Social conditions, social relationships and neighbourhood environment, the components of social capital, are important determinants of health. The objective of this study was to investigate the association of neighbourhood and individual social capital with consistent self-rated health in women between the first trimester of pregnancy and six months postpartum.A multilevel cohort study in 34 neighbourhoods was performed on 685 Brazilian women recruited at antenatal units in two cities in the State of Rio de Janeiro, Brazil. Self-rated health (SRH) was assessed in the 1st trimester of pregnancy (baseline) and six months after childbirth (follow-up). The participants were divided into two groups: 1. Good SRH – good SRH at baseline and follow-up, and, 2. Poor SRH – poor SRH at baseline and follow-up. Exploratory variables collected at baseline included neighbourhood social capital (neighbourhood-level variable), individual social capital , demographic and socioeconomic characteristics, health-related behaviours and self-reported diseases. A hierarchical binomial multilevel analysis was performed to test the association between neighbourhood and individual social capital and SRH, adjusted for covariates.The Good SRH group reported higher scores of social support and social networks than the Poor SRH group. Although low neighbourhood social capital was associated with poor SRH in crude analysis, the association was not significant when individual socio-demographic variables were included in the model. In the final model, women reporting poor SRH both at baseline and follow-up had lower levels of social support  [OR 0.82 (95% CI: 0.73-0.90)] and a lower likelihood of friendship social networks [OR 0.61 (95% CI: 0.37-0.99)] than the Good SRH group. The characteristics that remained associated with poor SRH were low level of schooling, Black and Brown ethnicity, more children, urinary infection and water plumbing outside the house.Low individual social capital during pregnancy, considered here as social support and social network, was independently associated with poor SRH in women whereas neighbourhood social capital did not affect women’s SRH during pregnancy and the months thereafter. From pregnancy and up to six months postpartum, the effect of individual social capital explained better the consistency of SRH over time than neighbourhood social capital. Social capital are features of social structure, such as interpersonal trust, networks and norms of mutual aid and reciprocity, which act as resources for individuals and facilitate cooperation and collective action . In geneCultural context and social rules influence reciprocity and mutual cooperation, and therefore affect the level of social capital. A report of the Brazilian Commission on Social Determinants of Health found that social trust is extremely poor among Brazilians . Citizenet al. and 39% [OR 0.61 (95% CI: 0.37-0.99)] lower chances of reporting poor SRH. Among socio-demographic variables, water supply outside the house [OR 1.85 (95% CI: 1.05-3.28)] and low schooling [OR 2.06 (95% CI: 1.15-3.71)] were also associated with poor SRH. Women from Black and Brown ethnicity were 2.11 and 2.02 times more likely to report poor SRH compared to White women. Women with 2 or 3 children and those with 4 or more children were 3.23 and 3.39 times more likely to report poor SRH compared to those with no or one other child. Women reporting urinary infection had a higher likelihood of reporting poor SRH [OR 2.11 (95% CI: 1.28-3.49)].In additional analysis that included the 222 women who changed SRH status, individual social capital was the major differential characteristic among the four groups. The mean score of affectionate and emotional supports  was higher among women with good SRH, and reduced significantly among those who changed from good SRH to poor SRH. Conversely, women who had poor SRH at baseline and changed to good SRH showed higher social support than those who remained with poor SRH at both assessments. In addition, in comparison to those with good SRH at baseline and poor SRH at follow-up, those with good SRH on both occasions had more relatives. However, no statistical differences were found concerning individual social capital between groups who changed their SRH status from good to poor, or from poor to good SRH. Data are presented in Additional file The interaction term, composed of neighbourhood social capital and individual social capital, was not statistically significant when added to the Model 1, and was therefore removed.Despite the claim that neighbourhood social capital may be a contextual influence on the health of residents , and thaEven though there is evidence of a moderate association between neighbourhood environment and health , some stet al.[et al.[Our findings differ from other similar studies in Brazil that found an association between neighbourhood characteristics and SRH. In a cohort study involving public university workers, neighbourhood characteristics were associated with SRH after controlling for individual factors such as age, ethnicity, income, education, social class, home, and health conditions ,93. Howeet al. also repl.[et al., social proxy measures or dimensions of neighbourhood social capital - social control, social trust, norms of reciprocity, feelings of safety (neighbourhood security), political efficacy and empowerment [A positive association between social capital and SRH has been frequently reported ,23,32,33owerment ,16,44. TAlthough there was no association between neighbourhood social capital and poor SRH in the present study, there was a positive association between social support and social network and SRH. Therefore, the latter hypothesis was confirmed. The lower the social support and social networks, the higher the likelihood of maintaining poor SRH during pregnancy and 6 months postpartum. That finding accords with previous findings ,60,95.The period between pregnancy and 6 months after childbirth is a unique one in most women’s life. Individual social capital appears to be important for women, especially during pregnancy and after delivery. The lack of social support constitutes an important risk factor for maternal well-being during pregnancy . Having The strong influence of social support and social networks on women’s health ,35 may bNeighbourhood social capital may be affected by aspects of individual social capital that are associated with health . Strong Women whose SRH status changed during the period of the study were not included in the multilevel analysis. By excluding them from the analysis, the study population may have been conditioned based on an effect of the exposure. However, the possibility of a ‘collider-stratification bias’ , a form Additional analysis showed that individual social capital differed between groups whose SRH status changed. In addition, the possible influence of individual social capital in women not changing from good to poor levels of SRH over a period of great stress and transition was not confirmed. Conversely, individual social capital was associated with consistent SRH. The findings indicate that it is unlikely that including women who changed their SRH status over time in the overall analysis would affect the results. The influence of individual and neighbourhood social capital on SRH in women whose pregnancies were aborted or miscarried should be considered in future studies.The findings of this study have several important implications, but are not without limitations. Recruiting pregnant women from public antenatal care units might affect the generalizability of our findings resulting in a homogeneous sample concerning some socioeconomic characteristics. This may explain the lack of association between social class and SRH. The number of women per neighbourhood was slightly below the ideal and might have affected the power of the study . Our stuA point to be taken into account when considering our findings is that urinary infection was prevalent in our sample (45.5%), and was statistically higher in women with poor SRH. The prevalence of urinary infection was slightly higher than in other studies ,106. ThePositive features of the study were the use of social support and social networks questionnaires with adequate psychometric properties for the Brazilian population. The neighbourhood social capital questionnaire had been used in Brazilian populations and had good reliability. The data collection was standardized and performed by trained interviewers reducing information bias. In addition, the response rate was high and losses to follow-up were low.per si. There is no consensus on how to measure social capital. Contextual social features have been routinely assessed using aggregated measures and indexes, like percentage of poverty or median of income, mainly those available in census databases [There are some points related to the social capital assessment that should to be considered in future studies. One is the measure atabases . In thisatabases .The present study enhances knowledge on social capital and health in several ways. Our findings indicate a relationship between individual social capital and SRH. In addition, multilevel analysis is an adequate statistical approach to evaluate simultaneously the role of neighbourhood-level and individual factors on health. Above all, more attention was given to low socioeconomic status women, where undesirable pregnancy outcomes rates remain relatively high in other low and middle income countries. Knowledge about how proximal and distal social determinants affect women’s health during pregnancy and the initial period of motherhood has important policy implications on the health and social areas. The identification on what level of social capital was more important to womenÂ´s health is relevant in developing interventions. Although social inequalities are viewed as a broad and unspecific determinants of health, our findings suggest that there are specific social risk factors during and after pregnancy that should be taken into account to improve both maternal and child health.Low individual social capital during pregnancy, such as social support and social networks, independently influenced women who had consistently poor SRH whereas neighbourhood social capital did not affect their SRH during pregnancy and the postpartum period. From pregnancy and up to six months after delivery the effect of individual social capital better explained consistent SRH in women than did neighbourhood social capital.The authors declare that they have no competing interests.GL was involved in design of the study, acquisition of data, analysis and interpretation of the data, interpretation of the results and drafted the manuscript. M do CL helped design the study, interpreted the data and reviewed the manuscript. AS was involved in interpretation of the data and revising the manuscript. MV was involved in the conception and design of the study, developed the statistical framework for data analysis, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/13/1/prepubSocial capital questionnaire.Click here for fileSocial networks questionnaire.Click here for fileSocioeconomic and demographic characteristics, health-related behaviours, and individual social capital scores in all groups of SRH.Click here for file


