Convergent reductive evolution of cyanobacteria in symbiosis with Dinophysiales dinoflagellates.

The diversity of marine cyanobacteria has been extensively studied due to their vital roles in ocean primary production. However, little is understood about the diversity of cyanobacterial species involved in symbiotic relationships. In this study, we successfully sequenced the complete genome of a cyanobacterium in symbiosis with Citharistes regius, a dinoflagellate species thriving in the open ocean. A phylogenomic analysis revealed that the cyanobacterium (CregCyn) belongs to the marine picocyanobacterial lineage, akin to another cyanobacterial symbiont (OmCyn) of a different dinoflagellate closely related to Citharistes. Nevertheless, these two symbionts are representing distinct lineages, suggesting independent origins of their symbiotic lifestyles. Despite the distinct origins, the genome analyses of CregCyn revealed shared characteristics with OmCyn, including an obligate symbiotic relationship with the host dinoflagellates and a degree of genome reduction. In contrast, a detailed analysis of genome subregions unveiled that the CregCyn genome carries genomic islands that are not found in the OmCyn genome. The presence of the genomic islands implies that exogenous genes have been integrated into the CregCyn genome at some point in its evolution. This study contributes to our understanding of the complex history of the symbiosis between dinoflagellates and cyanobacteria, as well as the genomic diversity of marine picocyanobacteria.

Distinct regulation of two flagella by calcium during chemotaxis of male gametes in the brown alga Mutimo cylindricus (Cutleriaceae, Tilopteridales).

Brown algal male gametes show chemotaxis to the sex pheromone that is released from female gametes. The chemotactic behavior of the male gametes is controlled by the changes in the beating of two flagella known as the anterior and posterior flagellum. Our previous study using Mutimo cylindricus showed that the sex pheromone induced an increment in both the deflection angle of the anterior flagellum and sustained unilateral bend of the posterior flagellum, but the mechanisms regulating these two flagellar waveforms were not fully revealed. In this study, we analyzed the changes in swimming path and flagellar waveforms with a high-speed recording system under different calcium conditions. The extracellular Ca2+ concentration at 10-3 M caused an increment in the deflection angle of the anterior flagellum only when ionomycin was absent. No sustained unilateral bend of the posterior flagellum was induced either in the absence or presence of ionomycin in extracellular Ca2+ concentrations below 10-2 M. Real-time Ca2+ imaging revealed that there is a spot near the basal part of anterior flagellum showing higher Ca2+ than in the other parts of the cell. The intensity of the spot slightly decreased when male gametes were treated with the sex pheromone. These results suggest that Ca2+-dependent changes in the anterior and posterior flagellum are regulated by distinct mechanisms and that the increase in the anterior flagellar deflection angle and sustained unilateral bend of the posterior flagellum may not be primarily induced by the Ca2+ concentration.

The Role of Soluble Adenylyl Cyclase in the Regulation of Flagellar Motility in Ascidian Sperm.

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca2+-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca2+ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca2+ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion.

Positive selection on ADAM10 builds species recognition in the synchronous spawning coral Acropora.

The reef-building coral Acropora is a broadcast spawning hermaphrodite including more than 110 species in the Indo-Pacific. In addition, many sympatric species show synchronous spawning. The released gametes need to mate with conspecifics in the mixture of the gametes of many species for their species boundaries. However, the mechanism underlying the species recognition of conspecifics at fertilization remains unknown. We hypothesized that rapid molecular evolution (positive selection) in genes encoding gamete-composing proteins generates polymorphic regions that recognize conspecifics in the mixture of gametes from many species. We identified gamete proteins of Acropora digitifera using mass spectrometry and screened the genes that support branch site models that set the "foreground" branches showing strict fertilization specificity. ADAM10, ADAM17, Integrin α9, and Tetraspanin4 supported branch-site model and had positively selected site(s) that produced polymorphic regions. Therefore, we prepared antibodies against the proteins of A. digitifera that contained positively selected site(s) to analyze their functions in fertilization. The ADAM10 antibody reacted only with egg proteins of A. digitifera, and immunohistochemistry showed ADAM10 localized around the egg surface. Moreover, the ADAM10 antibody inhibited only A. digitifera fertilization but not the relative synchronous spawning species A. papillare. This study indicates that ADAM10 has evolved to gain fertilization specificity during speciation and contributes to species boundaries in this multi-species, synchronous-spawning, and species-rich genus.

