Conservation of Protein Kinase A Substrates in the Cnidarian Coral Spermatozoa Among Animals and Their Molecular Evolution.

The coral Acropora spp., known for its reef-building abilities, is a simultaneous hermaphroditic broadcast spawning species. Acropora spp. release gametes into seawater, activating sperm motility. This activation is mediated by adenylyl cyclase (AC) and protein kinase A (PKA). Notably, membrane-permeable cAMP (8-bromo-cAMP) promotes sperm motility activation of Acropora florida. While the signal transduction for PKA-dependent motility activation is highly conserved among animals, the downstream signaling of PKA remains unclear. In this study, we used mass spectrometry (MS) analyses to identify sperm proteins in the coral Acropora digitifera, as well as the serine/threonine residues of potential PKA substrates, and then, we investigated the conservation of these proteins from corals to vertebrates. We identified 148 sperm proteins of A. digitifera with typical PKA recognition motifs, namely RRXT and RRXS. We subsequently used ORTHOSCOPE to screen for orthologs encoding these 148 proteins from corals to vertebrates. Among the isolated orthologs, we identified positive selection in 48 protein-encoding genes from 18 Acropora spp. Subsequently, we compared the conservation rates of the PKA phosphorylation motif residues between the orthologs under positive and purifying selections. Notably, the serine residues of the orthologs under positive selection were more conserved. Therefore, adaptive evolution might have occurred in the orthologs of PKA substrate candidates from corals to vertebrates, accompanied by phosphorylation residue conservation. Collectively, our findings suggest that while PKA signal transduction, including substrates in sperm, may have been conserved, the substrates may have evolved to adapt to diverse fertilization conditions, such as synchronous broadcast spawning.

Integrative taxonomic analyses reveal that rapid genetic divergence drives Acropora speciation.

Reef-building corals provide the structural basis for one of Earth's most spectacular and diverse but increasingly threatened ecosystems. The reef-building coral genus Acropora may have undergone substantial speciation during the Pleistocene climate and sea-level changes. Here, we aimed to evaluate the speciation history of four morphologically similar tabular Acropora species (Acropora aff. hyacinthus, A. cf. bifurcata, A. cf. cytherea, and A. cf. subulata) using an integrative approach with morphology, genetic, and reproduction methodology. Extensive morphological analyses showed that these four species are distinct and exhibited high gamete incompatibility, preventing hybridization. Furthermore, population structure and principal component analyses with SNPs (>60,000) indicated that these species were genetically distinct, and the ABBA-BABA test did not support introgression among these species. Many of their coding and noncoding RNA sequences showed high genetic variance at loci with high Fst values along the genome. Comparison of these orthologs with those of other Acropora species suggested that many of these genes are under positive selection, which could be associated with spawning time, gamete, and morphological divergence. Our findings show that the speciation of tabular Acropora occurred without hybridization, and the divergence accompanying the rapid evolution of genes in species-rich Acropora could be associated with speciation.

Trajectory and course of problematic alcohol use after the Great East Japan Earthquake: Eight-year follow-up of the Higashi-Matsushima cohort study.

BACKGROUND: Problematic alcohol use (PAU) after natural disasters is an unignorable public health issue. However, the long-term trajectory and course of PAU after an earthquake disaster remain unknown. METHODS: The Higashi-Matsushima cohort study was conducted between 2012 (time 1) and 2019 (time 8) in areas affected by the Great East Japan Earthquake in 2011. In the annual health checks, participants responded to self-report questionnaires on PAU, traumatic experiences (e.g., house damage), resources (e.g., social support), and other covariates (e.g., gender, psychological distress). The trajectory and course of PAU were estimated by latent growth model and latent class analyses. Risk factors for the long-term course of PAU were calculated by multinomial logistic regression analysis with multiple imputation. The analytical sample comprised 8929 residents who participated in at least one survey across the eight time points. RESULTS: The trajectory of PAU showed a sustained trend (slope <0.001). Three potential courses of PAU (No PAU course: 84.3%, Subthreshold PAU course: 12.4%, and Persistent PAU course: 3.4%) were estimated. The long-term course of PAU, especially the persistent PAU course, was predicted by house damage (OR = 1.43, 95% CI 1.06 to 1.92), less social support (OR = 0.71, 95% CI 0.53 to 0.96), gender (male) (OR = 16.86, 95% CI 9.42 to 30.20), and psychological distress (OR = 1.15, 95% CI 1.09 to 1.20). CONCLUSIONS: Long-term support is needed after an earthquake disaster, especially for residents who in early phases of the disaster suffer from PAU, males, and those in vulnerable situations resulting from conditions such as severe house damage, low social support, or high psychological distress.

