The appendicularian Oikopleura dioica can enhance carbon export in a high CO2 ocean.

Gelatinous zooplankton are increasingly recognized to play a key role in the ocean's biological carbon pump. Appendicularians, a class of pelagic tunicates, are among the most abundant gelatinous plankton in the ocean, but it is an open question how their contribution to carbon export might change in the future. Here, we conducted an experiment with large volume in situ mesocosms (~55-60 m3 and 21 m depth) to investigate how ocean acidification (OA) extreme events affect food web structure and carbon export in a natural plankton community, particularly focusing on the keystone species Oikopleura dioica, a globally abundant appendicularian. We found a profound influence of O. dioica on vertical carbon fluxes, particularly during a short but intense bloom period in the high CO2 treatment, during which carbon export was 42%-64% higher than under ambient conditions. This elevated flux was mostly driven by an almost twofold increase in O. dioica biomass under high CO2 . This rapid population increase was linked to enhanced fecundity (+20%) that likely resulted from physiological benefits of low pH conditions. The resulting competitive advantage of O. dioica resulted in enhanced grazing on phytoplankton and transfer of this consumed biomass into sinking particles. Using a simple carbon flux model for O. dioica, we estimate that high CO2 doubled the carbon flux of discarded mucous houses and fecal pellets, accounting for up to 39% of total carbon export from the ecosystem during the bloom. Considering the wide geographic distribution of O. dioica, our findings suggest that appendicularians may become an increasingly important vector of carbon export with ongoing OA.

Comparative Transcriptomic Analysis Reveals the Functionally Segmented Intestine in Tunicate Ascidian.

The vertebrate intestinal system consists of separate segments that remarkably differ in morphology and function. However, the origin of intestinal segmentation remains unclear. In this study, we investigated the segmentation of the intestine in a tunicate ascidian species, Ciona savignyi, by performing RNA sequencing. The gene expression profiles showed that the whole intestine was separated into three segments. Digestion, ion transport and signal transduction, and immune-related pathway genes were enriched in the proximal, middle, and distal parts of the intestine, respectively, implying that digestion, absorption, and immune function appear to be regional specializations in the ascidian intestine. We further performed a multi-species comparison analysis and found that the Ciona intestine showed a similar gene expression pattern to vertebrates, indicating tunicates and vertebrates might share the conserved intestinal functions. Intriguingly, vertebrate pancreatic homologous genes were expressed in the digestive segment of the Ciona intestine, suggesting that the proximal intestine might play the part of pancreatic functions in C. savignyi. Our results demonstrate that the tunicate intestine can be functionally separated into three distinct segments, which are comparable to the corresponding regions of the vertebrate intestinal system, offering insights into the functional evolution of the digestive system in chordates.

Contributions from both the brain and the vascular network guide behavior in the colonial tunicate Botryllus schlosseri.

We studied the function, development and aging of the adult nervous system in the colonial tunicate Botryllus schlosseri. Adults, termed zooids, are filter-feeding individuals. Sister zooids group together to form modules, and modules, in turn, are linked by a shared vascular network to form a well-integrated colony. Zooids undergo a weekly cycle of regression and renewal during which mature zooids are replaced by developing buds. The zooid brain matures and degenerates on this 7-day cycle. We used focal extracellular recording and video imaging to explore brain activity in the context of development and degeneration and to examine the contributions of the nervous system and vascular network to behavior. Recordings from the brain revealed complex firing patterns arising both spontaneously and in response to stimulation. Neural activity increases as the brain matures and declines thereafter. Motor behavior follows the identical time course. The behavior of each zooid is guided predominantly by its individual brain, but sister zooids can also exhibit synchronous motor behavior. The vascular network also generates action potentials that are largely independent of neural activity. In addition, the entire vascular network undergoes slow rhythmic contractions that appear to arise from processes endogenous to vascular epithelial cells. We found that neurons in the brain and cells of the vascular network both express multiple genes for voltage-gated Na+ and Ca2+ ion channels homologous (based on sequence) to mammalian ion channel genes.

Transcriptome landscapes that signify Botrylloides leachi (Ascidiacea) torpor states.

Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunolocalization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be 'aroused' to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.

An Efficient Adaptive Salp Swarm Algorithm Using Type II Fuzzy Entropy for Multilevel Thresholding Image Segmentation.