Calaxin is required for asymmetric bend initiation and propagation in sperm flagella.

Regulation of waveform asymmetry in flagella is critical for changes in direction when sperm are swimming, as seen during the chemotaxis of sperm towards eggs. Ca2+ is an important regulator of asymmetry in flagellar waveforms. A calcium sensor protein, calaxin, is associated with the outer arm dynein and plays a key role in the regulation of flagellar motility in a Ca2+-dependent manner. However, the underlying mechanism of regulating asymmetric waves by means of Ca2+ and calaxin remains unclear. To clarify the calaxin-dependent mechanism for generating Ca2+-dependent asymmetric flagellar waveforms, we analyzed the initial step of flagellar bend formation and propagation in the sperm of the ascidian Ciona intestinalis. Our experiment used demembranated sperm cells, which were then reactivated by UV flash photolysis of caged ATP under both high and low Ca2+ concentrations. Here, we show that initial bends in the flagella are formed at the base of the sperm and propagate towards the tip during waveform generation. However, the direction of the initial bend differed between asymmetric and symmetric waves. When a calaxin inhibitor (repaglinide) was applied, it resulted in the failure of asymmetric wave formation and propagation. This was because repaglinide had no effect on initial bend formation, but it significantly inhibited the generation of the subsequent bend in the reverse direction. Switching of dynein sliding activity by mechanical feedback is crucial for flagellar oscillation. Our results suggest that the Ca2+/calaxin mechanism plays an important role in the switching of dynein activity from microtubule sliding in the principal bend into the suppressed sliding in the reverse bend, thereby allowing the sperm to successfully change direction.

Spawning-Induced pH Increase Activates Sperm Attraction and Fertilization Abilities in Eggs of the Ascidian, Phallusia philippinensis and Ciona intestinalis.

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo fertilization in the gonoductal fluids. The body fluids of the ascidians, especially in the gonoducts, were much more acidic (pH 5.5-6.8) than seawater (pH 8.2), and the fertilization rate was low under such acidic conditions. Hence, we examined the effect of pH on gametes. Pre-incubation of gonoductal eggs at pH 8.2 prior to insemination increased fertilization rates, even when insemination was performed under low pH conditions. Furthermore, an increase in ambient pH induced an increase in the intracellular pH of the eggs. It was also found that an increase in ambient pH triggered the release of sperm attractants from the egg and is therefore necessary for sperm chemotaxis. Hence, acidic conditions in the gonoductal fluids keep the gametes, especially eggs, infertile, and the release of eggs into seawater upon spawning induces an increase in ambient pH, which enables egg fertilization.

Fertilization modes and the evolution of sperm characteristics in marine fishes: Paired comparisons of externally and internally fertilizing species.

Fertilization mode may affect sperm characteristics, such as morphology, velocity, and motility. However, there is little information on how fertilization mode affects sperm evolution because several factors (e.g., sperm competition) are intricately intertwined when phylogenetically distant species are compared. Here, we investigated sperm characteristics by comparing seven externally and four internally fertilizing marine fishes from three different groups containing close relatives, considering sperm competition levels. The sperm head was significantly slenderer in internal fertilizers than in external fertilizers, suggesting that a slender head is advantageous for swimming in viscous ovarian fluid or in narrow spaces of the ovary. In addition, sperm motility differed between external and internal fertilizers; sperm of external fertilizers were only motile in seawater, whereas sperm of internal fertilizers were only motile in an isotonic solution. These results suggest that sperm motility was adapted according to fertilization mode. By contrast, total sperm length and sperm velocity were not associated with fertilization mode, perhaps because of the different levels of sperm competition. Relative testis mass (an index of sperm competition level) was positively correlated with sperm velocity and negatively correlated with the ratio of sperm head length to total sperm length. These findings suggest that species with higher levels of sperm competition have faster sperm with longer flagella relative to the head length. These results contradict the previous assumption that the evolution of internal fertilization increases the total sperm length. In addition, copulatory behavior with internal insemination may involve a large genital morphology, but this is not essential in fish, suggesting the existence of various sperm transfer methods. Although the power of our analyses is not strong because of the limited number of species, we propose a new scenario of sperm evolution in which internal fertilization would increase sperm head length, but not total sperm length, and change sperm motility.