Bidirectional relationship of problematic Internet use with hyperactivity/inattention and depressive symptoms in adolescents: a population-based cohort study.

Problematic Internet use (PIU), hyperactivity/inattention, and depressive symptoms are comorbid problems in adolescence, but the causal relationships among these issues are unclear. To assess the relationships among PIU, hyperactivity/inattention, and depressive symptoms in adolescents in the general population. This longitudinal cohort study used data from the Tokyo Teen Cohort study in Tokyo, Japan, for two years between October 2012 and January 2015. Of the 3171 pairs of children and parents, 3007 pairs continued to participate in the second wave of the Tokyo Teen Cohort study. A total of 3007 children were included in the analysis (mean [standard deviation] age, 9.7 [0.4] years; 1418 women [47.2%]. Cross-lagged panel analysis revealed that PIU at timepoint 1 was significantly associated with hyperactivity/inattention at timepoint 2 (ß = 0.03; 95% confidence interval (CI) 0.01-0.06), and hyperactivity/inattention at timepoint 1 was also significantly associated with PIU at timepoint 2 (ß = 0.07; 95% CI 0.04-0.10), even after adjustments were made for depressive symptoms. Furthermore, PIU at timepoint 1 was significantly associated with depressive symptoms at timepoint 2 (ß = 0.05; 95% CI 0.01-0.12), and depressive symptoms at timepoint 1 were also significantly associated with PIU at timepoint 2 (ß = 0.05; 95% CI 0.02-0.07), even after adjustments were made for hyperactivity/inattention. These results support the bidirectional relationships among PIU, hyperactivity/inattention, and depressive symptoms. PIU may be a target to improve hyperactivity/inattention and depressive symptoms in adolescents.

Differences of Extracellular Cues and Ca2+ Permeable Channels in the Signaling Pathways for Inducing Amphibian Sperm Motility.

Low osmolality of freshwater and/or sperm motility-initiating substance (SMIS) induce amphibian sperm motility through increases in intracellular Ca2+. In the internally fertilizing newt Cynops pyrrhogaster, the sperm motility-initiating substance engages T type voltage-dependent Ca2 + channels and N-methyl D-aspartate-type glutamate receptors to initiate sperm motility and L type voltage-dependent Ca2+ channels to enhance motility. In the present study, differences in the usages of SMIS and Ca2+ permeable channels for sperm motility regulation were examined in amphibians that undergo different reproductive modes. Proteins of 14-17 kDa were detected by antibody against the active site peptide of SMIS in the oviduct secretion of internal fertilizers (C. pyrrhogaster, Cynops ensicauda, and Ambystoma mexicanum) and arboreal fertilizers (Rhacophorus arboreus and Rhacophorus schlegelii), but not in Buergeria japonica, an external fertilizer in freshwater. In the pharmacological study, a blocker of some transient receptor potential channels (RN1734) additionally suppressed enhancement of sperm motility in C. pyrrhogaster. In R. schlegelii, blockers of four types of channels differently suppressed sperm motility induced by low osmolality with or without the active site peptide of SMIS. Notably, blockers of L type voltage-dependent Ca2+ channels (nifedipine) and N-methyl D-aspartate-type glutamate receptors (MK801) suppressed sperm motility in the presence and the absence of the peptide, respectively. Low osmolality-induced sperm motility was suppressed by RN1734 and MK801 in B. japonica, but not in Xenopus laevis. These results reveal complex differences in the signaling pathways for inducing sperm motility that may be partly related to reproductive modes in amphibians.

