcis-regulatory sequence change that underlies the reduced expression ofM. occulta Ebfin motor neurons, but not in other tissues and cell types. This points to a potential mechanism for arresting larval motor neuron differentiation in the non-swimming larvae of this species.

An organismal perspective on C. intestinalis development, origins and diversification

The ascidian Ciona intestinalis, commonly known as a ‘sea squirt’, has become an important model for embryological studies, offering a simple blueprint for chordate development. As a model organism, it offers the following: a small, compact genome; a free swimming larva with only about 2600 cells; and an embryogenesis that unfolds according to a predictable program of cell division. Moreover, recent phylogenies reveal that C. intestinalis occupies a privileged branch in the tree of life: it is our nearest invertebrate relative. Here, we provide an organismal perspective of C. intestinalis, highlighting aspects of its life history and habitat—from its brief journey as a larva to its radical metamorphosis into adult form—and relate these features to its utility as a laboratory model.

Intergenerational transmission of symbiotic bacteria in oviparous and viviparous demosponges, with emphasis on intracytoplasmically-compartmented bacterial types

Recent molecular detection of vast microbial communities exclusively associated with sponges has made evident the need for a better understanding of the mechanisms by which these symbiotic microbes are handled and transferred from one sponge generation to another. This transmission electron microscopy (TEM) study investigated the occurrence of symbiotic bacteria in free-swimming larvae of two viviparous species (Haliclona caerulea and Corticium candelabrum) and spawned gametes of two oviparous species (Chondrilla nucula and Petrosia ficiformis). Complex microbial communities were found in these sponges, which in two cases included bacteria characterized by an intra-cytoplasmic membrane (ICM). When ICM-bearing and ICM-lacking bacteria co-existed, they were transferred following identical pathways. Nevertheless, the mechanism for microbial transference varied substantially between species. In C. nucula, a combination of intercellular symbiotic ICM-bearing and ICM-lacking bacteria, along with cyanobacteria and yeasts, were collected from the mesohyl by amoeboid nurse cells, then transported and transferred to the oocytes. In the case of Corticium candelabrum, intercellular bacteria did not enter the gametes, but spread into the division furrows of early embryos and proliferated in the central cavity of the free-swimming larva. Surprisingly, symbiotic bacteria were not vertically transmitted by P. ficiformis gametes or embryos, but apparently acquired from the environment by the juveniles of each new generation. This study failed to unravel the mechanism by which the intercellular endosymbiotic bacterium found in the central mesohyl of the H. caerulea larva got there. Nevertheless, the ultrastructure of this bacterial rod, which was characterized by a star-shaped cross section with nine radial protrusions, an ICM-bound riboplasm, and a putative membrane-bound acidocalcisome, suggested that it may represent a novel organization grade within the prokaryotes. It combines traits occurring in members of Poribacteria, Planctomycetes and Verrucomicrobia, emerging as one of the most complex prokaryotic architectures known to date.