Small RNAs are known to regulate diverse gene expression processes including translation, transcription, and splicing. Among small RNAs, the microRNAs (miRNAs) of 17 to 27 nucleotides (nts) undergo biogeneses including primary transcription, RNA excision and folding, nuclear export, cytoplasmic processing, and then bioactivity as regulatory agents. We propose that analogous hairpins from RNA molecules that function as part of the spliceosome might also be the source of small, regulatory RNAs .Deep sequencing technology has enabled discovery of a novel 16-nt RNA sequence in total RNA from human brain that we propose is derived from RNU1, an RNA component of spliceosome assembly. Bioinformatic alignments compel inquiring whether the novel 16-nt sequence or its precursor have a regulatory function as well as determining aspects of how processing intersects with the miRNA biogenesis pathway. Specifically, our preliminary in silico investigations reveal the sequence could regulate splicing factor Arg/Ser rich 1 (SFRS1), a gene coding an essential protein component of the spliceosome. All 16-base source sequences in the UCSC Human Genome Browser are within the 14 instances of RNU1 genes listed in wgEncodeGencodeAutoV3. Furthermore, 10 of the 14 instances of the sequence are also within a common 28-nt hairpin-forming subsequence of RNU1.An abundant 16-nt RNA sequence is sourced from a spliceosomal RNA, lies in a stem of a predicted RNA hairpin, and includes reverse complements of subsequences of the 3'UTR of a gene coding for a spliceosome protein. Thus RNU1 could function both as a component of spliceosome assembly and as inhibitor of production of the essential, spliceosome protein coded by SFRS1. Beyond this example, a general procedure is needed for systematic discovery of multiple alignments of sequencing, splicing, and RNA folding data. The numerous, very dissimilar types of bioinformatic data conspire to make integration a central problem for efficient and effective application of biological findings. Integration of data of three particular types is the goal of this paper. Gene splicing is the focus, held up as an example of how sequencing, splicing, and RNA folding data types might be used to guide research that could illuminate major mechanisms of cell biology such control of levels of ribonucleoprotein species.H, has a loop of four nts and a stem of 12 pairs of nts including eight C-G bonds (hence is very stable).Function and dysfunction of gene splicing impact embryogenesis, cell motility and viability, cell cycle arrest, and many other mechanisms of metazoan cell biology . This paH. We denote herein the 16-nt sequence as S.Our deep sequencing to detect small RNAs in three samples of post-mortem human prefrontal cortex produced abundant reads corresponding to a 16-nt sequence from the 3' side of the stem of Ciona intestinalis (sea squirt). Langenberger et al. [Giardia lambia, again with miRNA-like processing and apparent RNAi activity. Other non-miRNAs of about 16 nts that are subsequences of known miRNAs have been shown by Li et al. to participate in gene regulation, targeting the 3'UTRs of target genes as efficiently as sequentially enclosing miRNAs [Regarding small RNA context , Kawaji r et al.  also four et al.  first rer et al.  then four et al.  reportedr et al.  assayed r et al.  used seqg miRNAs . Importag miRNAs  discoverg miRNAs .Additional sources of small ncRNA are the vault RNAs, ~100-nt Pol III transcripts in the enigmatic vault organelles of eukaryotic cells. There are three described human vault RNAs from a cluster on chromosome 5 . StadlerIt seems quite likely that nature must put such abundant, selected subsequences of the above types to some purpose, implying unrevealed pathways that are presently without definitive annotations or even realisation . For exaRegarding neurological bioactivity, Smalheiser et al.  discoverS appear in every sample more than ten times as frequently as reads from the other three RNU1 hairpins and at frequencies comparable to those of abundant brain miRNAs. Further compounding interest in the 16-nt sequence S from hairpin H are, in the manner of miRNA target predictions, two putative target regions (lengths 9 and 11 nts) in the 3'UTR of splicing regulator gene SFRS1. Thus the 16-nt byproduct of RNU1 synthesis (from promotion of splicing) might also inhibit expression of SFRS1 (inhibition of splicing or at least inhibition of formation of spliceosome components). This might be a form of auto-regulation essential to homeostasis of splicing. Our neuroscience interests provide focus on SFRS1 protein product because it modulates several forms of synaptic plasticity considered to be involved in the very essence of memory [Consistent with the above prior work, we found that reads representing the 16-nt sequence f memory .in silico coincidences demand investigation of potential miRNA-like mechanisms involving the RNU1 hairpin H, especially with regard to SFRS1. Needed are functional validations of nuclear RNU1 targets. Considering the huge impact of splicing function in nature and dysfunction in disease, elucidation of splicing homeostasis would carry a significant potential for progress toward novel diagnostic tools and drug platforms.Thus there is a triple intersection of bioinformatics: annotated function of an ncRNA, abundance in brain of a small RNA evidently processed from the same ncRNA source, and sequence alignment of the complement of the same small RNA with the 3'UTR of a major gene having the same function. These S) were found to be bioactive, as mentioned above. Additionally, it has long been known that pre-mRNA splicing can be regulated both positively and negatively by reversible phosphorylation of spliceosomal SR proteins [Regarding RNU1 context, hairpins studied by O'Gormann et al.   of three persons who had no mental illness at time of death. Samples were generously made available from the Stanley Medical Research Institute. The spliceosome is involved in synaptic plasticity, critical to normal brain function. It is of interest that altered expression of SFRS1 has been reported in post-mortem schizophrenia studies involving variable isoforms of DISC1  or NCAM1S = TTCGCGCTTTCCCCTG or UUCGCGCUUUCCCCUG--in thousands of reads, and we confirmed by PCR the presence of S in commercially available neural stem cells derived from human embryonic stem cells. S includes 9 and 11 nucleotide subsequences that appear exactly as the reverse complements of two subsequences of the 3'UTR of the important splicing gene SFRS1. Furthermore, S is included as part of the loop and the 3' side of a well-formed 28-nt hairpin H from the snRNA RNU1. Parallel to other studies cited above, this level of complementary suggests that S could bind to the 3'UTR of SFRS1 and inhibit processing or accelerate sequestration or degradation of SFRS1 mRNA, possibly in an miRNA-like manner.We discovered a 16-nt sequence--herein denoted as S itself. Our algorithm was a modification of published methods [S), and 1% from mitochondrial tRNAs.As yet undiscovered regulatory small RNAs might function in normal human brain or might provide signatures of human brain diseases. Total RNA from dorsolateral prefrontal cortex was isolated using Trizol under the auspices of Stanley Medical Research Institute. We derived cDNA libraries using the Illumina Small RNA Sample Preparation Kit (San Diego CA), following the manufacturer's version 1.5 protocol. The cDNA libraries were sequenced with an Illumina Genome Analyzer at our university core facility. Resulting 35-base reads were first filtered to remove concatenated adaptors at the 3' ends. Specifically, we sought the first six or more bases of the Illumina 3' adapter TCTCGTATGCCGTCTTCTGCTTGAAA... in valid reads. Next a variable number of consecutive As were trimmed from 3' ends. Those remaining sequences with 16-29 bases were aligned with ClustalW2  and then methods ,32. SourS itself were 4736, 1317, 2453.We obtained raw read counts from three subjects as follows: 1,813,994; 2,276,814; 3,655,462. Counts of distinct core sequences were 1213, 1423, 1790. Counts of S and sought them among all raw reads of all three samples. Not one was found. The probability of finding not even one of 1000 16-base random sequences within a million, distinct, random, 30-base sequences is 0.0304 (but of course read sequences are not random). Thus while S itself is plentiful among our reads, distinct permutations of the bases corresponding to S yield random sequences that are consistently absent.We generated 1000 distinct, random permutations of the 16 bases corresponding to S to occur only when preceded by TGCA or TGCG and only in 14 genomic locations, all 14 listed within the RNU1 genes in wgEncodeGencodeAutoV3.We used BLAT in UCSC Human Genome Browser and found full 16-base sources of S. Cells used in these experiments were commercially available neural stem cells called hNP1 cells from ArunA Biomedical (Athens GA) derived from human embryonic WA09 cell lines. hNP1 cells grow as an adherent monolayer and are homogeneous, uniformly expressing various neural stem cell proteins  and low levels of embryonic stem cell proteins ) [Given our interest in neuroscience, we sought to determine if human embryo-derived neural stem cells also generated copies of s OCT4)) ,34. hNP1We cultured hNP1 cells according to the manufacturer's protocol. We harvested about 1 million passage 4 cells. Cytoplasm and nuclear lysate were separated using the Norgen Cytoplasmic and Nuclear RNA purification kit (Thorold ON). We verified partitioning with an Agilent Bioanalyzer (Santa Clara CA) by observing with a DNA chip a DNA:DNA concentration ratio of >100X of nucleus over cytoplasm and for the same samples processed with a Bioanalyzer RNA chip an RNA:RNA ratio of >10X for 18S peak in cytoplasm over nucleus. These two ratios for the same samples imply good nuclear and cytoplasmic partitioning.S in the samples. For negative control, we used a miScript primer for a 16-nt subsequence Arabidopsis thaliana miRNA-159a  and a 16-nt subsequence of human miRNA-128 . Input RNA templates were 0.76 pg/uL for nuclear extract and 1.01 pg/uL for cytoplasmic extract. All 12 samples were run simultaneously using an Applied Biosystems (Foster City CA) 7900HT Fast Real-Time PCR System. As shown in Figure S in both nuclear and cytoplasmic fractions from hNP1 cells. All negative controls were not detected or very weakly detected (cycle count >35).We used a Qiagen (Alameda CA) miScript PCR system with custom primer to determine the presence of in silico predicted targets for all ten of the seven-nt subsequences of S. Repeatedly appearing was SFRS1. This was because 11 consecutive nts from S were the exact reverse complement of a 3'UTR sequence of SFRS1; a second target was generated from 9 nts. How S might bind to the 3'UTR is shown in Figure Using TargetScan 5.1  we foundS is also the seed of any human miRNA  no seven-nt subsequence of e miRNAs ); thus ts et al. .SFRS1 protein is employed in protein-protein interactions and other processes and in particular recruits the U1 snRNP to the 5' splice site ,39. The S is also mounted in a nuclear RISC that includes Ago2.Importantly, Ohrt et al.  have repH with S as loop and 3' side; one full conformation is shown in Figure RNA hairpin structures are key features of RNA functions and processing generally, and miRNA processing in particular. The mfold engine uses dynamic programming and large tables of empirically derived binding affinities for small dsRNAs . mfold pH in isolation, the number is much stronger (.59) than that of any other RNU1 hairpin (average ~.35).Regarding stability of the various hairpins, the stability ratio defined by -Gibbs free energy divided by number of nts (length) of a hairpin is -dG/L. For the fourth hairpin Again, integration of biochemical and bioinformatic data to yield information about mechanisms of control of gene expression is the goal of this paper. To that end, the above example provides a paradigm for seeking causal relationships among biochemical concepts and bioinformatic concepts. Figure Deep sequencing applied to cDNA libraries constructed from small RNA molecules provided us with millions of reads of observed sequences. From tables of Illumina 35-base reads, we trimmed from 3' regions the 5' ends of adaptors (at least six bases) and zero to four As, and then retained only the results that appeared at least three times. In our procedure, this yielded thousands of distinct sequences of 16 to 29 bases, each with an instance count ranging from three to more than 100,000. The sequences were aligned with ClustalW2, and then commonly occurring subsequences of at least 16 bases were deduced, each with a total instance count; we called each such subsequence a "core." This procedure includes multiple tuned parameters and an inevitable degree of arbitrariness. Conclusions reached with an algorithm containing tunable parameters must always be tested for invariance with respect to reasonable retuning.In a separate line of investigation, tables of ncRNAs can be processed by submitting sliding windows of 50-nt subsequences to RNA folding engines such as mfold. Sought are simple RNA hairpins incorporating at least 25 nts and meeting certain stability criteria. For example, the above ratio of -Gibbs free energy  to length (number of nts) in a hairpin should be at least ~0.30. A more sophisticated approach might include the minimal folding free energy index (MFEI) defined by Zhang et al.  as:MFEI might be a criterion for finding RNA hairpins that are processed into mature miRNAs or other RNAs similar to mature miRNAs. A survey by Zhang et al. of 513 pre-miRNAs found average MFEI was 0.97, significantly higher than sampled mRNAs (0.65), tRNAs (0.64), or rRNAs (0.59). The distinguished hairpin in Figure Thus sequencing and hairpin searches can lead to comparisons of cores with sides of predicted hairpins to deduce potential agents of gene regulation, possibly processed in an miRNA-like manner.In a third line of investigation, ncRNAs can be considered for membership in well annotated ribonucleoprotein particles of many types with wide-ranging functions . The proThe complete flowchart of the proposed triple colligation is shown in Figure All three contributed to the preparation of the manuscript and to various applications of bioinformatic analysis. CDJ conceived an initial version of the program for finding read cores and aligned some cores with known ncRNAs including RNU1; DOP contributed design of sequencing procedures and verification methods; XG contributed invention of data processing methods and programs. All authors read and approved the final manuscript.


