Separate and combined effects of elevated pCO2 and temperature on the branching reef corals Acropora digitifera and Montipora digitata.

Ocean acidification (OA) and warming (OW) are major global threats to coral reef ecosystems; however, studies on their combined effects (OA + OW) are scarce. Therefore, we evaluated the effects of OA, OW, and OA + OW in the branching reef corals Acropora digitifera and Montipora digitata, which have been found to respond differently to environmental changes. Our results indicate that OW has a greater impact on A. digitifera and M. digitata than OA and that the former species is more vulnerable to OW than the latter. OW was the main stressor for increased mortality and decreased calcification in the OA + OW group, and the effect of OA + OW was additive in both species. Our findings suggest that the relative abundance and cover of M. digitata are expected to increase whereas those of A. digitifera may decrease in the near future in Okinawa.

Short-term improvement of heat tolerance in naturally growing Acropora corals in Okinawa.

Mass bleaching and subsequent mortality of reef corals by heat stress has increased globally since the late 20th century, due to global warming. Some experimental studies have reported that corals may increase heat tolerance for short periods, but only a few such studies have monitored naturally-growing colonies. Therefore, we monitored the survival, growth, and bleaching status of Acropora corals in fixed plots by distinguishing individual colonies on a heat-sensitive reef flat in Okinawa, Japan. The level of heat stress, assessed by the modified version of degree heating week duration in July and August, when the seawater temperature was the highest, was minimally but significantly higher in 2017 than in 2016; however, the same colonies exhibited less bleaching and mortality in 2017 than in 2016. Another study conducted at the same site showed that the dominant unicellular endosymbiotic algal species did not change before and after the 2016 bleaching, indicating that shifting and switching of the Symbiodiniaceae community did not contribute to improved heat tolerance. Colonies that suffered from partial mortality in 2016 were completely bleached at higher rates in 2017 than those without partial mortality in 2016. The present results suggest that either genetic or epigenetic changes in coral hosts and/or algal symbionts, or the shifting or switching of microbes other than endosymbionts, may have improved coral holobiont heat tolerance.

Phosphate bound to calcareous sediments hampers skeletal development of juvenile coral.

To test the hypothesis that terrestrial runoff affects the functions of calcareous sediments in coral reefs and hampers the development of corals, we analysed calcareous sediments with different levels of bound phosphate, collected from reef areas of Okinawajima, Japan. We confirmed that phosphate bound to calcareous sediments was readily released into ambient seawater, resulting in much higher concentrations of phosphorous in seawater from heavily polluted areas (4.3-19.0 µM as compared with less than 0.096 µM in natural ambient seawater). Additionally, we examined the effect of phosphate released from calcareous sediments on the development of Acropora digitifera coral juveniles. We found that high phosphate concentrations in seawater clearly inhibit the skeletal formation of coral juveniles. Our results demonstrate that calcareous sediments in reef areas play a crucial role in mediating the impact of terrestrial runoff on corals by storing and releasing phosphate in seawater.

Presence-absence polymorphisms of single-copy genes in the stony coral Acropora digitifera.

BACKGROUND: Despite the importance of characterizing genetic variation among coral individuals for understanding phenotypic variation, the correlation between coral genomic diversity and phenotypic expression is still poorly understood. RESULTS: In this study, we detected a high frequency of genes showing presence-absence polymorphisms (PAPs) for single-copy genes in Acropora digitifera. Among 10,455 single-copy genes, 516 (5%) exhibited PAPs, including 32 transposable element (TE)-related genes. Five hundred sixteen genes exhibited a homozygous absence in one (102) or more than one (414) individuals (n = 33), indicating that most of the absent alleles were not rare variants. Among genes showing PAPs (PAP genes), roughly half were expressed in adults and/or larvae, and the PAP status was associated with differential expression among individuals. Although 85% of PAP genes were uncharacterized or had ambiguous annotations, 70% of these genes were specifically distributed in cnidarian lineages in eumetazoa, suggesting that these genes have functional roles related to traits related to cnidarians or the family Acroporidae or the genus Acropora. Indeed, four of these genes encoded toxins that are usually components of venom in cnidarian-specific cnidocytes. At least 17% of A. digitifera PAP genes were also PAPs in A. tenuis, the basal lineage in the genus Acropora, indicating that PAPs were shared among species in Acropora. CONCLUSIONS: Expression differences caused by a high frequency of PAP genes may be a novel genomic feature in the genus Acropora; these findings will contribute to improve our understanding of correlation between genetic and phenotypic variation in corals.

