The evolutionary history of placodes: a molecular genetic investigation of the larvacean urochordate Oikopleura dioica

The evolutionary origin of vertebrate placodes remains controversial because divergent morphologies in urochordates, cephalochordates and vertebrates make it difficult to recognize organs that are clearly homologous to placode-derived features, including the olfactory organ, adenohypophysis, lens, inner ear, lateral line and cranial ganglia. The larvacean urochordate Oikopleura dioica possesses organs that morphologically resemble the vertebrate olfactory organ and adenohypophysis. We tested the hypothesis that orthologs of these vertebrate placodes exist in a larvacean urochordate by analyzing the developmental expression of larvacean homologs of the placode-marking gene families Eya, Pitx and Six. We conclude that extant chordates inherited olfactory and adenohypophyseal placodes from their last common ancestor, but additional independent proliferation and perhaps loss of placode types probably occurred among the three subphyla of Chordata.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

Simple procedure for sperm cryopreservation in the larvacean tunicate Oikopleura dioica

The larvacean tunicate Oikopleura dioica is an attractive organism for studies of the development, evolution, and physiology of chordates, showing considerable promise for genetic approaches given its short life cycle of five days. To facilitate future genetic studies, the development of protocols for the maintenance of individual strains is essential. Here we report a simple and practical protocol for the cryopreservation of sperm using liquid nitrogen (-196°C) and dimethyl sulfoxide (DMSO) as a protective agent. The quality of the frozen-thawed sperm was evaluated in terms of fertilizing ability and subsequent development of the fertilized eggs. We examined several parameters to optimize the efficiency of cryopreservation, such as the concentration of DMSO, the method for acclimation of sperm to DMSO before freezing, and for placing sperm in liquid nitrogen, as well as the pH of the seawater used in resuspending the thawed sperm. We confirmed that viable sperm were recovered after preservation for more than one year. In addition, mature animals, and even a subsequent generation, were obtained from eggs fertilized by the cryopreserved sperm. The present procedure seems to be simple and sufficiently practical for maintenance of future established lines of O. dioica using frozen sperm.     

Molecular patterning of the oikoplastic epithelium of the larvacean tunicate Oikopleura dioica

Appendicularia are protochordates that rely on a complex mucous secretion, the house, to filter food particles from seawater. A monolayer of cells covering the trunk of the animal, the oikoplastic epithelium, secretes the house. This epithelium contains a fixed number of cells arranged in characteristic patterns with distinct sizes and nuclear morphologies. Certain house structures appear to be spatially related to defined, underlying groups of cells in the epithelium. We show that the house is composed of at least 20 polypeptides, a number of which are highly glycosylated, with glycosidase treatments resulting in molecular mass shifts exceeding 100 kDa. Nanoelectrospray tandem mass spectrometric microsequencing of house polypeptides was used to design oligonucleotides to screen an adult Oikopleura dioica cDNA library. This resulted in the isolation of cDNAs coding for three different proteins, oikosin 1, oikosin 2, and oikosin 3. The latter two are novel proteins unrelated to any known data base entries. Oikosin 1 has 13 repeats of a Cys domain, previously identified as a subunit of repeating sequences in some vertebrate mucins. We also find one repeat of this Cys domain in human cartilage intermediate layer protein but find no evidence of this domain in any invertebrate species, including those for which entire genomes have been sequenced. The three oikosins show distinct and complementary expression patterns restricted to the oikoplastic epithelium. This easily accessible epithelium, with differential gene expression patterns in readily identifiable groups of cells with distinctive nuclear morphologies, is a highly attractive model system for molecular studies of pattern formation.     

Zooplankton growth rates: extraordinary production by the larvacean Oikopleura dioica in tropical waters

The growth of Oikopleura dioica was determined from microcosmsincubated in situ at 29°C in Kingston Harbour, Jamaica.Minimum generation times approached 1 day, with Oikopleura dailyspecific growth rates ranging from 3 to 23 and averaging 10.7-foldincreases in biomass over 24 h. This was an order of magnitudegreater than the copepod Paracalanus crassirostris, whose growthrates ranged from 0.3 to 1.2 and averaged 0.67 day^–1 fromthe same experiments A reassessment of previous data indicatesthat larvacean production approaches, and may exceed, the 693kJ m^–2 annual production of the copepods. Growth rateand recruitment of Oikopleura decreased as the biomass of thezooplankton community increased; both resource and interferencecompetition are probably occurring. The extraordinary productionpotential of the Larvacea emphasizes their understated importancein marine planktonic systems.     