The damage and reconstruction of the Kumamoto earthquake: an analysis on the impact of changes in expenditures with multi-regional input-output table for Kumamoto Prefecture.

The Kumamoto earthquake which occurred in April 2016 measured twice the maximum seismic intensity of 7, causing serious damage to the Kumamoto Prefecture. This study mainly focuses on the demand side of expenditures, estimating the monthly expenditures for 1 year before and after the earthquake. Then, using the multi-regional input-output table for Kumamoto Prefecture, we analyzes the ripple effects by region of the changes in monthly expenditures due to the earthquake. Expenditures in the prefecture in fiscal year 2016 by month decreased by a cumulative total of 592 billion yen because of the earthquake, which generated a value-added loss of 348 billion yen. On the other hand, expenditures increased by a cumulative total of 648 billion yen caused by reconstruction demand, inducing 375 billion yen in value-added gains. Thus, net increase of the value-added of 27 billion yen occupied 10.9% of net increase of the gross prefectural domestic product between fiscal years 2015-2016. The fluctuation of expenditures, induced production, and induced value-added caused by the earthquake is huge. Although the damage to the prefectural economy was severe, reconstruction demand exceeded it, resulting in a quick recovery. However, at the same time, there was a confirmed delay in restoration in industries that were almost unrelated to reconstruction and in regions with a heavy concentration of damage.

Two distinct compartments of a ctenophore comb plate provide structural and functional integrity for the motility of giant multicilia.

Comb plates are large ciliary structures uniquely seen in comb jellies (ctenophores).1,2,3 A comb plate is constructed from tens of thousands of cilia that are bundled together by structures called compartmenting lamellae (CLs).4,5,6 We previously reported the first component of the CL, CTENO64, and found that it was specifically found in ctenophores and was essential for the determination of ciliary orientation.3 However, CTENO64 is localized only in the proximal region of the CL; therefore, the molecular architecture of the CL over the entire length of a comb plate has not been elucidated. Here, we identified a second CL component, CTENO189. This ctenophore-specific protein was present in the distal region of comb plates, with a localization clearly segregated from CTENO64. Knockdown of the CTENO189 gene using morpholino antisense oligonucleotides resulted in complete loss of CLs in the distal region of comb plates but did not affect the formation of comb plates or the orientation of each cilium. However, the hexagonal distribution of cilia was disarranged, and the metachronal coordination of comb plates along a comb row was lost in the CTENO189 morphants. The morphant comb plate showed asymmetric ciliary-type movement in normal seawater, and in a high-viscosity solution, it could not maintain the normal waveforms but showed a symmetric flagellar-type movement. Our findings demonstrated two distinct compartments of a comb plate: the proximal CL as the building foundation that rigidly fixes the ciliary orientation, and the distal CL that reinforces the elastic connection among cilia to overcome the hydrodynamic drag of giant multiciliary plates.

Mass spectrometry of short peptides reveals common features of metazoan peptidergic neurons.

The evolutionary origins of neurons remain unknown. Although recent genome data of extant early-branching animals have shown that neural genes existed in the common ancestor of animals, the physiological and genetic properties of neurons in the early evolutionary phase are still unclear. Here, we performed a mass spectrometry-based comprehensive survey of short peptides from early-branching lineages Cnidaria, Porifera and Ctenophora. We identified a number of mature ctenophore neuropeptides that are expressed in neurons associated with sensory, muscular and digestive systems. The ctenophore peptides are stored in vesicles in cell bodies and neurites, suggesting volume transmission similar to that of cnidarian and bilaterian peptidergic systems. A comparison of genetic characteristics revealed that the peptide-expressing cells of Cnidaria and Ctenophora express the vast majority of genes that have pivotal roles in maturation, secretion and degradation of neuropeptides in Bilateria. Functional analysis of neuropeptides and prediction of receptors with machine learning demonstrated peptide regulation of a wide range of target effector cells, including cells of muscular systems. The striking parallels between the peptidergic neuronal properties of Cnidaria and Bilateria and those of Ctenophora, the most basal neuron-bearing animals, suggest a common evolutionary origin of metazoan peptidergic nervous systems.

Axonemal Growth and Alignment During Paraspermatogenesis in the Marine Gastropod Strombus luhuanus.

