Evolutionary Insights from the Mitochondrial Genome of Oikopleura dioica: Sequencing Challenges, RNA Editing, Gene Transfers to the Nucleus, and tRNA Loss.

Sequencing the mitochondrial genome of the tunicate Oikopleura dioica is a challenging task due to the presence of long poly-A/T homopolymer stretches, which impair sequencing and assembly. Here, we report on the sequencing and annotation of the majority of the mitochondrial genome of O. dioica by means of combining several DNA and amplicon reads obtained by Illumina and MinIon Oxford Nanopore Technologies with public RNA sequences. We document extensive RNA editing, since all homopolymer stretches present in the mitochondrial DNA correspond to 6U-regions in the mitochondrial RNA. Out of the 13 canonical protein-coding genes, we were able to detect eight, plus an unassigned open reading frame that lacked sequence similarity to canonical mitochondrial protein-coding genes. We show that the nad3 gene has been transferred to the nucleus and acquired a mitochondria-targeting signal. In addition to two very short rRNAs, we could only identify a single tRNA (tRNA-Met), suggesting multiple losses of tRNA genes, supported by a corresponding loss of mitochondrial aminoacyl-tRNA synthetases in the nuclear genome. Based on the eight canonical protein-coding genes identified, we reconstructed maximum likelihood and Bayesian phylogenetic trees and inferred an extreme evolutionary rate of this mitochondrial genome. The phylogenetic position of appendicularians among tunicates, however, could not be accurately determined.

Tracing Homopolymers in Oikopleura dioica's Mitogenome.

Oikopleura dioica is a planktonic tunicate (Appendicularia class) found extensively across the marine waters of the globe. The genome of a single male individual collected from Okinawa, Japan was sequenced using the single-molecule PacBio Hi-Fi method and assembled with NOVOLoci. The mitogenome is 39,268 bp long, featuring a large control region of around 22,000 bp. We annotated the proteins atp6, cob, cox1, cox2, cox3, nad1, nad4, and nad5, and found one more open reading frame that did not match any known gene. This study marks the first complete mitogenome assembly for an appendicularian, and reveals that A and T homopolymers cumulatively account for nearly half of its length. This reference sequence will be an asset for environmental DNA and phylogenetic studies.

A miniaturized nigrostriatal-like circuit regulating locomotor performance in a protochordate.

To gain insight into the evolution of motor control systems at the origin of vertebrates, we have investigated higher-order motor circuitry in the protochordate Oikopleura dioica. We have identified a highly miniaturized circuit in Oikopleura with a projection from a single pair of dopaminergic neurons to a small set of synaptically coupled GABAergic neurons, which in turn exert a disinhibitory descending projection onto the locomotor central pattern generator. The circuit is reminiscent of the nigrostriatopallidal system in the vertebrate basal ganglia, in which disinhibitory circuits release specific movements under the modulatory control of dopamine. We demonstrate further that dopamine is required to optimize locomotor performance in Oikopleura, mirroring its role in vertebrates. A dopamine-regulated disinhibitory locomotor control circuit reminiscent of the vertebrate nigrostriatopallidal system was thus already present at the origin of ancestral chordates and has been maintained in the face of extreme nervous system miniaturization in the urochordate lineage.

Gelatinous larvacean zooplankton can enhance trophic transfer and carbon sequestration.

Larvaceans are gelatinous zooplankton abundant throughout the ocean. Larvaceans have been overlooked in research because they are difficult to collect and are perceived as being unimportant in biogeochemical cycles and food-webs. We synthesise evidence that their unique biology enables larvaceans to transfer more carbon to higher trophic levels and deeper into the ocean than is commonly appreciated. Larvaceans could become even more important in the Anthropocene because they eat small phytoplankton that are predicted to become more prevalent under climate change, thus moderating projected future declines in ocean productivity and fisheries. We identify critical knowledge gaps and argue that larvaceans should be incorporated into ecosystem assessments and biogeochemical models to improve predictions of the future ocean.

The filter-house of the larvacean Oikopleura dioica. A complex extracellular architecture: From fiber production to rudimentary state to inflated house.