Nylanderia pubens (Forel) (Hymenoptera: Formicidae) workers for use in expression library construction, severe RNA degradation consistently occurred. This degradation was masked by spectrophotometric analysis but clearly evident by microfluidic-based assay. Although not specifically identified, the degrading entity was endogenous and localized to the abdomen  of adult ants. RNA degradation was not observed in preparations of larvae, non-melanized pupae, or eggs. Various RNase and protease inhibitors had no protective effect. However, the metal chelating agent ethylenediaminetetraacetic acid prevented RNA degradation and provides insight into the occurrence.During preparation of total RNA from  Nylanderia pubens (Forel) (Hymenoptera: Formicidae), previously Paratrechina pubens . During preparation of total RNA from adult N. pubens for use in expression library construction to screen for pathogens, severe RNA degradation was observed. This degradation was masked by spectrophotometric analysis but clearly evident by microfluidic-based assay. Thus, the objective of this paper was to document and characterize this endogenous RNA-degrading entity in order to make those interested in molecular-based studies with this ant species aware of its existence and the consequences of its presence in down-stream assays.a pubens , is an ia pubens . The rapa pubens . A deartN. pubens  using the guanidium isothiocyanate method  according the manufacturer's instructions). N. pubens were obtained from colonies collected from field sites located in Gainesville, Alachua County, Florida, and maintained in the laboratory. Colonies were housed in nesting tubes described by Oi and Williams . RNA was also extracted from tagma of adult ants: head and thorax, abdomen, and the last 3 to 4 terminal abdominal segments. Homogenization of whole adults was also conducted in different solutions (150 µl) just before the addition of Trizol. These solutions included ethylenediaminetetraacetic acid (EDTA) , neutralizing TRISHCl buffer , RNase Out , proteinase K (20 and 200 µg), diethyl pyrocarbonate , and formic acid (0.1 and 1 mM).RNA was extracted from different developmental stages of Williams  and rearhttp://www.home.agilent.com/), using the RNA 6000 Nano kit according to the manufacturer's directions. Microfluidic assays were completed immediately after RNA extraction using a 1 µl volume of purified sample. RNA size standards  and a positive RNA control  were included for each experiment. For comparison, RNA quality and quantity were determined spectrophotometrically on an ND-1000 spectrophotometer . The 260:280 nm ratio and quantity of RNA were determined.RNA quality was assessed by microfluidic analysis on an Agilent 2100 Bioanalyzer  from different preparations and life stages was digested with DNase I  for 10 minutes at 37° C according to the manufacturer's instructions. The DNase-digested RNA was reverse transcribed with Superscript III reverse transcriptase (Invitrogen) at 55° C for 30 minutes using oligonucleotide primer p1222 (5′ TGCAATAGCAATAGTGTCGTTGCTATAAACAGGT). PCR was subsequently conducted with Platinum Taq polymerase (Invitrogen) and oligonucleotide primers p1222 and p1221 (5′ TGCCTCAGTTAATGACACGTCAGAAAATTCGA) using the following program: 94°C for 2 minutes, 35 cycles of 94° C for 15 seconds, 62°C for 15 seconds, and 68° C for 30 seconds, followed by a polishing step of 68° C for 5 minutes. Amplicons were separated on a 1% Agarose gel and visualized with SYBR-safe (Invitrogen). DNase digestion was verified by including the undigested RNA as template in PCR. Non-template controls and positive controls  were included in each evaluation.RNA integrity was evaluated by its ability to serve as a template for transcription into cDNA and subsequent amplification by PCR. Oligonucleotide primers were designed for the N. pubens with Trizol reagent yielded consistent results; RNA from workers, queens, and alates was completely degraded, while RNA from larvae, pupae, and eggs was not . Indeed, RT-PCR of the housekeeping gene, ubiquitin of N. pubens, failed to generate an amplicon from DNase-treated RNA templates in adult workers, confirming the denatured state of this RNA  of the purification procedure  always resulted in degraded RNA from adult stages and intact RNA from immature stages. Interestingly, spectrophotometric analysis masked the degraded nature of the RNA . The speN. pubens exhibited a strong 18S fragment band, indicating that the integrity of the sample was intact  is comprised of a storage sac and long, sinuous filaments thought to collect molecular precursors for venom synthesis  is inhibitory to RNases , and livAlso considered was the possibility that endogenous formic acid was catalyzing the hydrolysis of RNA. However, neutralization of worker ant homogenates to pH 7 or 9 with Tris buffer also yielded degraded RNA . Indeed,A final series of experiments examined the effect of EDTA on RNA purification and integrity. Whole worker ants homogenized in at least 50 mM EDTA yielded intact RNA when subsequently extracted with Trizol reagent . InteresN. pubens workers. This entity does not appear to be a canonical RNase, as DEPC, RNase Out (both RNase inhibitors), and proteinase K did not provide protection from its RNA-degrading activity. It also does not appear that formic acid played a role, because addition of synthetic formic acid did not result in degraded RNA from larvae or abdomenless adult ants. Transesterification of RNA can be catalyzed by nitrogenous bases (2+ has been shown to serve as a Lewis acid promoting activation of the nucleophile RNA, causing its rapid degradation (Although not specifically identified, the RNA-degrading entity appears to be endogenous and liberated upon homogenization of us bases , a varieus bases . Howeverus bases . Indeed,radation .2+ can be a powerful catalyst for the degradation of RNA, and that EDTA inhibits its hydrolytic action. Mg2+ also plays an important role in ribozyme-catalyzed degradation of RNA (AbouHaidar and Ivanov  have shon of RNA .N. pubens causing RNA degradation, its presence is undeniable. Because N. pubens has emerged as an important pest ant species in recent years, and will likely be the focus of intense research effort in coming years, this study aimed to create awareness of the RNA-degrading entity in the adult stage. Fortunately, the RNA-degrading effect can be mitigated by the inclusion of EDTA in appropriate concentration. Unfortunately, EDTA carryover can inhibit subsequent RT-PCR and PCR analyses. Extraction of sound, intact RNA is a crucial first step in any downstream molecular analysis of genes. Stage-dependent degradation of RNA observed in N. pubens illustrates the importance of proper assessment of the integrity of nucleic acid purification processes, especially in untested organisms. Furthermore, proper techniques must be employed to make those determinations. For example, spectrophotometric examination alone may provide a deceiving assessment of the integrity of an RNA sample, as was observed in N. pubens workers.Although this study was unable to identify the entity or location in worker A recent paper by Gotzek et al.  has rena


Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.The marine plathyhelminth  Macrostomum lignano, presumably endemic to the Adriatic Sea, was described as a new species by Ladurner and colleagues in 2005 )lleagues . Three ylleagues  and Shenlleagues  developelleagues ,9. Agelalleagues ,10, whiclleagues ,12,13. Slleagues ,14.Cellular transport mechanisms, which export substances from the cellular interior, constitute an important defense line of cells and organisms against toxic compounds. Multidrug resistance transporters in eukaryotes belong to the superfamily of ABC-transporters, which are named after their most characteristic feature, the highly conserved ATP-binding cassette . The majet al. showed that Probenecid reversed multidrug resistance in a cancer cell line by the modulation of the MRP-mediated drug transport [Specific inhibitors for multidrug resistance transporters applicable in cancer patients, animals, and living cells are rare . Verapamransport . MK571, ransport . By measransport ,32,33,34et al. [Schistosoma mansoni. In aquatic animals, as well as in parasites, the whole body may be exposed to toxins or toxicants. The mechanisms of multidrug resistance in marine sponges, freshwater fish, and both freshwater and marine mussels are similar to multidrug resistance in human cancer cells [Human MRP and MDR homologues have been described in a wide variety of evolutionary early organisms, from plants, yeast, and different types of worms, including parasites. Sato et al.  demonstrer cells . Homologer cells ,37, fisher cells , musselser cells ,40,41.Prior to this study, experiments were performed to estimate the pH values in tissues and cells of marine transparent animals  by using the alkaloid Ageladine A as a pH sensitive fluorescence dye ,11. RemaM. lignano is easy to culture, and due to its small size and transparency, it is a good candidate for optical investigations of living, undisturbed animals. By using multiphoton scanning microscopy, we have investigated whether homologues of MDR1 and MRPs are functionally expressed in the flatworm and whether Ageladine A may function as an inhibitor or substrate of such transporters. For the latter question, rat astrocytes were used as an established MRP1-expressing model [M. lignano.ng model  in a comM. lignano individuals with the highest fluorescence coinciding with the rhabdites and some parts around the mouth .Presence of the MRP1 transport inhibitor MK571 increased the fluorescence of Fura-2, Fluo-3, and Calcein A–C, indior CMFDA D–F, suggAs Fluo-3 and Calcein are transported by MDR1 as well as by MRP transporters, we added Ageladine A to study a possible inhibitory effect of this compound on the transporters involved. However, Ageladine A had no effect on the accumulation of either fluorophore by the worms .In order to confirm the lack of effect of Ageladine A on MRP transporters, we used an established model to investigate the function of these transporters: the GSH export from cultured brain astrocytes . AgeladiM. lignano deals with exposure to toxins and toxicants. Using various fluorescent dyes as reporters with well-established pharmacologically inhibitors of multidrug transporters, we demonstrate the presence of functional multidrug resistance proteins in the worms that appear to have a pharmacological profile which resembles that of the mammalian transporters MDR1 and MRP1. These results demonstrate that the fluorescent dyes are taken up into the worms. We further ruled out the possible inhibition of multidrug resistance transporter-mediated dye export from M. lignano by the sponge derived secondary metabolite Ageladine A and showed that Ageladine A has no direct toxic or lytic effect on the worms for a minimum of a week.The question we addressed was how The flatworms appear to have the ability to quickly and sustainably remove acetoxymethyl ester dyes as xenobiotic surrogates. In many experiments, staining of animals with such dyes was weak in the absence of inhibitors of the multidrug resistance transporters, indicating efficient constitutive export activity. Functional expression and a baseline activity support the idea of an evolutionary adaptation to toxin exposure from the diatoms contained in the diet