Parasperm are non-fertilizing sperm that are produced simultaneously with fertile eusperm. They occur in several animal species and show considerable morphological diversity. We investigated the dynamics of axonemes during paraspermatogenesis in the marine snail S. luhuanus. Mature parasperm were characterized by two lateral undulating membranes for motility and many globular vesicles. Axonemes were first observed as brush-like structures that extruded from the anterior region. Multiple axonemes longer than the brush then started to extend inside the cytoplasm towards the posterior region. The mass of the axonemes separated into two lateral rows and formed an undulating membrane that drives bidirectional swimming in the mature parasperm. The central pair of axonemes was aligned in the undulating membrane, resulting in cooperative bend propagation. During paraspermatogenesis, centrioles were largely diminished and localized to the anterior region. CEP290, a major component of the transition zone, showed a broad distribution in the anterior area. Axonemes in the posterior region showed a 9 + 0 structure with both outer and inner arm dyneins. These observations provide a structural basis for understanding the physiological functions of parasperm in marine reproductive strategies.

JAMBIO and Its Coastal Organism Joint Surveys: Network of Marine Stations Explores Japanese Coastal Biota.

Marine stations have continued to contribute significantly to understanding the physiology, taxonomy, development, ecology, and evolution of animals. There are more than 50 marine stations of national universities in Japan, and historically their establishments were closely related to the initial stage of zoology in the country. More than 10 years ago, Japanese Association for Marine Biology (JAMBIO) was established to facilitate the collaboration among marine stations in the activities of research, education and administration. One of the successful activities of JAMBIO that contribute to zoology is the JAMBIO Coastal Organism Joint Surveys, in which scientists and students at multiple marine stations, as well as those from research institutes or museums, stay at a marine station for a few days, and collect and make a record of marine organisms. As of 2021, 22 surveys have been performed and new species have been reported from taxa such as Cnidaria, Nematoda, Platyhelminthes, Annelida, Mollusca, Arthropoda, and Echinodermata.

A New Species of Acoela Possessing a Middorsal Appendage with a Possible Sensory Function.

Acoels, belonging to Xenacoelomorpha, are small worms with a relatively simple body plan and are considered a critical clade for understanding the evolution of bilaterians. Despite acoels' importance, however, many undiscovered species are predicted to be present worldwide. Here, we describe a new marine acoel species, Amphiscolops oni sp. nov., based on materials collected from the intertidal and subtidal zones of rocky shores at several localities along the Japanese Pacific coast. The new species is approximately 3 mm long and shows typical characteristics of the family Convolutidae, such as the presence of eyespots, symbiosis with algae, position of the gonopores, morphology of the bursal nozzles, lack of central singlet microtubules in the axonemes of spermatozoa, and funnel-like posture of the anterior end. Based on morphology and the results of molecular phylogenetic analyses, we assign this species to the genus Amphiscolops. Interestingly, these worms show unique behaviors such as swimming by flapping the lateral sides and actively capturing prey by swinging the anterior funnel. Furthermore, they possess a dorsal appendage-a characteristic previously unreported in Xenacoelomorpha-representing an evolutionary novelty acquired by this species.

The Roles of Two CNG Channels in the Regulation of Ascidian Sperm Chemotaxis.

Spermatozoa sense and respond to their environmental signals to ensure fertilization success. Reception and transduction of signals are reflected rapidly in sperm flagellar waveforms and swimming behavior. In the ascidian Ciona intestinalis (type A; also called C. robusta), an egg-derived sulfated steroid called SAAF (sperm activating and attracting factor), induces both sperm motility activation and chemotaxis. Two types of CNG (cyclic nucleotide-gated) channels, Ci-tetra KCNG (tetrameric, cyclic nucleotide-gated, K+-selective) and Ci-HCN (hyperpolarization-activated and cyclic nucleotide-gated), are highly expressed in Ciona testis from the comprehensive gene expression analysis. To elucidate the sperm signaling pathway to regulate flagellar motility, we focus on the role of CNG channels. In this study, the immunochemical analysis revealed that both CNG channels are expressed in Ciona sperm and localized to sperm flagella. Sperm motility analysis and Ca2+ imaging during chemotaxis showed that CNG channel inhibition affected the changes in flagellar waveforms and Ca2+ efflux needed for the chemotactic turn. These results suggest that CNG channels in Ciona sperm play a vital role in regulating sperm motility and intracellular Ca2+ regulation during chemotaxis.

Morphological differences in tardigrade spermatozoa induce variation in gamete motility.