While cellulose is the most abundant macromolecule in the biosphere, most animals are unable to produce cellulose with the exception of tunicates. Some tunicates have evolved the ability to secrete a complex house containing cellulosic fibers, yet little is known about the early stages of the house building process. Here, we investigate the rudimentary house of Oikopleura dioica for the first time using complementary light and electron microscopic techniques. In addition, we digitally modeled the arrangement of chambers, nets, and filters of the functional, expanded house in three dimensions based on life-video-imaging. Combining 3D-reconstructions based on serial histological semithin-sections, confocal laser scanning microscopy, transmission electron microscopy, scanning electron microscopy (SEM), and focused ion beam (FIB)-SEM, we were able to elucidate the arrangement of structural components, including cellulosic fibers, of the rudimentary house with a focus on the food concentration filter. We developed a model for the arrangement of folded structures in the house rudiment and show it is a precisely preformed structure with identifiable components intricately correlated with specific cells. Moreover, we demonstrate that structural details of the apical surfaces of Nasse cells provide the exact locations and shapes to produce the fibers of the house and interact among each other, with Giant Fol cells, and with the fibers to arrange them in the precise positions necessary for expansion of the house rudiment into the functional state. The presented data and hypotheses advance our knowledge about the interrelation of structure and function on different biological levels and prompt investigations into this astonishing biological object.

Spermatogenesis as a tool for staging gonad development in the gonochoric appendicularian Oikopleura dioica Fol 1872.

Oikopleura dioica, the only gonochoric species among appendicularians, has a spematozoon with a mid-piece and a conspicuous acrosome that, during fertilisation, undergoes a reaction forming an acrosomal process. To provide more insight into the spermatogenesis of a holoplanktonic tunicate species that completes its life cycle in three to five days, changes in the testis during individual growth have been examined. Spermatogenesis has been subdivided into seven stages based on ultrastructural features during the formation and organisation of the male gonad and the relationships between its macroscopic anatomy and the events of sperm differentiation. Gametes undergo highly synchronised differentiation due to the presence of widespread syncytial structures. Both meiosis and spermiogenesis are brief, and the passage from spermatocytes to spermatids involves a progressive segregation of the germ cells from the syncytial mass with the formation of large cytoplasmic bridges and volume reduction for nucleus compacting and cytoplasmic material changing. The nucleus is small and penetrated anteriorly by a complex acrosome and posteriorly by the distal centriole and part of the flagellum. In spermatids, the single, large mitochondrion appears laterally to the nucleus, and finally, in spermatozoa, it migrates into the mid-piece, wrapping the proximal portion of the axoneme. Because this mitochondrial position is reached only in the late phases of spermatogenesis, it suggests that appendicularians have derived oligopyrenic sperms in which the small nucleus results from adaptation to the assembly of numerous spermatozoa inside the narrow space of the testis compacted in the genital cavity. The formulation of a staging system of gonad development in a model tunicate species known for having the most compacted genome in chordates led to a comparison of histological observations with recent molecular data, improving the characterisation of its biology and life cycle in light of evolutionary implications.

Analysis of the Appendicularia class (subphylum Urochordata) as a possible tool for biomonitoring four estuaries of the tropical region.

This study aims to characterize the differences between physical and chemical parameters of water quality indicators and eutrophication in four estuaries of the state of Rio de Janeiro and relate them to full density of the species that belong to the Appendicularia class with the purpose of seeking bioindicators for estuarine areas. The estuaries studied were from the rivers Macaé, São João, Bracuí, and Perequê-Açu. The physicochemical parameters monitored for evaluation of the water bodies and used for the correlation with the appendicularians were pH, temperature, nitrite, nitrate, N-ammonia, total nitrogen, phosphate, total phosphorus, dissolved oxygen (DO), suspended particulate matter (SPM), chlorophyll a, heterotrophic bacteria, and fecal coliform. The estuaries Bracuí and São João showed enhanced qualities of water and lower degrees of eutrophication than Macae and Perequê-Açu estuaries. The species of the Appendicularia class found in this study were Oikopleura longicauda (Vogt, 1854), Oikopleura dioica Fol, 1872, Oikopleura fusiformis Fol, 1872, and Fritillaria haplostoma Fol, 1872. The density of appendicularians and O. longicauda was inversely related to the nutrient values and SPM; nonetheless, no species of appendicularians could be used as a bioindicator of any estuary in the current study. Studies on the quality of estuary waters and the search for other bioindicators belonging to other zooplankton groups are recommended to confirm the obtained results and to expand the knowledge of these coastal environments.