and from additional uptake of anthropogenic pollutants at the Adriatic coast.M. lignano [M. lignano that have a similar pharmacological profile as those known in mammals. Typical substrates of these transporters are hydrophobic or amphipatic compounds reaching 300–2000 Da in mass [The structure and function of MDR1 and MRP1 are highly conserved in evolution ,47,48,49 lignano  annotate lignano . Now, ou in mass  fitting i [Seelig  comparedi .Seawater and F/2 medium have a pH value around 8.1 and in this neutral to slightly basic environment Ageladine A is uncharged and hydrophobic, turning it into a membrane-permeable dye . Inside The MRP transporters are described as organic anion transporters. Jedlitschky  and cowoAgeladine A does not seem to either reduce the ATPase activity or compete against normal substrates for an interaction with multidrug resistance transporters. Furthermore, our results indicate that Ageladine A cannot be considered as a catalytic substrate. The assays of our study only show the retention of the protonated fluorescent micro-species of Ageladine A. A possible extrusion of uncharged lipophilic micro-species of Ageladine A cannot be assessed through the use of this assay. Our results indicate that the protonated form of Ageladine A is a non-substrate of multidrug resistance transporters that interacts neither with the ATPase activity nor with the substrate binding domains.M. lignano, which is not closely related to mammals. The study of Scherer et al. [Thalassiosira rotula. Our results support the fact that multidrug resistance transporters are highly conserved structures in nature.The multidrug resistance transporter inhibitors used in our study were developed for medical purposes. MK571, for example, was discovered as a competitive leukotriene D4 receptor inhibitor in human tumor cells . Probener et al.  showed aSarkadi and Müller introduced Calcein am as “the dye of choice” for fluorescent reporter assays . The hydRhodamine B was additionally used as a reported dye. The literature is controversial about the function of Rhodamine B in MDR1- or MRP-mediated transport. Minier and Moore  suggest M. lignano.CMFDA is not mentioned to be a substrate of multidrug resistance transporters in the literature and is a good long-term cell tracker. The cell-impermeant S-conjugate does not leak out of cells . In our M. lignano (DV-1 line) was cultured at 20 °C with 95% humidity and a 14/10 day-night cycle in glass dishes with nutrient-enriched artificial seawater (Guillard’s F2 medium) as previously described by Ladurner et al. [Nitzschia sp.The plathyhelminth gnano DV- line was2. Triplicate experiments were performed on two-week-old cell cultures.Astroglia-rich primary cell cultures were obtained from the brains of neonatal Wistar rats. The cells were obtained as previously described  and seedSix different fluorescent dyes were used. Ageladine A  and the calcium indicators Calcein acetoxymethylester , Fura-2 acetoxymethylester , and Fluo-3 acetoxymethylester  were used at a common concentration of 5 μM. All of the three calcium indicator dyes are known to be substrates of MDR1 ,43,44 anCell Tracker Green 5-chloromethylfluorescein diacetate  and Rhodamine B  were also used.MK571 (Biomol) was applied in a final concentration of 50 μM. It has been considered to be a specific inhibitor of MRP1 transporters . ProbeneM. lignano was monitored with a confocal laser scanning microscope Leica TCS SP5. The microscope was equipped with a multi-photon laser , an argon-laser (488 nm), and a helium-neon-laser (561 nm). For analyzing differences in fluorescence, the region-of-interest function of the software Leica LAS AF was used, with five transects drawn through each animal’s body covering all tissues.The fluorescence in M. lignano. The experimental animals were transferred to Petri dishes containing fresh F/2 medium (without food) to avoid fluorescence interference from diatoms, etc. Animals were placed on slides filled with 100 μL F/2 medium containing the respective fluorescent dye. After 1.5 h of incubation, animals were transferred to fresh F/2 medium without fluorescent dyes. To avoid contamination and evaporation of the medium, slides were kept in plastic boxes sealed with foil. To measure the fluorescence intensity animals were transferred into Utermöhl chambers containing 100 μL F/2 medium with 5 µL of a relaxing solution  to facilitate the scanning.The fluorescence intensity of six dyes was investigated for up to nine days in stained The MDR1-inhibitor Verapamil and the MRP1 inhibitors MK571 or Probenecid were added to the F/2 medium containing one of the listed fluorescent dyes . An inhibition of the transporters should cause a stronger accumulation of fluorescence substrates in the worms by inhibiting dye export. Changes in fluorescence intensity were measured with a Leica SP5 confocal microscope after 1.5 h of incubation. Furthermore, Ageladine A was added to the medium containing fluorescent dyes to test for transporter inhibition of this compound. Ageladine A and Fura-2 am have overlapping excitation and emission ranges, which could lead to fluorescence interference among dyes. Therefore, combined staining of Fura-2 together with Ageladine A was avoided.The extracellular amount of glutathione ) was analyzed to test for a potential effect of Ageladine A on MRP1-mediated GSH export. Astrocytes were incubated in incubation buffer without (control) or with 1% DMSO (solvent control), 50 μM MK571, 1 μM, 10 μM, or 100 μM Ageladine A for up to 3 h as previously described and extracellular GSH was determined as described previously . Cell viT-tests were performed, using the software IBM SPSS 20.Fluorescence intensities from five regions of interest per animal were pooled, averaged, and considered as one value. Each treatment group consisted of 5–10 individuals. Results are presented as means ± SE. Data generated by lactate dehydrogenase and GSH assays measurements represent means ± SD of three independent experiments. General data processing was conducted with Microsoft Excel 2010. The statistical tests ANOVA and nonparametric M. lignano, and provides evidence for the presence of at least two classes of highly evolutionary conserved multidrug resistance transporters that have the pharmacological profile of MDR1 and MRP1. The results shown disprove our original hypothesis that Ageladine A may have an inhibitory effect on MDR1- and/or MRP-mediated export processes. Nevertheless, the lack of export of Ageladine A together with the apparent low toxicity of this compound make Ageladine A a highly useful tool for life imaging of marine transparent animals and cells.The presented work is a multidrug resistance study on the living flatworms


A maximum habitat loss of 43% by 2100 was predicted in RCP4.5 and 82% in RCP8.5. When the model is trained solely with environmental data from the Caribbean/Atlantic, 83% of global habitat was lost by 2100 for RCP4.5 and 88% was lost for RCP8.5. Similarly, global runs trained only with Pacific Ocean reefs estimated that 60% of suitable habitat would be lost by 2100 in RCP4.5 and 90% in RCP8.5. When Maxent was trained solely with Indian Ocean reefs, suitable habitat worldwide increased by 38% in RCP4.5 by 2100 and 28% in RCP8.5 by 2050. Global habitat loss by 2100 was just 10% for RCP8.5. This projection suggests that shallow tropical sites in the Indian Ocean basin experience conditions today that are most similar to future projections of worldwide conditions. Indian Ocean reefs may thus be ideal candidate regions from which to select the best strands of coral for potential re-seeding efforts. Coral reef ecosystems are threatened by both climate change and direct anthropogenic stress. Climate change will alter the physico-chemical environment that reefs currently occupy, leaving only limited regions that are conducive to reef habitation. Identifying these regions early may aid conservation efforts and inform decisions to transplant particular coral species or groups. Here a species distribution model (Maxent) is used to describe habitat suitable for coral reef growth. Two climate change scenarios  from the National Center for Atmospheric Research’s Community Earth System Model were used with Maxent to determine environmental suitability for corals (order Scleractinia). Environmental input variables best at representing the limits of suitable reef growth regions were isolated using a principal component analysis. Climate-driven changes in suitable habitat depend strongly on the unique region of reefs used to train Maxent. Increased global habitat loss was predicted in both climate projections through the 21 Anthropogenic climate change will alter many physical and chemical characteristics that comprise the niches of marine species and ecosystem habitats. Changes in these physico-chemical conditions are already leading to shifts in the habitat ranges of some marine species , and extAs climate change alters oceanographic conditions, the geographic range of ocean habitat suitable for the growth of coral reef ecosystems is shifting. Corals of the order Scleractinia provide the foundation of shallow-water coral reef ecosystems. Scleractinia corals secrete calcium carbonate skeletons, which accumulate as reef structures that in turn support highly biodiverse communities. All shallow-water, tropical coral reefs are defined by the same broad ecological functional groups, but they develop across a wide array of ocean environments . The meaIncreasing sea surface temperatures and extreme temperature excursions are considered a major threat to coral reef ecosystems primarily because they have been shown to be the major factor behind the recent global increase in coral bleaching ,6. OceanThis article considers the geographical change in physico-chemical environments appropriate for coral reef ecosystems, as projected by the National Climate and Atmospheric Research (NCAR) center’s Community Earth System Model Version 1 (CESM1) in a suite of climate change scenarios. Suitable reef habitat was defined using a niche model, Maxent , throughPotential coral reef habitat was modeled using Maxent, a maximum entropy niche model that performs well with presence-only data for species or communities . Maxent 2, HCO3-, CO32-, and alkalinity), which are then used to calculate calcium carbonate (aragonite) saturation state [CESM1 is a global atmosphere-ocean, fully coupled climate model that includes global carbon cycling. CESM1 is built on the Community Climate System Model version 4 (CCSM4), with a nominal horizontal resolution of 1° by 1° which is enhanced in the tropics and at high latitudes . The oceon state . th century run (1985–2005) and two 21st Century scenario runs described by unique representative carbon pathways (RCPs): RCP4.5 (2005–2100) and RCP8.5 (2005–2100). The scenarios yield differences of approximately 2°C in mean global air temperature. Ocean data were retrieved only from the surface layer (the upper 10 m), as most shallow-water coral growth occurs in the upper littoral zone . The absolute increase in mean sea surface temperature for the domain considered is 0.73° C for RCP4.5 and 2.10° C for RCP8.5. The variables extracted from CESM1 included sea surface temperature, sea surface salinity, phosphate concentration at the sea surface (PO4), average light availability in the surface layer as PAR, surface horizontal current