Responses of branching reef corals Acropora digitifera and Montipora digitata to elevated temperature and pCO2.

Anthropogenic emission of CO2 into the atmosphere has been increasing exponentially, causing ocean acidification (OA) and ocean warming (OW). The "business-as-usual" scenario predicts that the atmospheric concentration of CO2 may exceed 1,000 µatm and seawater temperature may increase by up to 3 °C by the end of the 21st century. Increases in OA and OW may negatively affect the growth and survival of reef corals. In the present study, we separately examined the effects of OW and OA on the corals Acropora digitifera and Montipora digitata, which are dominant coral species occurring along the Ryukyu Archipelago, Japan, at three temperatures (28 °C, 30 °C, and 32 °C) and following four pCO2 treatments (400, 600, 800, and 1,000 µatm) in aquarium experiments. In the OW experiment, the calcification rate (p = 0.02), endosymbiont density, and maximum photosynthetic efficiency (Fv/Fm) (both p < 0.0001) decreased significantly at the highest temperature (32 °C) compared to those at the lower temperatures (28 °C and 30 °C) in both species. In the OA experiment, the calcification rate decreased significantly as pCO2 increased (p < 0.0001), whereas endosymbiont density, chlorophyll content, and Fv/Fm were not affected. The calcification rate of A. digitifera showed greater decreases from 30 °C to 32 °C than that of M. digitata. The calcification of the two species responded differently to OW and OA. These results suggest that A. digitifera is more sensitive to OW than M. digitata, whereas M. digitata is more sensitive to OA. Thus, differences in the sensitivity of the two coral species to OW and OA might be attributed to differences in the endosymbiont species and high calcification rates, respectively.

Bleaching and post-bleaching mortality of Acropora corals on a heat-susceptible reef in 2016.

In 2016, global temperatures were the highest on record, and mass coral bleaching occurred world-wide. However, around Sesoko Island, Okinawa, southwestern Japan, the heat stress assessed by degree heating week (DHW) based on local temperature measurements was moderate in 2016; in 1998, DHW was three times higher than in 2016 (10.6 vs. 3.3 in September in respective years). On a reef flat of Sesoko Island where the effect of severe coral bleaching on coral assemblage was monitored in 1998, significant coral bleaching occurred in 2016. Bleaching of the heat stress sensitive Acropora corals began in July 2016 on the reef flat as seawater temperature rose. We observed the bleaching and post-bleaching mortality status of individual colonies of Acropora spp. in 2016 in fixed plots on the reef flat. In total, 123 Acropora colonies were followed for six months after seawater temperature became normal by multiple surveys. At the beginning of September 2016, 99.2% of colonies, were either completely (92.7%) or partially (6.5%) bleached. Of those, the dominant species or species groups were A. gemmifera (Ag), A. digitifera (Ad), and tabular Acropora (tA). For all Acropora colonies, the overall whole and partial mortality was 41.5% and 11.4%, respectively. Whole mortality rate differed significantly among species; 72.5%, 17.9%, and 27.8% in Ag, Ad, and tA, respectively. Mortality rates at the end of the surveys were similar in smaller (≤10 cm in diameter) and larger Ag, but the former suffered mortality earlier than the latter. Higher survival of smaller colonies was observed only in tA (100%), which may be associated with large morphological differences between smaller and larger colonies. Some of the dominant Acropora colonies had survived without partial mortality including 15.0% survival of the most vulnerable Ag at the end of the surveys. These results suggest that moderate heat stress may have a potential for selecting heat-tolerant genotypes. A longer period of mortality lasting for six months, was observed in Ag in addition to immediate whole mortality after bleaching, due to the continuous loss of living tissue by partial mortality. This highlights the need for multiple surveys at least during several months to accurately assess the impact of thermal stress event to corals. In contrast to DHW based on local measurements, DHW obtained from satellite data were similar between 1998 and 2016. Although satellite-based measurement of sea surface temperature is very useful to reveal variations in heat stress at a large spatial scale, temperature should be measured on site when variations at smaller spatial scales are of interest.