Population-neuroscience study of the Tokyo TEEN Cohort (pn-TTC): Cohort longitudinal study to explore the neurobiological substrates of adolescent psychological and behavioral development.

AIM: Adolescence is a crucial stage of psychological development and is critically vulnerable to the onset of psychopathology. Our understanding of how the maturation of endocrine, epigenetics, and brain circuit may underlie psychological development in adolescence, however, has not been integrated. Here, we introduce our research project, the population-neuroscience study of the Tokyo TEEN Cohort (pn-TTC), a longitudinal study to explore the neurobiological substrates of development during adolescence. METHODS: Participants in the first wave of the pn-TTC (pn-TTC-1) study were recruited from those of the TTC study, a large-scale epidemiological survey in which 3171 parent-adolescent pairs were recruited from the general population. Participants underwent psychological, cognitive, sociological, and physical assessment. Moreover, adolescents and their parents underwent magnetic resonance imaging (MRI; structural MRI, resting-state functional MRI, and magnetic resonance spectroscopy), and adolescents provided saliva samples for hormone analysis and for DNA analysis including epigenetics. Furthermore, the second wave (pn-TTC-2) followed similar methods as in the first wave. RESULTS: A total of 301 parent-adolescent pairs participated in the pn-TTC-1 study. Moreover, 281 adolescents participated in the pn-TTC-2 study, 238 of whom were recruited from the pn-TTC-1 sample. The instruction for data request is available at: http://value.umin.jp/data-resource.html. CONCLUSION: The pn-TTC project is a large-scale and population-neuroscience-based survey with a plan of longitudinal biennial follow up. Through this approach we seek to elucidate adolescent developmental mechanisms according to biopsychosocial models. This current biomarker research project, using minimally biased samples recruited from the general population, has the potential to expand the new research field of population neuroscience.

Variations in the breeding behavior of cichlids and the evolution of the multi-functional seminal plasma protein, seminal plasma glycoprotein 120.

BACKGROUND: Seminal plasma proteins are associated with successful fertilization. However, their evolutionary correlation with fertilization mechanisms remains unclear. Cichlids from Lake Tanganyika show a variety-rich spawning behavior that is associated with the transfer of the sperm to the egg for fertilization. One of these behaviors, called "oral fertilization," emerged during their speciation. In oral fertilization, females nuzzle the milt from male genitalia and pick up the released eggs in their mouths, which are then fertilized inside the oral cavity. Thus, the success of the fertilization is dependent on the retention of sperm in the oral cavity during spawning. Sperm aggregation and immobilization in viscous seminal plasma may help retain the sperm inside the oral cavity, which ultimately determines the success of the fertilization. Seminal plasma glycoprotein 120 (SPP120) is one of the major seminal plasma proteins present in cichlids. SPP120 has been implicated to immobilize sperm and increase the milt viscosity. However, the functional linkage between oral fertilization and seminal plasma proteins has not been investigated. RESULTS: During trials of simulated oral fertilization, it was observed that milt viscosity contributed to fertilization success by facilitating longer retention of the milt inside the mouth during spawning. Glycosylation of SPP120 was associated with high milt viscosity. Its glycosylation was specifically present in the milt of cichlid species exhibiting oral fertilization. Moreover, recombinant SPP120 from several the oral fertilization species strongly immobilized/aggregated sperm. Therefore, the functions of SPP120 (immobilization/aggregation and its glycosylation) may contribute to success of oral fertilization, and these functions of SPP120 are more prominent in oral fertilization species. In addition, comparative phylogenetic analyses showed a positive evolutionary correlation between SPP120 function and oral fertilization. Hence, these evolutions may have occurred to keep up with the transition in the mode of fertilization. In addition, rapid evolution in the molecular sequence might be associated with functional modifications of SPP120. CONCLUSION: These results suggest that SPP120 might be associated with oral fertilization. In other words, reproductive traits that define the interaction between sperms and eggs could be the evolutionary selective force that cause the rapid functional modification of the fertilization-related reproductive protein, SPP120.