velocities, and carbonate system parameters. Since concentrations of the nutrients nitrate, phosphate, and silicate are strongly correlated in seawater [Model data were extracted from the CESM1 BEC 20seawater , only phth century (for training Maxent), and the two 21st century simulations RCP4.5 and RCP8.5, were interpolated to a grid with 1° by 1° resolution. The monthly data were averaged over 20-year time periods, from 1985–2005 for the 20th century model and as overlapping time periods in the 21st century runs  until 2100. As the 21st century runs begin at year 2005, data were also collected for the ten year time period 2006–2015 to calculate an average for 2010. These files were converted to Matlab® structure files for processing in Matlab® (R2011a). This process is described in more detail below in Section 2.3. Monthly output from the CESM1 biogeochemistry model simulations of the 20Coral-reef ecosystems presently exist in limited regions of the tropical and subtropical oceans. Data regarding the specific locations of these ecosystems are available from ReefBase (www.reefbase.org). ReefBase locations are provided by the Millennium Coral Reef Mapping Project . These sarag) was calculated as the ratio of carbonate ion concentration and carbonate ion concentration at Ωarag =1, from the CESM1 carbon-system components. The mean, maximum, minimum, and annual range for current speed, Ωarag, sea surface temperature, sea surface salinity, sea surface phosphate concentration, and surface PAR were obtained for each of the 20-year time periods. Averaging across a 20-year time period provided an estimate of the climatological state from CESM1. All variables are normalized prior to subsequent analyses.Surface current speeds were calculated from the horizontal velocity components of the CESM1 data. Aragonite saturation state (ΩFrom temperature (SST), further calculations were performed to estimate cumulative thermal stress (CTS), as shown in (1). CTS is similar to the degree heating week measure e.g. , and is x represents latitude, y represents longitude, t represents time, SST represents sea surface temperature, and σMMM represents the standard deviation of the MMM. MMM data were calculated from the 1985–2005 20th century run, and CTS was then calculated and summed over each 20-year simulation period. These data are not a direct analog to degree heating weeks, but rather represent the overall thermal stress for each 20-year time period considered in this study. where st century . Each of these runs yielded two dominant (25-element) structure functions that combined explain at least 60% of the total variance among the spatial locations, and each generated similar structure functions that revealed correlations among the variables when plotted along perpendicular axes  for three time periods in the 21lar axes . Six groCTS:  provides an indication of the duration and magnitude of temperature excursions that exceed the tolerance of coral organisms. Temperature stress is strongly correlated with coral bleaching [leaching , which oPhosphate maximum:  is also considered representative of nitrate and silicate levels in this study. Nutrient enrichment is often associated with coral-reef ecosystem degradation as it can lead to a shift in community structure towards one dominated by fleshy macroalgae [croalgae . ClimateCurrent speed maximum:  is an indication of the hydrodynamic energy in the surrounding environment. Currents are important to coral-reef ecosystems because they provide a well-oxygenated environment that enhances food availability and provides flushing of reef waters. High current speeds have been correlated with reduced heat stress and thus less coral bleaching and mortality in several previous studies [ studies -22. Salinity minimum: . Most reef building coral organisms are intolerant of salinities less than 25 parts per thousand (ppt), [d (ppt), , and no d (ppt), . Climatearag minimumΩ:  is a measure of the degree of aragonite saturation in seawater. Both the skeletal growth rate in individual coral colonies and the rate of increase in the quantity of reef-building framework have been correlated with Ωarag [arag , regions of high Ωarag are contracting in area, mostly equatorward [2 levels double in comparison to preindustrial levels (estimated occur at approximately 2050 in RCP 8.5), the combined effects of coral bleaching and decreased Ωarag will decrease net calcification on reefs to the point that most will shift from net reef-building to net dissolution [th Ωarag . Becauseatorward ,26. Oncesolution .PAR minimum:  represents the amount of radiation available to organisms for photosynthesis. As coral polyps house photosynthesizing symbiotic dinoflagellates, PAR is critical for their survival. Minimum PAR levels can be limiting to suitable reef habitat area, particularly at high latitudes where persistently low PAR can limit coral structures to very shallow regions [ regions . Climate regions , which iarag, nutrients, and light [Maxent is a maximum entropy niche model that uses environmental variables  combined with species presence data to determine the likelihood of suitable habitat for that species at each grid cell within a geographic domain ,29. Maxend light . Here, cnd light ) might cMaxent version 3.3.3 (http://www.cs.princeton.edu/~schapire/maxent/) was first trained using the world-wide distribution of coral reefs and the six selected environmental parameters from the CESM1 model output for the time period 1985–2005. The model was trained using 75% of reef locations, selected randomly, and testing of model performance was implemented by predicting the remaining 25% of reef locations. For the first case considering all reefs, 894 locations were used for training and 298 additional locations were used for testing. For the Indian Ocean case, 146 sites were used for training and 48 for testing; for the Pacific Ocean case, 569 sites were used for training and 189 for testing; and for the Caribbean case 135 locations were used for training and 45 additional sites were used for testing. For each of the 72 sets of Maxent runs, the mean AUC score (an indicator of model performanc) was greater than 0.8 with the ‘Clamping’ function enabled. st Century CESM1 output for the overlapping 20-year time periods in both RCP scenarios. Projections were first run based on training with the global distribution of coral reefs. Projections were also run based on training restricted to three separate coral reef domains corresponding to the Caribbean/Atlantic, Pacific, and Indian Ocean basins. For these three cases, the Maxent model was trained using a subset of coral reef distribution that only included regions where corals are found within each ocean basin. These basin-specific projections were run to examine how the suitable environmental area in which coral reefs specific to each domain reside would shift geographically both within and outside each domain during the specified climate change scenarios. Predictions outside of the ‘home’ domain enabled Maxent to estimate where coral reef habitat, as represented in each basin, would theoretically exist within other ocean basins. To project how the spatial distribution of coral reef habitat may change in the future, Maxent was run with the 21These projections of suitable habitat areas for coral reef growth in the future do not take into account bathymetry. That is, suitable habitat illustrates regions where the phsyico-chemical conditions are adequate for coral reef growth, but in reality reef growth within these habitats will be restricted to water depths of approximately 30 m or less.Compared with Maxent’s prediction of the 1985–2005 distribution of reefs, projected coral-reef habitat declined with time in both the RCP4.5 and RCP8.5 climate scenarios . PercentWhen Maxent was trained separately for each ocean region using the basin-specific physico-chemical preferences associated with the presence of coral reefs, the resulting projections of suitable habitat area for both the Pacific and Caribbean/Atlantic reefs were similar to when the global distribution of reefs was used . The envarag minimum in the climatological variables calculated from CESM1 (20 year averages) from the 2050 mean to 2060 mean. The average for the entire study area is 2.48 in 2050, 1.87 in 2060, and recovers to 2.15 in 2070. Suitable habitat in the Caribbean/Atlantic had already decreased by over 70% in 2050 and does not show the same drop. The global runs, as well as those considering only Indian reefs and only Pacific reefs, all experience a precipitous drop in percent of suitable habitat from 2050 to 2060 in the RCP8.5 scenario only. This is mainly a result of a dramatic change in ΩSpatial distributions of the relationship between physico-chemical variables and the existence of present-day of coral reefs indicate marked differences in the “habitat space” occupied by reefs from different basins. In addition, model projections suggest markedly different basin-specific shifts in those habitats ,4,5,6. BUnder both climate change scenarios, the region of suitable habitat for Pacific reefs is reduced in a relatively uniform manner across all ocean regions. Apart from the Pacific, some suitable habitat was identified in both the Indian and the Atlantic Oceans but not in the Caribbean . RegionsThese bioclimate modeling results indicate that climate change and ocean acidification will impact the distribution of suitable coral reef habitat in the future, for both the RCP4.5 and the RCP8.5 scenarios. Globally the envelope of oceanographic conditions favorable to coral reef development will decrease by 43% and 82% by the year 2100 for the RCP4.5 and RCP8.5 scenarios, respectively. The decreases in actual reef habitat will further depend, in part, on the existence of shallow substrate within the envelope. arag have both been shown to limit coral reef development at high latitudes [arag has been associated with a decreased ability of coral communities to construct coral reefs [arag to shift habitat to ‘marginal’ by the mid- to late-21st century, but those results also indicate that regions of habitat within the margins of present-day coral reef habitation persist for at least the next 100 years [The global runs also identify where potential new coral habitats may emerge in the future. For example, rising sea surface temperatures are expected to shift the suitable habitat envelope poleward in both hemispheres, and some coral species have indeed begun to colonize at higher latitudes ,38. The atitudes  and declal reefs ,39. One 00 years . st century. The projected envelopes of suitable habitat for Caribbean/Atlantic reefs, however, nearly disappear under both climate change scenarios. This striking difference for Caribbean/Atlantic reefs remained robust across multiple iterations using Maxent, and persisted even when removing each of the six variables individually from training. Thus the susceptibility of Caribbean/Atlantic reefs to future conditions does not appear to be an artifact of a single variable skewing the results. The modeled vulnerability of reefs in this region may simply reflect the smaller number of reef locations in the Caribbean/Atlantic used to train Maxent, which may narrow the range of environmental conditions presently experienced by reefs across the domain. The difference may also be due to the unique physico-chemical environment of the Caribbean. Regardless