Repetitive sex change in the stony coral Herpolitha limax across a wide geographic range.

Sex change has been widely studied in animals and plants. However, the conditions favoring sex change, its mode and timing remain poorly known. Here, for the first time in stony corals, we report on a protandrous (youngest individuals are males) repetitive sex change exhibited by the fungiid coral Herpolitha limax across large spatial scales (the coral reefs of Japan, Jordan and Israel) and temporal scales (2004-2017). In contrast to most corals, this species is a daytime spawner (08:00-10:00 AM) that spawned at the same time/same date across all the study sites. The sporadically scattered populations of H. limax among the coral reefs of Eilat (Israel) and Aqaba (Jordan) exhibited significantly slower growth, earlier sex change, and lower percentages of reproduction and sex change in comparison to the densely aggregated populations in Okinawa (Japan). At all sites, sex ratio varied among years, but was almost always biased towards maleness. Growth rate decreased with size. We conclude that comparable to dioecious plants that display labile sexuality in response to energetic and/or environmental constraints, the repetitive sex change displayed by H. limax increases its overall fitness reinforcing the important role of reproductive plasticity in the Phylum Cnidaria in determining their evolutionary success.

Effects of moderate thermal anomalies on Acropora corals around Sesoko Island, Okinawa.

Over the past several decades, coral reef ecosystems have experienced recurring bleaching events. These events were predominantly caused by thermal anomalies, which vary widely in terms of severity and spatio-temporal distribution. Acropora corals, highly prominent contributors to the structural complexity of Pacific coral reefs, are sensitive to thermal stress. Response of Acropora corals to extremely high temperature has been well documented. However, studies on the effects of moderately high temperature on Acropora corals are limited. In the summer of 2016, a moderate coral bleaching event due to moderately high temperature was observed around Sesoko Island, Okinawa, Japan. The objective of this study was to examine thermal tolerance patterns of Acropora corals, across reefs with low to moderate thermal exposure (degree heating weeks ~2-5°C week). Field surveys on permanent plots were conducted from October 2015 to April 2017 to compare the population dynamics of adult Acropora corals 6 months before and after the bleaching events around Sesoko Island. Variability in thermal stress response was driven primarily by the degree of thermal stress. Wave action and turbidity may have mediated the thermal stress. Tabular and digitate coral morphologies were the most tolerant and susceptible to thermal stress, respectively. Growth inhibition after bleaching was more pronounced in the larger digitate and corymbose coral morphologies. This study indicates that Acropora populations around Sesoko Island can tolerate short-term, moderate thermal challenges.

Presence-Absence Polymorphisms of Highly Expressed FP Sequences Contribute to Fluorescent Polymorphisms in Acropora digitifera.