Acid secretion by the boring organ of the burrowing giant clam, Tridacna crocea.

The giant clam Tridacna crocea, native to Indo-Pacific coral reefs, is noted for its unique ability to bore fully into coral rock and is a major agent of reef bioerosion. However, T. crocea's mechanism of boring has remained a mystery despite decades of research. By exploiting a new, two-dimensional pH-sensing technology and manipulating clams to press their presumptive boring tissue (the pedal mantle) against pH-sensing foils, we show that this tissue lowers the pH of surfaces it contacts by greater than or equal to 2 pH units below seawater pH day and night. Acid secretion is likely mediated by vacuolar-type H+-ATPase, which we demonstrate (by immunofluorescence) is abundant in the pedal mantle outer epithelium. Our discovery of acid secretion solves this decades-old mystery and reveals that, during bioerosion, T. crocea can liberate reef constituents directly to the soluble phase, rather than producing sediment alone as earlier assumed.

A Unique Seminal Plasma Protein, Zona Pellucida 3-Like Protein, has Ca2+ -Dependent Sperm Agglutination Activity.

Identification of seminal proteins provides a means of investigating their roles. Despite their importance in the study of protein function, such as regulation of sperm motility, it is difficult to select candidates from the large number of proteins. Analyzing the rate of molecular evolution is a useful strategy for selecting candidates, and expressing the protein allows the examination of its function. In the present study, we investigated seminal plasma proteins of the cichlid Oreochromis mossambicus, which exhibits a unique mode of fertilization and a rapidly evolving gene that encodes a seminal plasma protein, zona-pellucida 3-like (ZP3-like), which does not belong to the same molecular family as other ZPs. Seminal plasma proteins of O. mossambicus were separated by two-dimensional electrophoresis, and 19 major proteins were identified by mass spectrometry (MALDI-Tof Mass). Because proteins that are under positive selection often impact sperm function, the rates of molecular evolution of these proteins were analyzed in terms of non-synonymous/synonymous substitutions (ω). Among the 19 proteins, positive selection was supported for five genes; functional assays were carried out on four of the proteins encoded by these genes. Of the four positively selected proteins, only ZP3-like protein agglutinated sperm in a dose- and Ca2+ -dependent manner. The other three proteins did not affect sperm motility. Because of the unique fertilization type, in which fertilization occurs in the buccal cavity, the need to retain sperm within the cavity during spawning, and the agglutination of sperm, which may be partly assisted by ZP3-like protein, may contribute to fertilization success. Fertilization in the buccal cavity may be related to its rapid molecular evolution.

Sperm motility initiating substance may be insufficient to induce forward motility of Cynops ensicauda sperm.

Sperm motility-initiating substance (SMIS) is a key protein for internal fertilization of the newt, Cynops pyrrhogaster, and commonly enhances forward sperm motility in some amphibian species, including external fertilizers. SMIS action varies among different species in correlation with a species-specific reproductive environment. In the present study, we identified the gene of C. ensicauda SMIS (CeSMIS) and examined the mechanism of SMIS action with reference to that of the closely related Cynops species. The CeSMIS was identified by a 176-amino acid sequence including seven amino acids critical for the initiation of sperm motility. The amino acid sequence showed 91% homology to the whole sequence of C. pyrrhogaster SMIS (CpSMIS). By immunostaining with an anti-CpSMIS antibody, CeSMIS was shown to be localized in the outer layer of the egg jelly. A peptide presenting the active site of SMIS was observed to bind to the axial rod of the midpiece in C. ensicauda sperm. The localization and binding patterns of CeSMIS were fundamentally similar to those of CpSMIS. However, the SMIS peptide did not induce forward motility of C. ensicauda sperm, although it induced a fast wave of the undulating membrane. Forward sperm motility was induced in the egg jelly extract containing CeSMIS. These results suggest that the mechanism of initiation of sperm motility is differentiated between C. ensicauda and C. pyrrhogaster.