of the cause, these projections suggest very different fates for coral reefs that presently exist in the three major ocean basins.The bioclimatic envelopes amenable to coral reef growth are defined slightly differently for each ocean basin. The changes in the projected envelopes of suitable habitat for Pacific Ocean reefs are similar to those for the global projections, while those for Indian Ocean reefs are maintained across many regions, in spite of the significant changes in modeled ocean conditions throughout the 21arag, light, nutrients and current speeds. Among these variables, projected temperature and Ωarag change markedly in the future, so that Maxent’s projection of future reef habitat strongly reflects the net effect of 1) a poleward shift of suitable habitat due to increasing temperature, and 2) a shift of suitable habitat toward the equator due to decreasing Ωarag. In both the global and basin-scale projections, certain regions stand out for their retention of suitable reef habitat under future climate scenarios through the year 2050 and beyond. These include mostly equatorial regions in the northern Indian Ocean, the Coral Triangle region, French Polynesia, and the northeast Brazilian shelf east of the Amazon River. These regions are where the CESM1 projections estimate suitable temperatures, Ωst century. One previous study argues that coral reefs will face serious decline unless atmospheric CO2 is limited to 350 ppm [st century under the RCP4.5 scenario [arag similarly predict a loss of at least 43% of habitable area worldwide under the RCP4.5 scenario.Our work is in line with previous predictions of coral reef decline in the 21 350 ppm . Anotherscenario . Our resThe results also indicate which basins presently include reef habitats that are “best conditioned” to spread to new regions. Present-day coral reefs in the Indian Ocean basin experience conditions that are most similar to future climate projections in the Indian as well as the Pacific and Atlantic Oceans . AnotherPorites lutea in the Pacific and Acropora palmata in the Caribbean/Atlantic. The responses of these two species alone were tested using Maxent, and the results were similar to the composite reef results. The main difference in both tests was a more constricted range, presumably as a result of the smaller number of locations used for model training, resulting in a more restricted suitable habitat envelope. While projections of suitable habitat area for A. palmata showed that suitable regions essentially disappeared in the future, there is some indication that hypothetically suitable habitat for P. lutea could persist in some portions of the Caribbean/Atlantic in addition to the Pacific and Indian basins through most of this century  are required to create models that are statistically more robust. Furthermore, these results do not consider the current ecological state of any coral-reef ecosystems, direct anthropogenic stressors such as pollution or fishing, the influence of invasive species, or molecular and ecological resilience of particular corals or coral-reef communities. 2 forcing is neither linear nor strictly latitudinal when considering a suite of representative variables known to influence coral ecology. Coral-reef ecosystems in different regions may respond in unique ways to the same forcing, and results suggest particular regions can be more suitable or less – an effect that remains consistent despite perturbations to model parameters. When individual basins are considered, conditions in which shallow-water coral-reef ecosystems presently exist in the Indian Ocean are shown to be most similar to future projections of global, tropical, physico-chemical ocean conditions. Coral reefs in this region may be most suited for persistence worldwide in future climate states. In summary, this study suggests that the response of shallow tropical coral reefs to increased COList S1Groups of highly correlated variables from principal components analysis.Variable selected for subsequent analyses is indicated in bold. PAR = photosynthetically active radiation; CS = current speed; Ωarag = aragonite saturation state.(DOCX)Click here for additional data file.Table S1Correlation matrix of variables selected for species distribution model analysis.Variables are listed along the top and left side, with ρ-correlations for each variable pair given in the table. CTS = cumulative thermal stress; PAR = photosynthetically active radiation; CS = current speed; Ωarag = aragonite saturation state.(DOCX)Click here for additional data file.


Genogroup II.4 norovirus (NoV) remains the predominant NoV strain in food- and water-borne outbreaks. Capsid integrity as well as viral RNA persistence were determined for GII.4 NoV by real-time RT-PCR after 1–14 freeze/thaw (F/T) cycles (−80 °C/+22 °C) or after −80 °C storage for up to 120 days. In both cases, capsid integrity and viral RNA titers remained stable. RNase was exogenously added after 1–14 F/T cycles, but did not alter the amount of genomic NoV RNA detected, indicating that capsids remained intact. Presumptive NoV infectivity was evaluated in functional studies by a porcine gastric mucin binding assay. Viruses frozen and thawed up to 14× bound similarly to porcine mucin, suggesting no reduction in virus infectivity. Overall, this study shows that a) NoV particles retain their integrity for at least 14 F/T cycles, b) long-term (120 day) frozen storage does not decrease NoV RNA titers, and c) capsid binding to receptor-like glycoprotein moieties remains unaltered after 14 F/T cycles. This work indicates that freezing and thawing of foods or beverages would not be a practical processing intervention to reduce NoV contamination. Likewise, repeated freezing and thawing, as might be encountered during winter months, is not expected to inactivate NoV in the environment. Results do show that laboratory samples destined for molecular biological analyses or for use as positive controls may be repeatedly frozen and thawed without any anticipated reduction in NoV RNA titers. This study documents the cryostability of NoV capsids and RNA to freezing and thawing and to the possible retention of virus infectivity. Human norovirus (NoV) is the principle cause of food-borne illness in the United States with an incidence recently estimated at 5.5 million cases annually  potential food processing techniques to eliminate NoV in foods and beverages, b) environmental conditions conducive to the inactivation of NoV, and c) methods for the preservation of viruses for use in NoV testing laboratories. Butot et al.  showed mNo studies have been performed to evaluate the persistence of GII.4 NoV after repeated freeze–thaw (F/T) cycles; therefore, the effects of freezing and thawing on NoV, as would be encountered under certain environmental conditions, are unclear. It is possible that NoV in lakes, streams, and coastal areas may be affected by freezing and thawing although a strikingly higher incidence of NoV illness occurs during the winter months and is what led to the earlier name for NoV illness as “winter vomiting disease”  as determined by the method described in Richards et al. . These NoV “filtered stocks” were maintained at 4 °C until use.Fresh stools were provided to the Delaware Division of Public Health Laboratory for routine norovirus screening after outbreaks of gastroenteritis in long-term care facilities. All stools were sequence confirmed to contain GII.4 NoV (New Orleans) and the high-titered stools were reserved at 4 °C for this study. Stools used in this study showed high titer by end-point dilution RT-PCR  thaw at room temperature (about 22 °C). Thawing of all tubes was conducted at room temperature in styrofoam racks to insure slow thawing and to maximize damage of viral capsids from ice crystals. Complete thawing was achieved within 30 min. The RNA was extracted from samples, and the extracts were tested in triplicate by real-time RT-PCR, as described below.A second set of 14 tubes was repetitively frozen at −80 °C and slowly thawed at room temperature at weekly intervals over the course of 14 weeks such that tubes underwent from 1–14 F/T cycles. At the designated weekly interval, the repeatedly frozen and thawed samples were thawed as a group. Tubes were transferred to another rack for storage at −80 °C when their designated number of F/T cycles was reached. Remaining tubes from that group were refrozen at −80 °C to await additional F/T cycles. Thawing was performed for 30 min for each time interval. One final thaw was performed on all samples at the end of the study and samples were subjected to viral RNA extraction and analysis by RT-PCR.Using 140 μl of stool filtrate, NoV RNA was extracted, column purified, and eluted in 60 μl of water using a Qiamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Positive controls consisted of similar NoV RNA extracts from the same stool filtrates, except samples were never frozen. For samples frozen and thawed, 140 μl were similarly extracted, concentrated to 60 μl, and assayed by real-time RT-PCR.2O, and 1 μl viral RNA extract (template). Reverse transcription was performed at 50 °C for 30 min, followed by Taq activation for 15 min at 95 °C and then 50 cycles of PCR amplification at 95 °C for 15 s and 60 °C for 1 min. Negative controls consisted of master mix including primers with water substituted for the NoV stool extracts. Positive controls were as described above. Norovirus RNA titers of the diluted stool filtrates were monitored by real-time RT-PCR. Cycle threshold (Ct) values were measured at an arbitrary threshold of 30 fluorescence units at the end of each 60 °C extension step. All aliquots were compared among each other for relative Ct values. All extracts were tested by real-time RT-PCR in triplicate.Real-time RT-PCR was performed on a Smart Cycler  using the one step RT-PCR Kit (Qiagen). Primers were as follows: forward primer QNIF2 (5′-ATGTTCAGRTGGATGAGRTTCTCWGA-3′)  to each tube to stop the reaction, and the samples were again mixed and spun for 2 s. After 5 min, the tubes were added to ice pending RNA extraction by Qiamp Viral RNA Mini Kit (Qiagen) and assay by RT-PCR. This trial was performed in duplicate and each extract was tested by RT-PCR in duplicate (n = 4). Previously, this RNase treatment was demonstrated to effectively eliminate all traces of NoV RNA in RNA extracts derived from the filtered NoV stock.The integrity of the NoV capsid to repeated (1–14) F/T cycles was monitored after the addition of exogenous RNase , which contained 2 μg/μl of RNase A and 4 U/μl of nonspecific RNase from t values by real-time RT-PCR, which was performed in duplicate.Fourteen 0.5-ml tubes were inoculated with 150 μl of filtered NoV stock. Tubes were frozen at −80 °C and slowly thawed at room temperature 1–14×, as described above. Porcine gastric mucin-conjugated magnetic beads (PGM-MBs) were prepared as previously described  was not significantly different (P > 0.05) from the mean titers of the samples frozen once (Ct = 20.25) or up to 14× (Ct = 20.36).The effect of storage duration at −80 °C was evaluated using an initial freeze and then a single, room temperature thaw at weekly or bi-weekly intervals for 17 weeks (120 days), followed by immediate viral RNA extraction and real-time RT-PCR analysis. The relative viral RNA titers of the extracts remained essentially the same over the 17-week trial