Salp swarm algorithm (SSA) is an innovative contribution to smart swarm algorithms and has shown its utility in a wide range of research domains. While it is an efficient algorithm, it is noted that SSA suffers from several issues, including weak exploitation, convergence, and unstable exploitation and exploration. To overcome these, an improved SSA called as adaptive salp swarm algorithm (ASSA) was proposed. Thresholding is among the most effective image segmentation methods in which the objective function is described in relation of threshold values and their position in the histogram. Only if one threshold is assumed, a segmented image of two groups is obtained. But on other side, several groups in the output image are generated with multilevel thresholds. The methods proposed by authors previously were traditional measures to identify objective functions. However, the basic challenge with thresholding methods is defining the threshold numbers that the individual must choose. In this paper, ASSA, along with type II fuzzy entropy, is proposed. The technique presented is examined in context with multilevel image thresholding, specifically with ASSA. For this reason, the proposed method is tested using various images simultaneously with histograms. For evaluating the performance efficiency of the proposed method, the results are compared, and robustness is tested with the efficiency of the proposed method to multilevel segmentation of image; numerous images are utilized arbitrarily from datasets.

Combined effect of cell geometry and polarity domains determines the orientation of unequal division.

Cell division orientation is thought to result from a competition between cell geometry and polarity domains controlling the position of the mitotic spindle during mitosis. Depending on the level of cell shape anisotropy or the strength of the polarity domain, one dominates the other and determines the orientation of the spindle. Whether and how such competition is also at work to determine unequal cell division (UCD), producing daughter cells of different size, remains unclear. Here, we show that cell geometry and polarity domains cooperate, rather than compete, in positioning the cleavage plane during UCDs in early ascidian embryos. We found that the UCDs and their orientation at the ascidian third cleavage rely on the spindle tilting in an anisotropic cell shape, and cortical polarity domains exerting different effects on spindle astral microtubules. By systematically varying mitotic cell shape, we could modulate the effect of attractive and repulsive polarity domains and consequently generate predicted daughter cell size asymmetries and position. We therefore propose that the spindle position during UCD is set by the combined activities of cell geometry and polarity domains, where cell geometry modulates the effect of cortical polarity domain(s).

Cardiopharyngeal deconstruction and ancestral tunicate sessility.

A central question in chordate evolution is the origin of sessility in adult ascidians, and whether the appendicularian complete free-living style represents a primitive or derived condition among tunicates1. According to the 'a new heart for a new head' hypothesis, the evolution of the cardiopharyngeal gene regulatory network appears as a pivotal aspect to understand the evolution of the lifestyles of chordates2-4. Here we show that appendicularians experienced massive ancestral losses of cardiopharyngeal genes and subfunctions, leading to the 'deconstruction' of two ancestral modules of the tunicate cardiopharyngeal gene regulatory network. In ascidians, these modules are related to early and late multipotency, which is involved in lineage cell-fate determination towards the first and second heart fields and siphon muscles. Our work shows that the deconstruction of the cardiopharyngeal gene regulatory network involved the regressive loss of the siphon muscle, supporting an evolutionary scenario in which ancestral tunicates had a sessile ascidian-like adult lifestyle. In agreement with this scenario, our findings also suggest that this deconstruction contributed to the acceleration of cardiogenesis and the redesign of the heart into an open-wide laminar structure in appendicularians as evolutionary adaptations during their transition to a complete pelagic free-living style upon the innovation of the food-filtering house5.

Selective feeding in Southern Ocean key grazers-diet composition of krill and salps.

Over the past decades, two key grazers in the Southern Ocean (SO), krill and salps, have experienced drastic changes in their distribution and abundance, leading to increasing overlap of their habitats. Both species occupy different ecological niches and long-term shifts in their distributions are expected to have cascading effects on the SO ecosystem. However, studies directly comparing krill and salps are lacking. Here, we provide a direct comparison of the diet and fecal pellet composition of krill and salps using 18S metabarcoding and fatty acid markers. Neither species' diet reflected the composition of the plankton community, suggesting that in contrast to the accepted paradigm, not only krill but also salps are selective feeders. Moreover, we found that krill and salps had broadly similar diets, potentially enhancing the competition between both species. This could be augmented by salps' ability to rapidly reproduce in favorable conditions, posing further risks to krill populations.

Nkx2-1 and FoxE regionalize glandular (mucus-producing) and thyroid-equivalent traits in the endostyle of the chordate Oikopleura dioica.