Checklist da classe appendicularia (Chordata: Tunicata) do Estado de São Paulo, Brasil / Checklist of class appendicularia (Chordata: Tunicata) from São Paulo State, Brazil

Os tunicados da classe Appendicularia são organismos pelágicos, exclusivamente marinhos, caracterizados pela ausência da cavidade branquial e da cloaca, bem como pela retenção de notocorda e cauda muscular no estágio adulto. O corpo, delicado e transparente, é formado pelo tronco que raramente excede os 5 mm e pela cauda, várias vezes mais longa que o tronco. São hermafroditas protândricos, exceção feita à espécie Oikopleura dioica, que na época da reprodução lançam os gametas na água onde ocorre a fecundação. Os apendiculários se alimentam basicamente de pico-nanoplâncton, bem como de pequenas diatomáceas, vírus, bactérias e material coloidal. Durante a alimentação utilizam a casa ou "house", secretada pelo epitélio glandular do tronco, que possui filtros internos para concentrar as partículas antes de serem selecionadas e ingeridas. Na teia alimentar marina, são elo importante entre o pico-nanoplâncton e os níveis tróficos superiores já que servem de alimento para os outros componentes do zooplâncton, incluindo peixes de interesse comercial. Sua elevada taxa de fecundidade e de crescimento, aliada à capacidade de produzir quantidades significativas de matéria orgânica representada pela casas descartadas e pelotas fecais, os tornam peça fundamental no fluxo de energia. A neve marinha produzida por estes organismos, representa uma importante fonte de carbono para as camadas mais profundas. Os apendiculários são encontrados em todas as regiões neríticas e oceânicas do mundo, sendo mais abundantes na camada dos 100 a 200 m, embora algumas espécies habitem as regiões meso e batipelágicas. A maior diversidade de espécies foi registrada em águas quentes. Das 82 espécies identificadas até o momento em todo o mundo, 43 foram citadas para o Oceano Atlântico Sul, 29 para as águas brasileiras e 20 espéies para o Estado de São Paulo.

The appendicularians are planktonic tunicates exclusively marine, characterized by the presence of the notochord in the adult stage and the lack of the peribranchial cavity and cloacae. The body is transparent and divided into two regions: a trunk, exceptionally exceeding 5 mm, and a tail which is generally several times longer than the trunk. These organisms, with exception of Oikopleura dioica, are hermaphroditic protandric, and release their gametes directly to the water column. They have both very high fecundity and growth rate. The primarily food item of appendicularians is pico-nannoplankton, virus, bacteria and mucoid substances. They feed by means of a complex mucous structure, the "house" which is secreted by the trunk and used as tangential flow filter to concentrate ambient food particles prior to their collection by the pharyngeal filter. The appendicularians are important food item of others components of the zooplankton, including larval and adult fish. On the other hand, the marine snow produced by them contribute substantially to the carbon turn over time. The sinking rates of their faecal pellets and discharged houses can reach deep waters and their repackaging activity may play a significant role in channeling short lived carbon toward sequestration. The Appendicularians are conspicuous members of marine zooplankton, occurring in both neritic and oceanic regions of all oceans. High number of species is epipelagic, being most abundant around 100-200 m. However, some species inhabit the meso-and bathypelagic regions. The highest diversity of Appendicularians has been reported from warmest waters. At the moment 82 species belonging to the Oikopleuridae, Fritillariidae and Kowalesvkiidae families were identified, and from these, 43 species have been recorded in South Atlantic and 29 species in Brazilian waters. In São Paulo State twenty species were found along the coastal waters.