Global Registry of Acute Coronary Events risk score predicts mortality and neurological outcome in out-of hospital cardiac arrest.

PURPOSE: The aim of this study was to assess the usefulness of the Global Registry of Acute Coronary Events (GRACE) risk score in predicting in-hospital mortality and neurological outcome of patients resuscitated after out-of-hospital cardiac arrest (OHCA). METHODS: We retrospectively analyzed the data of patients admitted to our hospital between October 2009 and October 2015 with OHCA and shockable initial cardiac rhythm who were resuscitated via conventional cardiopulmonary resuscitation. We calculated the GRACE risk score on admission and assessed its usefulness in predicting in-hospital mortality and neurological outcome. RESULTS: Among 91 patients, 42 (46%) had acute myocardial infarction (AMI), 19 (21%) died in-hospital, and 52 (57%) had favorable neurological outcome. Among all the study patients, GRACE risk score was lower in survivors than in non-survivors (median 211 [interquartile range 176-240] vs. 266 [219-301], p<0.001, respectively) and in favorable than in unfavorable neurological outcome group (202 [167-237] vs. 242 [219-275], p<0.001, respectively). Multivariate analysis showed significant association between GRACE risk score and favorable neurological outcome (odds ratio, 0.975; 95% confidence interval, 0.961-0.990). Areas under receiver-operating characteristic curves, that describe the accuracy of GRACE risk score in predicting in-hospital mortality and favorable neurological outcome, were both 0.79. CONCLUSION: GRACE risk score may predict the in-hospital mortality and neurological outcome associated with resuscitated patients with OHCA and shockable initial cardiac rhythm, regardless of the cause of arrest.

The reef-building coral Acropora conditionally hybridize under sperm limitation.

Multi-specific synchronous spawning risks both sperm limitation, which reduces fertilization success, and hybridization with other species. If available sperm of conspecifics are limited, hybridization with heterospecific sperm could be an alternative. Some species of the reef-building coral Acropora produce hybrid offspring in vitro, and therefore hybridization between such species does sometimes occur in nature. Here, we report that the interbreeding species Acropora florida and A. intermedia preferentially bred with conspecifics at optimal gamete concentrations (10(6) cells ml(-1)), but when sperm concentration was low (10(4) cells ml(-1)), A florida eggs displayed an increased incidence of fertilization by sperm of A intermedia However, A intermedia eggs never crossed with heterospecific sperm, regardless of gamete concentrations. It appears that A florida eggs conditionally hybridize with heterospecific sperm; in nature, this would allow A florida to cross with later-spawning species such as A intermedia These results indicate that hybridization between some Acropora species could occur in nature according to the number of available sperm, and the choice of heterospecific sperm for fertilization could be one of the fertilization strategies in the sperm-limited condition.

Self/non-self recognition mechanisms in sexual reproduction: new insight into the self-incompatibility system shared by flowering plants and hermaphroditic animals.