The endostyle is a ventral pharyngeal organ used for internal filter feeding of basal chordates and is considered homologous to the follicular thyroid of vertebrates. It contains mucus-producing (glandular) and thyroid-equivalent regions organized along the dorsoventral (DV) axis. Although thyroid-related genes (Nkx2-1, FoxE, and thyroid peroxidase (TPO)) are known to be expressed in the endostyle, their roles in establishing regionalization within the organ have not been demonstrated. We report that Nkx2-1 and FoxE are essential for establishing DV axial identity in the endostyle of Oikopleura dioica. Genome and expression analyses showed von Willebrand factor-like (vWFL) and TPO/dual oxidase (Duox)/Nkx2-1/FoxE as orthologs of glandular and thyroid-related genes, respectively. Knockdown experiments showed that Nkx2-1 is necessary for the expression of glandular and thyroid-related genes, whereas FoxE is necessary only for thyroid-related genes. Moreover, Nkx2-1 expression is necessary for FoxE expression in larvae during organogenesis. The results demonstrate the essential roles of Nkx2-1 and FoxE in establishing regionalization in the endostyle, including (1) the Nkx2-1-dependent glandular region, and (2) the Nkx2-1/FoxE-dependent thyroid-equivalent region. DV axial regionalization may be responsible for organizing glandular and thyroid-equivalent traits of the pharynx along the DV axis.

Current status (as of end of 2020) of marine alien species in Turkey.

The 2020's update of marine alien species list from Turkey yielded a total of 539 species belonging to 18 taxonomic groups, 404 of which have become established in the region and 135 species are casual. A total of 185 new alien species have been added to the list since the previous update of 2011. The present compilation includes reports of an ascidian species (Rhodosoma turcicum) new to the marine fauna of Turkey and range extensions of six species. Among the established species, 105 species have invasive characters at least in one zoogeographic region, comprising 19% of all alien species. Mollusca ranked first in terms of the number of species (123 species), followed by Foraminifera (91 species), Pisces (80 species) and Arthropoda (79 species). The number of alien species found in seas surrounding Turkey ranged from 28 (Black Sea) to 413 (Levantine Sea). The vectoral importance of the Suez Canal diminishes when moving from south to north, accounting for 72% of species introductions in the Levantine Sea vs. only 11% of species introductions in the Black Sea. Most alien species on the coasts of Turkey were originated from the Red Sea (58%), due to the proximity of the country to the Suez Canal. Shipping activities transported 39% of alien species, mainly from the Indo-Pacific area (20%) and the Atlantic Ocean (10%). Misidentified species (such as Pterois volitans, Trachurus declivis, etc.) and species those classified as questionable or cryptogenic were omitted from the list based on new data gathered in the last decade and expert judgements. The documented impacts of invasive species on socio-economy, biodiversity and human health in the last decade as well as the legislation and management backgrounds against alien species in Turkey are presented.

Phylogenomic and morphological relationships among the botryllid ascidians (Subphylum Tunicata, Class Ascidiacea, Family Styelidae).

Ascidians (Phylum Chordata, Class Ascidiacea) are a large group of invertebrates which occupy a central role in the ecology of marine benthic communities. Many ascidian species have become successfully introduced around the world via anthropogenic vectors. The botryllid ascidians (Order Stolidobranchia, Family Styelidae) are a group of 53 colonial species, several of which are widespread throughout temperate or tropical and subtropical waters. However, the systematics and biology of this group of ascidians is not well-understood. To provide a systematic framework for this group, we have constructed a well-resolved phylogenomic tree using 200 novel loci and 55 specimens. A Principal Components Analysis of all species described in the literature using 31 taxonomic characteristics revealed that some species occupy a unique morphological space and can be easily identified using characteristics of adult colonies. For other species, additional information such as larval or life history characteristics may be required for taxonomic discrimination. Molecular barcodes are critical for guiding the delineation of morphologically similar species in this group.

3D reconstruction of structures of hatched larva and young juvenile of the larvacean Oikopleura dioica using SBF-SEM.