period  from the titer after 14 F/T cycles . Some increases in Ct values (suggesting about a 1-log and 0.5-log decrease in titer) were observed at 7 and 8 F/T cycles in one of the trials, but are attributed to the variability in column extraction of the NoV RNA since Ct values remained low and nearly constant from 2 to 6 and 9–14 F/T cycles  and slowly thawed +22 °C) from 1–14× followed by the addition of commercially obtained RNase cocktail after the designated number of F/T cycles. After incubation for 30 min, RNase inhibitor was added and viral RNA was extracted and assayed. Unfrozen NoV stock filtrate served as a control. Results confirm no loss of NoV RNA titers over 14 F/T cycles  in binding efficiency over 14 F/T cycles  contained essentially the same levels of viral RNA as samples frozen and thawed up to 14×. Freezing followed by slow thawing at room temperature would be expected to exert maximum damage to viral capsids from prolonged exposure to ice crystals. If F/T damage had substantially affected the capsid’s integrity, then the viral RNA would have been destroyed by ubiquitous RNases present in stool filtrates. These RNases would include a variety of endo- and exonucleases from both human and bacterial cells in the gut. While RNases are considered very hardy enzymes, it is conceivable that RNases from the stool filtrates could be damaged and inactivated after repeated F/T cycles. Consequently, we performed additional tests where a commercially obtained RNase cocktail was added to NoV after 1–14 F/T cycles. It showed that there was also no reduction in virus titer after repeated freezing and thawing.Another line of evidence for capsid integrity is predicated on the ability of viable viruses to bind to porcine gastric mucin. It is well established that the detection of viral RNA does not indicate the presence of infectious viruses, because virus infectivity also depends on the retention of capsid protein conformation Richards . For a NOverall, we conclude that NoV GII.4 is highly resistant to repeated F/T cycles and to long-term storage. It is uncertain if other genogroups or clades of NoV offer this same cryostability. Our results, showing the persistence of NoV to long-term freezing, are consistent with a NoV volunteer study which demonstrated that GI.1 NoV  retained infectivity after frozen storage for more than two decades (Teunis et al. The persistence of NoV to freezing and thawing is unwelcome news in food processing and for the elimination of NoV from the environment. For foods potentially contaminated with NoV, such as produce and shellfish, there is no evidence to suggest that freezing and thawing will have a beneficial effect on reducing virus infectivity. In fact, food matrix and proteins would be expected to further stabilize NoV over water alone. Thus, we conclude that freezing and thawing are unsuitable as a food processing intervention for NoV. Likewise, our results indicate that winter freezing and thawing events are not likely to contribute to the inactivation of environmental NoVs. However, this persistence of NoV to freezing and thawing is welcome news in the laboratory, where samples destined for analysis or to be used as positive controls may be repeatedly frozen and thawed without any anticipated reduction in viral RNA titers.


Acanthurus triostegus (Acanthuridae) from new settler to adult stages at Moorea Island (French Polynesia). Specifically, we tested the relationship between diet and habitat shifts and changes in overall body shape during the ontogeny of A. triostegus using a combination of geometric morphometric methods, stomach contents and stable isotope analysis.Many coral reef fishes undergo habitat and diet shifts during ontogeny. However, studies focusing on the physiological and morphological adaptations that may prepare them for these transitions are relatively scarce. Here, we explored the body shape variation related to ontogenetic shifts in the ecology of the surgeonfish A. triostegus is highly allometric. The allometric shape changes mainly concern cephalic and pectoral regions. The head becomes shorter and more ventrally oriented during growth. Morphological changes are directly related to the diet shift given that a small mouth ventrally oriented is particularly suited for grazing activities at the adult stage. The pectoral fin is more anteriorely and vertically positioned and its basis is larger in adults than in juveniles. This shape variation had implications for swimming performance, manoeuvrability, turning ability and is related to habitat shift. Acanthurus triostegus achieves its main transformation of body shape to an adult-like form at size of 35–40 mm SL.After reef settlement, stable isotope composition of carbon and nitrogen revealed a change from a zooplanktivorous to a benthic algae diet. The large amount of algae (> 75% of stomach contents) found in the digestive tract of small juveniles (25–30 mm SL) suggested the diet shift is rapid. The post-settlement growth of Most of the shape changes occurred after the reef colonization but before the transition between juvenile habitat (fringing reef) and adult habitat (barrier reef). A large amount of allometric variation was observed after diet shift from zooplankton to benthic algae. Diet shift could act as an environmental factor favouring or inducing morphological changes. On the other hand, the main shape changes have to be achieved before the recruitment to adult populations and start negotiating the biophysical challenges of locomotion and feeding in wave- and current-swept outer reef habitat. Ontogenetic shifts in diet and habitat are the norm for demersal marine fishes. The majority of coral reef fishes have stage-structured life histories with two main distinct stages including a pelagic larval stage capable of long-distance dispersal and a demersal stage  . The traEcomorphology may assist in our better understanding of the relationships between morphological and ecological changes of an organism during ontogenetic shifts ,8. Most Acanthurus triostegus  was examined. This species is a common coral reef fish in the Indo-Pacific region . The timing of metamorphosis in coral reef fishes varies according to fish groups and is directly linked to species-specific ecological transition periods [A. triostegus achieve their metamorphosis to an adult-like form at a size of 35–40 mm SL (i.e. approximately one month after settlement to the reef).Metamorphosis in fish requires radical changes in external and internal morphology, and is often accompanied by ecological or habitat changes . GeneralcCormick  highlighdy depth . After 5dy depth . More reh [et al. highlighturnover . Knowingilibrium . On the habitats . Indeed,possible . During ychaetes ,22. UnfoA. triostegus live a calm inshore environment when adults swim in wave- and current-swept outer reef habitat. Fulton [A. triostegus use essentially their pectoral fins for swimming  detail quantitatively and qualitatively the complete ontogenetic shape changes of a surgeonfish occurring after reef settlement and to (2) highlight a mismatch between morphological changes and ecological shifts in coral reef fishes. Indeed, a large amount of allometric shape changes remains to be done after the diet shift when most of the shape changes are completed before the second habitat shift from beach zone to barrier reef. Our morphological study shows that cephalic and pectoral regions undergo a large amount of allometric shape changes through post-settlement ontogeny. The number of works focusing on the various modes of post-settlement transition in coral reef fishes is growing ,52 but sThe authors declare that they have no competing interests.BF and DL conceived of the study and both collected specimens in the field. BF carried out dissections, morphometric analyses and analysed the data. BF, OC and GL carried out stomach content and stable isotope analysis. BF wrote the paper with input from DL, OC and GL. All authors read and approved the final manuscript.Box plot illustrating body size distribution within settling larvae, juveniles and adults. Median, percentiles 25–75%, maximum and minimum values are illustrated.Click here for fileSampling locations in Moorea Island . White cross and star refer to sampling area for juveniles and adults, respectively.Click here for fileRelationships between (A–B) δ13C values and fish size (CS), and (C–D) δ13C values and shape (PC1) inAcanthurus triostegusliving in the juvenile habitat. Regressions were produced with all juveniles (A & C) and excluding the four largest juveniles (B & D). Equation of regression models: (A) δ13C = 0.7CS-19.4; (B) δ13C = 1.8CS-23.8; (C) δ13C = 34.3PC1–16.0; (D) δ13C = 40.5PC1–15.8.Click here for file


We also demonstrate that a relatively simple one-dimensional analytical model can provide a good description of the functional dependence of reef-water carbonate chemistry on benthic metabolism, wave forcing, sea level, reef flat morphology, and total system frictional resistance. Importantly, our results indicate that any long-term (weeks to months) net offsets in reef-water pCO2 relative to offshore values should be modest for reef systems with narrow and/or deep lagoons. Thus, the long-term evolution of water column pCO2 in many reef environments remains intimately connected to the regional-scale oceanography of offshore waters and hence directly influenced by rapid anthropogenically driven increases in pCO2.We present a three-dimensional hydrodynamic-biogeochemical model of a wave-driven coral-reef lagoon system using the circulation model ROMS  coupled with the wave transformation model SWAN (Simulating WAves Nearshore). Simulations were used to explore the sensitivity of water column carbonate chemistry across the reef system to variations in benthic reef metabolism, wave forcing, sea level, and system geomorphology. Our results show that changes in reef-water carbonate chemistry depend primarily on the ratio of benthic metabolism to the square root of the onshore wave energy flux as well as on the length and depth of the reef flat; however, they are only weakly dependent on channel geometry and the total frictional resistance of the reef system. Diurnal variations in  Thus, predicting the future response of reef calcifiers to changing atmospheric pCO2 is still uncertain. Furthermore, there are additional interactive effects of increased pCO2 on the ecology of reef biota that extend beyond rates of skeletal growth, yet these still remain poorly understood Rapidly rising levels of atmospheric COpCO2 of reef waters are generally not in equilibrium with the atmosphere on short timescales, a direct outcome of community metabolism driving diurnal variations in water column carbonate chemistry . Numerous subsequent studies have shown that the biological activity of reef communities can drive variations in pCO2 that range from levels below a pre-industrial atmosphere (<285 µatm) to greater than predicted for the earth's atmosphere by the year 2100 under one of the most severe IPCC forecasts  that act as individual ‘control volumes’ through which net inputs and outputs of mass and momentum are budgeted. More recently, the inclusion of benthic metabolism within such models have provided the additional means for modeling spatial and temporal changes in nutrient concentrations and carbonate chemistry within coral reef systems Field studies of reef water