Despite many hypotheses regarding the roles of fluorescent proteins (FPs), their biological roles and the genetic basis of FP-mediated color polymorphisms in Acropora remain unclear. In this study, we determined the genetic mechanism underlying fluorescent polymorphisms in A. digitifera. Using a high-throughput sequencing approach, we found that FP gene sequences in FP multigene family exhibit presence-absence polymorphism among individuals. A few particular sequences in short-to-middle wavelength emission and middle-to-long wavelength emission clades were highly expressed in adults, and different sequences were highly expressed in larvae. These highly expressed sequences were absent in the genomes of individuals with low total FP gene expression. In adults, presence-absence differences of the highly expressed FP sequences were consistent with measurements of emission spectra of corals, suggesting that presence-absence polymorphisms of these FP sequences contributed to the fluorescent polymorphisms. The functions of recombinant FPs encoded by highly expressed sequences in adult and larval stages were different, suggesting that expression of FP sequences with different functions may depend on the life-stage of A. digitifera. Highly expressed FP sequences exhibited presence-absence polymorphisms in subpopulations of A. digitifera, suggesting that presence-absence status is maintained during the evolution of A. digitifera subpopulations. The difference in FPs between adults and larvae and the polymorphisms of highly expressed FP genes may provide key insight into the biological roles of FPs in corals.

The stoichiometry of coral-dinoflagellate symbiosis: carbon and nitrogen cycles are balanced in the recycling and double translocation system.

Symbioses between microalgae and animal hosts have the advantage of acquiring and sharing autotrophically produced organic carbon (C) as their energy source. However, the stoichiometry and turnover rates of biological elements in symbioses are not fully understood because of complicated metabolic interactions. We report the first comprehensive and simultaneous measurement of C and nitrogen (N) flows through coral-dinoflagellate symbiosis by using the unique approach of dual-isotope labeling with 13C and 15N, in situ chasing, and isotope-mixing models. The coral autotrophy occurred with much lower C:N ratios than previously thought, and the autotrophically produced N-rich organic matter was efficiently transferred to the animal host through two different pathways. In contrast to the dynamic N cycles within the symbiosis, the N uptake from the ambient seawater was extremely limited, which enabled the coral symbiosis to sustain N with a long turnover time (1 year). These findings suggest that coral endosymbionts are not under N limitation but are actively producing organic N and driving microscale N cycles in the reef ecosystem. The present techniques could be applied to further quantify the C and N cycles in other symbiotic interactions and reveal their ecological advantages.

Calcification process dynamics in coral primary polyps as observed using a calcein incubation method.

Calcification processes are largely unknown in scleractinian corals. In this study, live confocal imaging was used to elucidate the spatiotemporal dynamics of the calcification process in aposymbiotic primary polyps of the coral species Acropora digitifera. The fluorophore calcein was used as a calcium deposition marker and a visible indicator of extracellular fluid distribution at the tissue-skeleton interface (subcalicoblastic medium, SCM) in primary polyp tissues. Under continuous incubation in calcein-containing seawater, initial crystallization and skeletal growth were visualized among the calicoblastic cells in live primary polyp tissues. Additionally, the distribution of calcein-stained SCM and contraction movements of the pockets of SCM were captured at intervals of a few minutes. Our experimental system provided several new insights into coral calcification, particularly as a first step in monitoring the relationship between cellular dynamics and calcification in vivo. Our study suggests that coral calcification initiates at intercellular spaces, a finding that may contribute to the general understanding of coral calcification processes.

Intraspecific variations in responses to ocean acidification in two branching coral species.

Ocean acidification is widely recognised to have a negative impact on marine calcifying organisms by reducing calcifications, but controversy remains over whether such organisms could cope with ocean acidification within a range of phenotypic plasticity and/or adapt to future acidifying ocean. We performed a laboratory rearing experiment using clonal fragments of the common branching corals Montipora digitata and Porites cylindrica under control and acidified seawater (lower pH) conditions (approximately 400 and 900µatm pCO2, respectively) and evaluated the intraspecific variations in their responses to ocean acidification. Intra- and interspecific variations in calcification and photosynthetic efficiency were evident according to both pCO2 conditions and colony, indicating that responses to acidification may be individually variable at the colony level. Our results suggest that some corals may cope with ocean acidification within their present genotypic composition by adaptation through phenotypic plasticity, while others may be placed under selective pressures resulting in population alteration.

Decentralized control mechanism underlying interlimb coordination of millipedes.