Development of the central nervous system in the larvacean Oikopleura dioica and the evolution of the chordate brain

In non-vertebrate chordates, central nervous system (CNS) development has been studied in only two taxa, the Cephalochordata and a single Class (Ascidiacea) of the morphologically diverse Urochordata. To understand development and molecular regionalization of the brain in a different deeply diverging chordate clade, we isolated and determined the expression patterns of orthologs of vertebrate CNS markers (otxa, otxb, otxc, pax6, pax2/5/8a, pax2/5/8b, engrailed, and hox1) in Oikopleura dioica (Subphylum Urochordata, Class Larvacea). The three Oikopleura otx genes are expressed similarly to vertebrate Otx paralogs, demonstrating that trans-homologs converged on similar evolutionary outcomes by independent neo- or subfunctionalization processes during the evolution of the two taxa. This work revealed that the Oikopleura CNS possesses homologs of the vertebrate forebrain, hindbrain, and spinal cord, but not the midbrain. Comparing larvacean gene expression patterns to published results in ascidians disclosed important developmental differences and similarities that suggest mechanisms of development likely present in their last common ancestor. In contrast to ascidians, the lack of a radical reorganization of the CNS as larvaceans become adults allows us to relate embryonic gene expression patterns to three subdivisions of the adult anterior brain. Our study of the Oikopleura brain provides new insights into chordate CNS evolution: first, the absence of midbrain is a urochordate synapomorphy and not a peculiarity of ascidians, perhaps resulting from their drastic CNS metamorphosis; second, there is no convincing evidence for a homolog of a midbrain-hindbrain boundary (MHB) organizer in urochordates; and third, the expression pattern of "MHB-genes" in the urochordate hindbrain suggests that they function in the development of specific neurons rather than in an MHB organizer.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Gut ultrastructure of the appendicularian Oikopleura dioica (Tunicata)

Abstract. The appendicularians, planktonic tunicates, possess a specialized, external filtering system that captures food particles <1 μm in size. In this work the alimentary canal of Oikopleura dioica has been studied by serial sections of whole animals and ultrastructure. The gut includes a dorsal esophagus, a bilobed saccular stomach, and a curved intestine, divided into vertical, mid-, and distal intestine (or rectum). No multicellular glands or cellular proliferative centers were found. Three main cell types were recognized, ciliated microvillar cells, globular cells and gastric band cells, with specializations reflecting different physiological roles in the various regions. Ciliated microvillar cells, the most diffuse, are considered to be involved in food propulsion, fecal pellet formation, absorption, and nutrient storage. Pinocytotic features and vacuoles suggest that absorption of macromolecules and intracellular digestion occur in the globular cells of the stomach and rectum. The large gastric band cells of the left lobe have typical features of intense protein synthesis and probably produce enzymes for extracellular digestion. Diffuse interdigitations of many cells enormously increase the plasmalemma surface and may be involved in liquid/ion exchange. Despite the apparent structural simplicity of the gut epithelium, O. dioica efficiently processes food to fulfill the energy requirements of its exceptionally rapid life-cycle.     

A Rostral Sensory Mechanism in Oikopleura dioica (Appendicularia)

The ventral sense organ, below the mouth, is composed of 30 primary sensory cells situated in a row perpendicular to the long axis of the animal. Each cell carries one long and slender, modified cilium which arises from an apical pocket in the cell. The sensory cells project 15 axons at each side of the pharynx to the brain, which is rostrally paired and terminates in bulb-like swellings. Each of these bulbs contains four cell bodies, which, according to their fine structure, as well as the synaptic connections with receptor and brain fibers, belong to three different types. It is suggested that the sense organ is a chemosensor and that its remarkable similarity to the vertebrate mechanisms for olfaction makes it probable that the appendicularian Oikopleura dioica possesses a ‘protochordate’ counterpart to the craniate olfactory apparatus.     