BACKGROUND: Fertilization is an event at the beginning of ontogeny. Successful fertilization depends on strategies for uniting female and male gametes that developed throughout evolutionary history. In some species of tardigrades, investigations of reproduction have revealed that released spermatozoa swim in the water to reach a female, after which the gametes are stored in her body. The morphology of the spermatozoa includes a coiled nucleus and a species-specific-length acrosome. Although the mating behaviour and morphology of tardigrades have been reported, the motility of male gametes remains unknown. Here, using a high-speed camera, we recorded the spermatozoon motilities of two tardigrades, Paramacrobiotus sp. and Macrobiotus shonaicus, which have longer and shorter spermatozoa, respectively. RESULTS: The movement of spermatozoa was faster in Paramacrobiotus sp. than in M. shonaicus, but the beat frequencies of the tails were equal, suggesting that the long tail improved acceleration. In both species, the head part consisting of a coiled nucleus and an acrosome did not swing, in contrast to the tail. The head part of Paramacrobiotus sp. spermatozoa swung harder during turning; in contrast, the tail of M. shonaicus moved more widely than the head. Finally, after mating, the spermatozoa that reached the female aggregated around the cloaca while waiting to enter her body in both tested species. CONCLUSIONS: This study provides results for the first observations and analyses of individual spermatozoon motility in tardigrades. A comparison of the spermatozoon movements of the two tardigrades suggested that the motilities of the male gametes were affected by morphological differences, where the longer spermatozoa swam faster and the shorter ones showed more stable swimming. Swimming was mainly induced by tail movement, but the long head of Paramacrobiotus sp. spermatozoa might be especially important for turning. In addition, observations of mated female cloacae suggested that the head parts of the spermatozoa were required for aggregation around the cloaca of a mated female.

A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light.

Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.

Region-Specific Loss of Two-Headed Ciliary Dyneins in Ascidian Endostyle.

A mucous secreting organ in ascidians, the endostyle, consists of several epithelial zones with different ciliary length, density, and beating direction. Here we found by transmission electron microscopy that long cilia in endostyle zone 1 showed 9 + 2 axonemal structures but completely lacked the outer arm dynein. In contrast, cilia in other zones bore both outer and inner dynein arms. Western blotting and immunofluorescence microscopy further revealed that zone 1 appeared to lack not only outer arm dynein but also two-headed inner arm dynein. These results suggest a mechanism for a region-specific gene suppression that causes the limited loss of two-headed axonemal dyneins in the endostyle epithelium. The loss of these dyneins in zone 1 is considered to contribute to the generation of undulating ciliary movement that is essential for a unique circuit of mucus flow in the endostyle.

Structural diversity and distribution of cilia in the apical sense organ of the ctenophore Bolinopsis mikado.

The apical organ of ctenophores is the center of sensory information that controls locomotion. Previous studies have described several types of cilia in this organ. However, detailed ciliary structures, particularly axonemal structures, have not been extensively investigated. Here, we reported that the apical organ of the ctenophore Bolinopsis mikado contains six types of cilia with different axonemal structures. These include the typical "9 + 2" motile axonemes, with both outer and inner dynein arms, only the inner dynein arm, or no dynein arm; axonemes with electron-dense structures in the A-tubules; "9 + 0" axonemes lacking the central pair of microtubules; and axonemes with compartmenting lamellae. Considering that "9 + 2" axonemal structures with both dynein arms are thought to be ancestral forms of cilia, the apical organ of ctenophores would comprise an elaborate assembly of modified ciliary forms that sense and transmit extracellular stimuli and generate various fluid flows.

Changes in fish communities due to benthic habitat shifts under ocean acidification conditions.

Ocean acidification will likely change the structure and function of coastal marine ecosystems over coming decades. Volcanic carbon dioxide seeps generate dissolved CO2 and pH gradients that provide realistic insights into the direction and magnitude of these changes. Here, we used fish and benthic community surveys to assess the spatio-temporal dynamics of fish community properties off CO2 seeps in Japan. Adding to previous evidence from ocean acidification ecosystem studies conducted elsewhere, our findings documented shifts from calcified to non-calcified habitats with reduced benthic complexity. In addition, we found that such habitat transition led to decreased diversity of associated fish and to selection of those fish species better adapted to simplified ecosystems dominated by algae. Our data suggest that near-future projected ocean acidification levels will oppose the ongoing range expansion of coral reef-associated fish due to global warming.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.