Legged animals exhibit adaptive and resilient locomotion through interlimb coordination. The long-term goal of this study is to clarify the relationship between the number of legs and the inherent decentralized control mechanism for interlimb coordination. As a preliminary step, the study focuses on millipedes as they represent the species with the greatest number of legs among various animal species. A decentralized control mechanism involving local force feedback was proposed based on the qualitative findings of behavioural experiments in which responses to the removal of part of the terrain and leg amputation were observed. The proposed mechanism was implemented in a developed millipede-like robot to demonstrate that the robot can adapt to the removal of the part of the terrain and leg amputation in a manner similar to that in behavioural experiments.

Decentralized control scheme for myriapod robot inspired by adaptive and resilient centipede locomotion.

Recently, myriapods have attracted the attention of engineers because mobile robots that mimic them potentially have the capability of producing highly stable, adaptive, and resilient behaviors. The major challenge here is to develop a control scheme that can coordinate their numerous legs in real time, and an autonomous decentralized control could be the key to solve this problem. Therefore, we focus on real centipedes and aim to design a decentralized control scheme for myriapod robots by drawing inspiration from behavioral experiments on centipede locomotion under unusual conditions. In the behavioral experiments, we observed the response to the removal of a part of the terrain and to amputation of several legs. Further, we determined that the ground reaction force is significant for generating rhythmic leg movements; the motion of each leg is likely affected by a sensory input from its neighboring legs. Thus, we constructed a two-dimensional model wherein a simple local reflexive mechanism was implemented in each leg. We performed simulations by using this model and demonstrated that the myriapod robot could move adaptively to changes in the environment and body properties. Our findings will shed new light on designing adaptive and resilient myriapod robots that can function under various circumstances.

An aposymbiotic primary coral polyp counteracts acidification by active pH regulation.

Corals build their skeletons using extracellular calcifying fluid located in the tissue-skeleton interface. However, the mechanism by which corals control the transport of calcium and other ions from seawater and the mechanism of constant alkalization of calcifying fluid are largely unknown. To address these questions, we performed direct pH imaging at calcification sites (subcalicoblastic medium, SCM) to visualize active pH upregulation in live aposymbiotic primary coral polyps treated with HCl-acidified seawater. Active alkalization was observed in all individuals using vital staining method while the movement of HPTS and Alexa Fluor to SCM suggests that certain ions such as H+ could diffuse via a paracellular pathway to SCM. Among them, we discovered acid-induced oscillations in the pH of SCM (pHSCM), observed in 24% of polyps examined. In addition, we discovered acid-induced pH up-regulation waves in 21% of polyps examined, which propagated among SCMs after exposure to acidified seawater. Our results showed that corals can regulate pHSCM more dynamically than was previously believed. These observations will have important implications for determining how corals regulate pHSCM during calcification. We propose that corals can sense ambient seawater pH via their innate pH-sensitive systems and regulate pHSCM using several unknown pH-regulating ion transporters that coordinate with multicellular signaling occurring in coral tissue.

Acropora digitifera Encodes the Largest Known Family of Fluorescent Proteins that Has Persisted during the Evolution of Acropora Species.

Fluorescent proteins (FPs) are well known and broadly used as bio-imaging markers in molecular biology research. Many FP genes were cloned from anthozoan species and it was suggested that multi-copies of these genes are present in their genomes. However, the full complement of FP genes in any single coral species remained unidentified. In this study, we analyzed the FP genes in two stony coral species. FP cDNA sequences from Acropora digitifera and Acropora tenuis revealed the presence of a multi-gene family with an unexpectedly large number of genes, separated into short-/middle-wavelength emission (S/MWE), middle-/long-wavelength emission (M/LWE), and chromoprotein (CP) clades. FP gene copy numbers in the genomes of four A. digitifera colonies were estimated as 16-22 in the S/MWE, 3-6 in the M/LWE, and 8-12 in the CP clades, and, in total, 35, 31, 33, and 33 FP gene copies per individual shown by quantitative PCR. To the best of our knowledge, these are the largest sets of FP genes per genome. The fluorescent light produced by recombinant protein products encoded by the newly isolated genes explained the fluorescent range of live A. digitifera, suggesting that the high copy multi-FP gene family generates coral fluorescence. The functionally diverse multi-FP gene family must have existed in the ancestor of Acropora species, as suggested by molecular phylogenetic and evolutionary analyses. The persistence of a diverse function and high copy number multi-FP gene family may indicate the biological importance of diverse fluorescence emission and light absorption in Acropora species.