Sexual reproduction is an essential process for generating a genetic variety in the next generation. However, most flowering plants and hermaphroditic animals potentially allow self-fertilization. Approximately 60% of angiosperms possess a self-incompatibility (SI) system to avoid inbreeding. The SI system functions at a process of interaction between pollen (or pollen tube) and the pistil. These SI-responsible factors (S-determinants) in pollen and the pistil are encoded by highly polymorphic multiallelic genes in the S-locus, which are tightly linked making a single haplotype. Different taxonomic families utilize different types of S-determinant proteins. In contrast to the plant system, the mechanisms of SI in simultaneously hermaphroditic animals are largely unknown. Among them, promising candidates for SI in ascidians (primitive chordates) were recently identified. The SI system in the ascidian Cionaintestinalis was found to be very similar to those in flowering plants: The products of sperm- and egg-side multiallelic SI genes, which are tight linked and highly polymorphic, appear to be responsible for the SI system as revealed by genetic analysis. These findings led us to speculate that the SI systems in plants and animals evolved in a manner of convergent evolution. Here, we review the current understanding of the molecular mechanisms of the SI system in flowering plants, particularly Brassicacea, and in ascidians from the viewpoint of common mechanisms shared by plants and animals.

Territorial males can sire more offspring in nests with smaller doors in the cichlid Lamprologus lemairii.

To examine how territorial males counter reproductive parasites, we examined the paternity of broods guarded by territorial males using 5 microsatellite loci and factors that determine siring success in a wild population of the Lake Tanganyika cichlid Lamprologus lemairii. Females enter rock holes (nests) and spawn inside, and territorial males release milt over the nest openings. Sneakers attempt to dart into the nests, but territorial males often interrupt the attempt. The body size of territorial males (territorial defense ability) and the size of nest opening (the ability to prevent sneakers from nest intrusions) are predicted to be factors that affect paternity at the premating stage, whereas milt quality traits are factors that affect paternity at the postmating stage. Parentage analyses of 477 offspring revealed that most clutches have few or no cuckolders, and territorial males sired >80% of eggs in 7 of the 10 analyzed clutches. Larger territorial males that spawned in nests with narrower openings had greater siring success. In contrast, none of the milt traits affected the siring success. These suggest that territorial male L. lemairii adopt premating strategies whereby they effectively prevent reproductive parasitism.

Cryopreservation of Acropora digitifera sperm with use of sucrose and methanol based solution.

Coral biodiversity has recently been considered an important topic in environmental studies. Biodiversity could be preserved with successful cryopreservation of endangered species gametes or embryos. Herein, we developed cryopreservation protocols for Acropora digitifera sperm with use of sucrose and methanol based extender. We studied cryopreservation of A. digitifera sperm with floating frames, allowing the placement of 250 µl French straws 4 cm above the liquid nitrogen surface, resulting in a 40 °C/min freezing rate. This method enabled the successful cryopreservation of sperm in 0.9 M sucrose supplemented with 20% methanol. In this protocol, we used a 1:3 (sperm:extender) dilution ratio. The fertilization ratios of freezing:thawed sperm were similar to the control and reached 63%. This method might be a valuable option in the formation of A. digitifera gene banking. Further studies are needed to explore possibilities of using this method in cryopreservation of other coral's sperm.

Beneficial effects of intracoronary nicorandil on microvascular dysfunction after primary percutaneous coronary intervention: demonstration of its superiority to nitroglycerin in a cross-over study.

PURPOSE: In patients undergoing primary percutaneous coronary intervention (PCI) for the treatment of ST-segment elevation myocardial infarction (STEMI), coronary microvascular dysfunction is associated with poor prognosis. Coronary microvascular resistance is predominantly regulated by ATP-sensitive potassium (KATP) channels. The aim of this study was to clarify whether nicorandil, a hybrid KATP channel opener and nitric oxide donor, may be a good candidate for improving microvascular dysfunction even when administered after primary PCI. METHODS: We compared the beneficial effects of nicorandil and nitroglycerin on microvascular function in 60 consecutive patients with STEMI. After primary PCI, all patients received single intracoronary administrations of nitroglycerin (250 µg) and nicorandil (2 mg) in a randomized order; 30 received nicorandil first, while the other 30 received nitroglycerin first. Microvascular dysfunction was evaluated with the index of microcirculatory resistance (IMR), defined as the distal coronary pressure multiplied by the hyperemic mean transit time. RESULTS: As a first administration, nicorandil decreased IMR significantly more than did nitroglycerin (median [interquartile ranges]: 10.8[5.2-20.7] U vs. 2.1[1.0-6.0] U, p=0.0002).As a second administration, nicorandil further decreased IMR, while nitroglycerin did not (median [interquartile ranges]: 6.0[1.3-12.7] U vs. -1.4[-2.6 to 1.3] U, p<0.0001). The IMR after the second administration was significantly associated with myocardial blush grade, angiographic TIMI frame count after the procedure, and peak creatine kinase level. CONCLUSION: Intracoronary nicorandil reduced microvascular dysfunction after primary PCI more effectively than did nitroglycerin in patients with STEMI, probably via its KATP channel-opening effect.