The Ciliated Brain Duct of Oikopleura dioica (Tunicata,Appendicularia)

Transmission electron microscopy revealed varying morphology of the dorsal part of the duct indicating the occurrence of different stages of activity. In some animals the tip cavity contains an amorphous substance which, at another phase of activity, is released into the surrounding hemocel through interstices in the walls of the tip. A transitional zone separates the dorsal tip from the large ventral part of the duct, where the wall, near the transitional zone, consists of a single layer of thin cells joined by tight junctions. Ventrally, the large ciliated cells lie in two levels near the opening into the buccal cavity. They bear numerous, tightly packed cilia which beat vigorously in living animals. The organization of this part resembles that of protonephridia. An excretory function is suggested for this part of the duct.     

The house of Oikopleura dioica (Tunicata,Appendicularia): Structure and functions

Summary Using appropriate techniques, we have studied in detail the structure and functions of the house of the appendicularian Oikopleura dioica Fol, 1872. In particular, we describe the structure of the inlet funnel, the reinforcing chamber on each side of the tail chamber and its function, the role and the structure of the valves located between the dorsal and water exit chambers and the structure and the role of the exit sphincter. In conclusion, we show that in addition to filtration of particles, movement of the tail also helps location of a favorable particulate environment in the sea.     

Productivity and grazing impact of Oikopleura dioica (Tunicata, Appendicularia) in Tokyo Bay

To estimate the productivity and grazing impact of a commonappendicularian species Oikopleura dioica in Tokyo Bay, monthlyobservations on its abundance and vertical distribution wereconducted during 2000. The abundance peaked in February andOctober, but was low during summer. Seasonal fluctuations inproductivity were similar to those of the abundance, with maximumvalues of 92, 134 and 508 mg C m^–2 day^–1 for somatic,new house and discarded house productivity in October and 206mg C m^–2 day^–1 for fecal pellet productivity inFebruary, respectively. The averaged biomass of O. dioica wasonly 2.5% of that of copepods; however, the secondary productivityof the former corresponded to 12.4% of the latter. Daily grazingimpact on particulate organic carbon ranged from 0.05% to 5%,which is close to the impact by copepods. These results implythat in Tokyo Bay, where small copepods and jellyfish are abundant,O. dioica is an important component of the ecosystem becauseit bridges between small primary producers and higher consumers.     

The Oral Gland Cells of Oikopleura dioica (Tunicata Appendicularia)

Light and electron microscopic studies showed that the oral gland cells have two quite different zones. Medially, the basal zone is in contact with body fluids and the endostyle. Its strongly pyroninophile cytoplasm contains the extremely digitated nucleus and numerous small mitochondria. Laterally, the apical zone contacts the epidermis and it may also send a process between epidermal cells and deliver cell fragments into the primordium of the new house. This cell zone contains numerous membranes. It is concluded that the oral gland cells are light producing glands and that the membrane-rich cell fragments which are incorporated into the house wall are the source of the bioluminiscence which has been reported from empty houses. The ontogenetically related subchordal cells have a similar structure and it is possible that also these cells are light producers.     

House Production by Oikopleura dioica (Tunicata, Appendicularia) Under Laboratory Conditions

We examined the generation time and the house renewal rate ofOikopleura dioica under various conditions. Animals were fedtwo flagellates, Isochrysis galbana and Tetraselmis sp., withconcurrent determination of the carbon contents of body andhouse to estimate house production. The generation time was6 days at 15°C, 4 days at 20°C and 3 days at 25°Cat both 25 and 30 p.s.u. with a food concentration of 4 x 10⁴cells ml^–1. The carbon content of newly secreted housesranged from 0.5 to 0.8 µg, corresponding to 15.3 ±4.8% of body carbon. The house renewal rates increased withincreasing temperature and decreasing salinity. Food concentrationsranging from 100 to 16 x 10⁴ cells ml^–1, body size andlight condition had no effect on house renewal rate. Cloggingof the inlet filter by adding the large diatom Ditylum sol causedan increase in house renewal rates. The total number and carboncontent of houses during an animal's lifetime ranged from 46to 53 houses and from 6.5 to 10.4 µg, respectively. Sincedaily house production calculated for the O. dioica populationcorresponded to 130–290% of its biomass and daily discardedhouse materials corresponded to 490–1100% of the biomass,this organism must play an important role as a producer of macroscopicaggregates.