The larvacean Oikopleura dioica is a planktonic chordate and an emerging model organism with a short life cycle of 5 days that belongs toTunicata (Urochordata), the sister clade of vertebrates. It is characterized by the rapid development of a tadpole-shaped body. Organ formation in the trunk proceeds within 7 h after the hatching of the tailbud larvae at 3 h after fertilization (hpf) and is completed at 10 hpf, giving rise to fully functional juveniles as miniature adult form. Serial block face scanning electron microscopy was used to acquire ~ 2000 serial transverse section images of a 3 hpf larva and a 10 hpf juvenile to characterize the structures and cellular composition of the trunk and organs using 3D images and movies. Germ cells were found to fuse and establish a central syncytial cell in the gonad as early as 10 hpf. Larval development gave rise to functional organs after several rounds of cell division through trunk morphogenesis. The feature would make O. dioica ideal for analyzing cellular behaviors during morphogenetic processes using live imaging. The detailed descriptions of the larvae and juveniles provided in this study can be utilized as the start and end points of organ morphogenesis in this rapidly developing organism.

High crystallinity of tunicate cellulose nanofibers for high-performance engineering films.

Tunicate cellulose nanofibers (CNFs) have received widespread attention as renewable and eco-friendly engineering materials because of their high crystallinity and mechanical stiffness. Here, we report the effects of disintegration process conditions on structure-property relationships of tunicate CNFs. By varying the hydrolysis time, we could establish a correlation between crystallinity of the CNFs with linearity and stiffness, which produces different molecular ordering within their nanostructured films. Despite having identical raw materials, tensile strength and thermal conductivity of the resulting layered films varied widely, ranging from 95.6 to 205 MPa and from 1.08 to 2.37 W/mK respectively. Furthermore, nanolayered CNF films provided highly anisotropic thermal conductivities with an in- and through-plane ratio of 21.5. Our systematic investigations will provide general and practical strategies in tailoring material properties for emerging engineering applications, including flexible paper electronics, heat sink adhesives and biodegradable, implantable devices.

Identification of DNA (de)methylation-related genes and their transcriptional response to environmental challenges in an invasive model ascidian.

Marine invasive species are constantly challenged by acute or recurring environmental stresses during their range expansions. DNA methylation-mediated stress memory has been proposed to effectively affect species' response and enhance their overall performance in recurring environmental challenges. In order to further test this proposal in marine invasive species, we identified genes in the DNA methylation and demethylation processes in the highly invasive model species, Ciona robusta, and subsequently investigated the expression patterns of these genes under recurring salinity stresses. After a genome-wide comprehensive survey, we found a total of six genes, including two genes of DNA methyltransferase 3a (DNMT3a1 and DNMT3a2), and one gene of DNA methyltransferase 1 (DNMT1), methyl-CpG-binding domain protein 2 (MBD2), methyl-CpG-binding domain protein 4 (MBD4) and ten-eleven-translocation protein 1 (TET1). Phylogenetic reconstruction and domain arrangement analyses showed that the deduced proteins of the identified genes were evolutionarily conserved and functionally similar with their orthologs. All genes were constitutively expressed in all four tested tissues. Interestingly, we found time-dependent and stress-specific gene expression patterns under high and low salinity stresses. Under the recurring high salinity stresses, DNMT3a1 and TET1 conformed to the definition of memory genes, while under the recurring low salinity stresses, two DNMT3a paralogues were identified as the memory genes. Altogether, our results clearly showed that the transcriptional patterns of (de)methylation-related genes were significantly influenced by environmental stresses, and the transcriptional memory of some (de)methylation-related genes should play crucial roles in DNA methylation-mediated stress memory during the process of biological invasions.

Phylogenetic comparison of egg transparency in ascidians by hyperspectral imaging.

The transparency of animals is an important biological feature. Ascidian eggs have various degrees of transparency, but this characteristic has not yet been measured quantitatively and comprehensively. In this study, we established a method for evaluating the transparency of eggs to first characterize the transparency of ascidian eggs across different species and to infer a phylogenetic relationship among multiple taxa in the class Ascidiacea. We measured the transmittance of 199 eggs from 21 individuals using a hyperspectral camera. The spectrum of the visual range of wavelengths (400-760 nm) varied among individuals and we calculated each average transmittance of the visual range as bio-transparency. When combined with phylogenetic analysis based on the nuclear 18S rRNA and the mitochondrial cytochrome c oxidase subunit I gene sequences, the bio-transparencies of 13 species were derived from four different families: Ascidiidae, Cionidae, Pyuridae, and Styelidae. The bio-transparency varied 10-90% and likely evolved independently in each family. Ascidiella aspersa showed extremely high (88.0 ± 1.6%) bio-transparency in eggs that was maintained in the "invisible" larva. In addition, it was indicated that species of the Ascidiidae family may have a phylogenetic constraint of egg transparency.