Acclimation potential of Acropora to mesophotic environment.

Mesophotic coral ecosystems may serve as a refuge for reef-building corals to survive the ongoing climate change. Distribution of coral species changes during larval dispersal. However, the acclimation potential in the early life stages of corals at different depths is unknown. This study investigated the acclimation potential of four shallow Acropora species at different depths via the transplantation of larvae and early polyps settled on tiles to 5, 10, 20, and 40 m depths. We then examined physiological parameters, such as size, survival, growth rate, and morphological characteristics. The survival and size of juveniles of A. tenuis and A. valida at 40 m depth were significantly higher than those at other depths. In contrast, A. digitifera and A. hyacinthus showed higher survival rates at shallow depths. The morphology (i.e., size of the corallites) also varied among the depths. Collectively, the shallow coral larvae and juveniles displayed substantial plasticity at depth.

Reactions of juvenile coral to three years of consecutive thermal stress.

As global temperatures continue to rise, corals are being exposed to increasing heat stress throughout their early life stages; however, the impact of this phenomenon is poorly understood. We exposed the reef-building coral Acropora tenuis juveniles to ∼26-28 °C (control) and ∼ 31 °C (heat stress) for one week per year over three consecutive years. In the first year of heat stress, >96 % of juveniles survived despite symbiotic algal densities in juvenile corals declining. In comparison, survival rates in the third year of heat stress declined to 50 %. Survival rates under natural conditions after stress also gradually decreased in the stressed groups. The rate in the reduction of survivorship was prominent in the consecutive thermally stressed groups (juveniles stressed twice in two years). Symbiotic algal density and photosynthetic activity (Fv/Fm) also declined in stressed juvenile groups. However, heat stress did not significantly affect the growth of juveniles. In the third year of heat stress, temperature negatively affected the physiology of juveniles in terms of survivorship, brightness (an indicator of bleaching), symbiotic algal density, and photosynthetic efficiency. Stress across consecutive years appeared to cause the survivorship of juvenile corals to decline, with three years of stress contributing to the severe decline of a reef. In conclusion, A. tenuis juveniles are not able to acclimatize to heat stress, with successive heat waves of <7 days in the summer potentially negatively affecting resilience.

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.

Response of resistant larvae of the coral Acropora tenuis to future thermal stress.

Seawater temperatures are rising rapidly and severely due to climate change, negatively affecting coral reef communities. The persistence of coral populations depends on their success during the early life stages. Thermal conditioning during the larval stage can increase coral larvae's ability to tolerate high temperatures in subsequent stages. We studied the response of resistant larvae of Acropora tenuis to thermal stress to increase their thermal tolerance during the juvenile stage. Larvae were exposed to ambient (∼26 °C) and thermal stress (∼31 °C) temperatures. Then, settlement success on preconditioned tiles was determined. After 28 days at ambient temperature, the juveniles were exposed to thermal stress for 14 days, and their survival was assessed. Our results showed that thermal stress in the larval stage did not alter the thermal tolerance of juveniles, and they could not acclimate to heat stress. As a result, the summer's heat waves could potentially threaten their resilience.