Unexpectedly complex gradation of coral population structure in the Nansei Islands, Japan.

To establish effective locations and sizes of potential protected areas for reef ecosystems, detailed information about source and sink relationships between populations is critical, especially in archipelagic regions. Therefore, we assessed population structure and genetic diversity of Acropora tenuis, one of the dominant stony coral species in the Pacific, using 13 microsatellite markers to investigate 298 colonies from 15 locations across the Nansei Islands in southwestern Japan. Genetic diversity was not significant among sampling locations, even in possibly peripheral locations. In addition, our results showed that there are at least two populations of A. tenuis in the study area. The level of genetic differentiation between these populations was relatively low, but significant between many pairs of sampling locations. Directions of gene flow, which were estimated using a coalescence-based approach, suggest that gene flow not only occurs from south to north, but also from north to south in various locations. Consequently, the Yaeyama Islands and the Amami Islands are potential northern and southern sources of corals. On the other hand, the Miyako Islands and west central Okinawa Island are potential sink populations. The Kerama Islands and the vicinity of Taketomi Island are potential contact points of genetic subdivision of coral populations in the Nansei Islands. We found that genetic population structure of A. tenuis in the Nansei Islands is more complex than previously thought. These cryptic populations are very important for preserving genetic diversity and should be maintained.

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness.

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had "negative-type" mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.

Comparison of the effects of thermal stress and CO2-driven acidified seawater on fertilization in coral Acropora digitifera.

Global warming (GW) and ocean acidification (OA) have been recognized as severe threats for reef-building corals that support coral reef ecosystems, but these effects on the early life history stage of corals are relatively unknown compared with the effects on calcification of adult corals. In this study, we evaluated the effects of thermal stress and CO2-driven acidified seawater on fertilization in a reef-building coral, Acropora digitifera. The fertilization rates of A. digitifera decreased in response to thermal stress compared with those under normal seawater conditions. In contrast, the changes of fertilization rates were not evident in the acidified seawater. Generalized Linear Mixed Model (GLMM) predicted that sperm/egg crosses and temperature were explanatory variables in the best-fitted model for the fertilization data. In the best model, interactions between thermal stress and acidified seawater on the fertilization rates were not selected. Our results suggested that coral fertilization is more sensitive to future GW than OA. Taking into consideration the previous finding that sperm motility of A. digitifera was decreased by acidified seawater, the decrease in coral cover followed by that of sperm concentration might cause the interacting effects of GW and OA on coral fertilization.

Responses of calcification of massive and encrusting corals to past, present, and near-future ocean carbon dioxide concentrations.

In this study, we report the acidification impact mimicking the pre-industrial, the present, and near-future oceans on calcification of two coral species (Porites australiensis, Isopora palifera) by using precise pCO2 control system which can produce acidified seawater under stable pCO2 values with low variations. In the analyses, we performed Bayesian modeling approaches incorporating the variations of pCO2 and compared the results between our modeling approach and classical statistical one. The results showed highest calcification rates in pre-industrial pCO2 level and gradual decreases of calcification in the near-future ocean acidification level, which suggests that ongoing and near-future ocean acidification would negatively impact coral calcification. In addition, it was expected that the variations of parameters of carbon chemistry may affect the inference of the best model on calcification responses to these parameters between Bayesian modeling approach and classical statistical one even under stable pCO2 values with low variations.