Role of PB1 Midbody Remnant Creating Tethered Polar Bodies during Meiosis II.

Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. Using ascidians, we observed that PB1 becomes tethered to the fertilized egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small structure around 1 µm in diameter protruded from the cortical outpocket that will form the future PB2, which we define as the "polar corps". As emission of PB2 progressed, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 outpocket was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in fertilized eggs that had PB1 removed suggested that the midbody remnant remained within the fertilized egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 since our data are correlative in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in several species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.

Characterization of vanadium of biological origin for possible applications in physics experiments.

For the first time, vanadium of biological origin, extracted from centrifugal fraction of vanadium-storing blood cells of the Ascidia sydneiensis samea species, was characterized as regards its isotopic composition and content of natural radioactive elements potassium (K), thorium (Th) and uranium (U). The natural abundance of vanadium isotopes has been confirmed with high accuracy, thus excluding a possible selectivity within bio-chemical reactions of vanadium concentration in blood cells from seawater. A large potassium concentration (up to 5500 × 10-6 g g-1) was found in the blood cell samples. The concentration of thorium was determined to be about 30 × 10-9 g g-1, while the uranium concentration was about 150 × 10-9 g g-1. Hence, a highly efficient two-stage purification approach with a total vanadium recovery of better than 70% was developed and applied. The final concentrations of K < 100 × 10-6 g g-1 and of U/Th < 0.5 × 10-9 g g-1 in the purified vanadium-containing samples were achieved. Vanadium extracted from centrifugal fraction of vanadium-storing blood cells after two-stage purification approach could be utilized in various applications, where a high chemical purity compound is required. However, to be used as a source of radiopure vanadium in ultra-low-background experiment aimed to search for 50V beta decay, it should be further purified by Electron Beam Melting against residual potassium.

Signatures of local adaptation in the spatial genetic structure of the ascidian Pyura chilensis along the southeast Pacific coast.

The highly heterogeneous Humboldt Current System (HCS) and the 30°S transition zone on the southeast Pacific coast, represent an ideal scenario to test the influence of the environment on the spatial genomic structure in marine near-shore benthic organisms. In this study, we used seascape genomic tools to evaluate the genetic structure of the commercially important ascidian Pyura chilensis, a species that exhibits a low larval transport potential but high anthropogenic dispersal. A recent study in this species recorded significant genetic differentiation across a transition zone around 30°S in putatively adaptive SNPs, but not in neutral ones, suggesting an important role of environmental heterogeneity in driving genetic structure. Here, we aim to understand genomic-oceanographic associations in P. chilensis along the Southeastern Pacific coast using two combined seascape genomic approaches. Using 149 individuals from five locations along the HCS, a total of 2,902 SNPs were obtained by Genotyping-By-Sequencing, of which 29-585 were putatively adaptive loci, depending on the method used for detection. In adaptive loci, spatial genetic structure was better correlated with environmental differences along the study area (mainly to Sea Surface Temperature, upwelling-associated variables and productivity) than to the geographic distance between sites. Additionally, results consistently showed the presence of two groups, located north and south of 30°S, which suggest that local adaptation processes seem to allow the maintenance of genomic differentiation and the spatial genomic structure of the species across the 30°S biogeographic transition zone of the Humboldt Current System, overriding the homogenizing effects of gene flow.

Wettability and Substrate Selection in the Larval Settlement of the Solitary Ascidian Phallusia philippinensis (Phlebobranchia: Ascidiidae).

For marine benthic animals, the selection of substrate by larvae is important for their survival, with early post-settlement mortality often being affected by the microenvironment where they settle. We tested the substrate preference of the larvae of the ascidian Phallusia philippinensis toward nine commercially available substrates. In the present assay, the larvae settled on one of four substrates for seven substrate combinations with different wettabilities; we counted the number of settled larvae on each of the four substrates, and Manly's selection indices were compared to determine the preference rank of each substrate. Larvae significantly preferred more hydrophobic and oleophilic substrates to hydrophilic and oleophobic ones. While it is uncertain how the larvae detect the properties of the substrate surface, they might be able to sense the physical force, such as stickiness and repellent force. Although a hydrophobic surface is not common in a natural marine environment, the use of hydrophobic materials (as flypaper-like tools) for ascidian larvae might help to prevent the settlement of non-indigenous ascidians in aquaculture facilities.