chemistry and derived rates of benthic community metabolism are thus complicated by ever-changing circulation patterns and water residence times. The application of numerical hydrodynamic models within coral reef systems has provided us with one means to overcome this limitation While prior efforts have used numerical models for the study of specific reef systems, the range of biogeochemical changes that an individual reef system can exhibit is still constrained by its particular geomorphology, benthic composition, and by the specific oceanic and atmospheric conditions driving internal circulation and exchange with offshore waters. This constrained range of observable behavior is often further limited by the finite duration of many field studies . Long-term monitoring efforts can overcome some of these time-dependent limitations, but such programs generally come at great cost and are therefore limited in number. To overcome the limitation of examining a single reef system under pre-defined conditions, we developed a flexible model reef system with simple but representative geomorphologies and rates of benthic metabolism that can be subject to a wide range of hydrodynamic forcing. We show that this approach offers the same level of focus and control in system-scale studies that experimental mesocosms accomplish in organism-scale studies. Both approaches complement more realistic but less-controlled field studies by exploring the effects of specific forcing variables on the response of a system (or organism) over a broader range of controlled conditions. If designed properly, the hydrodynamics and biogeochemistry of these experimental idealized reefs can be remarkably similar to real reef systems pCO2 that have been observed in natural reef systems. Finally, we discuss the utility of our simplified model to simulate larger reef systems as well as its implications for future studies of ocean acidification in coral reef systems.The present study is aimed at exploring how hydrodynamic factors (wave forcing and sea level), benthic metabolism, and reef geomorphology act in combination to fundamentally influence water column carbonate chemistry across representative wave-driven reef systems under a realistic range of input variables. The wealth of data on benthic net production, benthic net calcification, wave transformations, circulation, and morphology already available in the literature provide us with ample means to constrain our model domain within realistic limits  and 3. IP), respiration (R), and net calcification (Gnet) for entire reef flats tended to cluster around typical or ‘standard’ values of 580, 580, and 110 mmol C m−2 d−1, respectively. These rates were found to be largely independent of latitude and longitude (at least across the Indo-Pacific) despite seasonal and spatial variations in light, ocean sea surface temperature, and carbonate mineral saturation state; although seasonality in Gnet appeared to be stronger in higher-latitude reefs P≈300 to 1200 vs. 640 mmol C m−2 d−1 and Gnet≈50 to 250 vs. 130 mmol C m−2 d−1, P∶R ∼1.1, In a seminal review of reef community carbon metabolism, Kinsey 1985 −1 resulting in cross-reef transports 2 s−1Although there have been fewer studies of wave transformations and/or wave-driven circulation across shallow reef systems, it is nonetheless clear that the geomorphology and hydrodynamic forcing of many reef systems also fall within well-defined ranges . The arcT is the wave period, g is the gravitational acceleration constant , the water depth, and the horizontal transportforcing factor determined by the ratio of benthic metabolism or bottom flux to the square root of the onshore wave energy flux reef flat geometry factor determined by the length and depth of the reef flat total resistance of the reef system to wave-driven flow Assuming a vertically well-mixed water column, the change in concentration of a reactive species with space the reef . Thirdlyecreases ; thus, iecreases . Fourthlecreases , and theecreases . The tothemistry .H0, P, Gnet, Plag, hr, Lr, Llag, hc, Wc, and the offshore sea level We define a central model case based on median and average rates of benthic metabolism, morphological dimensions, and wave forcing reported in the literature see  and 3. W−1 is chosen to simulate offshore momentum boundary conditions similar to those both observed and used in prior modeling studies http://ivec.org.au/) using 132 processors.The hydrodynamic and biogeochemical processes were simulated using the three-dimensional ocean circulation model ROMS  The model domain consisted of one complete shallow reef flat bounded by a channel and one-half a reef flat to the north and to the south with a lagoon to the east  based onchannel  , Fig. 1aulations . This reulations . We needefs 1∶20  and moreefs 1∶20 . The maxp) and net calcification (gnet) were constrained to equal daily integrated rates over a 24-hour period:np) were calculated as the difference between r−2 d−1, respectively Plag = 330 mmol C m−2 d−1 and Hourly rates of gross production (:(10)see . Hourly gnet and np aspCO2 varied between undersaturated and oversaturated conditions with respect to the atmosphere over a diurnal cycle, and that air-sea CO2 fluxes are generally very small relative to the combined influence of benthic net production and calcification .Local benthic fluxes of Total Alkalinity (TA) and Dissolved Inorganic Carbon (DIC) were then calculated from hourly rates of tion <1% ; we ignopCO2 = 390 µatm), and having a sea surface TA typical of tropical surface waters worldwide  in order for diurnal changes in water column carbonate chemistry to reach an approximate steady state (−1) under the imposed constant hydrodynamic and forcing and diurnal variations in benthic metabolism.Offshore waters were assumed to be at a uniform temperature of 25°C and salinity of 35, in equilibrium with the atmosphere  are driven by their benthic uptake from net production and net calcification; behavior consistent with numerous observations reported in the literature . Therefore, in the interest of economy, most of the results and discussion that follows will focus on variations in pCO2 since this is the primary variable of interest with regards to future changes in atmospheric composition. Nonetheless, time-elapsed movies demonstrating hourly changes in ΔTA, ΔDIC, ΔpCO2, ΔpH and ΔΩ across the entire reef domain over a 24-hr period are also provided (see Movies S1 through S5).Changes in TA, DIC, terature . Water ipCO2 on the forereef appeared to follow quasi-sinusoidal variations . It is possible that this effect could be even more pronounced when alongshore currents, including the associated horizontal mixing, on the forereef are particularly weak. However, reducing the magnitude of the offshore current by one-half relative to the central case had only a modest effect on diurnal profiles of forereef water column carbonate chemistry. Furthermore, prior field observations (not shown) suggest that our reef-scale models if anything slightly overestimate the observed variability in the chemistry of forereef waters. This is likely the result of reef-scale models being unable to capture larger-scale hydrodynamic processes  that may also contribute significantly to cross-shelf mixing and transport.Diurnal changes in Δriations , whereasbehavior . This wabehavior , 4, 5. CA) of diurnal variation in a given carbonate chemistry parameter based on the temporal variance in that parameter valuecarb represents either TA, DIC, pCO2, pH, or Ωar and pCO2, pH, or Ωar in reef waters relative to offshore waters (Δcarb) averaged over a 24-hour periodx, y, z, t) into two-dimensional variables that vary only with location  through depth- and time-averaging.Since diurnal minima and maxima in carbonate chemistry parameters can be both complex and short-lived for many locations , we defined a statistically representative amplitude  as well as decreased the depth of the lagoon by one-half (hc = 3 vs. 6 m) to simulate the high level of metabolic activity observed in some shallow lagoons pCO2 across the reef flat itself; specifically those identified in Diurnal changes in backreef . This rebackreef . However−r to a maximum equal to 2 requires accounting for the fact that calcification releases less than one mol of CO2 for each mol of CaCO3 precipitated 22 released per mol of CaCO3 precipitated and equal to ∼0.6 for the representative tropical seawater used here ; however, Ψ can vary between approximately 0.5 and 0.7 in tropical reef systems depending on temperature, salinity, and pCO2pCO2 are in constant proportion to changes in dissolved CO2 gas concentration pCO2, pH, or Ωar on the ratio of metabolic to wave forcing, reef flat dimensions, and system frictional resistance is provided in To calculate the ratio of metabolic forcing to wave forcing, it is first necessary to establish a representative benthic flux on the reef flat lculate  . For exalculate  ; therefos curves  and gives curves , it is ms curves . TherefopCO2 were highly dependent on the depth and width of the reef flat. However, backreef hr = 0.5) deviated positively from the relationship defined by other simulations where Changes in water column carbonate chemistry simulated by the three-dimensional model were in close agreement with predictions made by the analytical one-dimensional model. fication . Backreenchanged . Finally on  or  .pCO2. For example, changing the ratio of Gnet∶P by ±100%  caused pCO2, pH, and Ω in most coral reef systems. However, net calcification does appear to play an influential role in driving the net offset pCO2, pH, and Ω over time scales of a day or more. Results from the central case model indicated that the uptake of CO2 by net production appear to balance the release of CO2 by net calcification over a 24-hour period such that Gnet∶P to 40% increased the 2 source was over-riding the ability of reef flat net autotrophy to act as a CO2 sink . This is largely due to the influence of the data from Bora Bay exhibiting 2 uptake measured in that reef system –660+610 or just +30 mmol CO2 m−2 d−1; a value consistent with prior estimates made for coral reefs worldwide . These changes are modest in comparison with the hundreds of µatm increase in pCO2 anticipated to occur over the 21st century.Our simulations focused mainly on coastally bound reef-lagoon systems with modest lagoon sizes  the release of CO2 from net community calcification being mostly offset by the uptake of CO2 from net community production, and 2) physically constrained limits on wave-driven circulation, the length and depth of the reef flat, and the overall frictional resistance of the entire reef system. More importantly, our results demonstrate that it is not possible to properly interpret observed spatial and temporal variations in water column carbonate chemistry, nor to determine the significance of these observations with respect to reef systems in general, without adequate hydrodynamic and morphological data. We suggest that future field studies and monitoring programs aimed at tracking the in situ impacts of ocean acidification on coral reef systems collect such physical information in addition to detailed measurements of water column chemistry. The analytical model we present here thus provides an additional tool to advance these efforts in wave-driven coral reef systems worldwide.The main objective of the present study was to demonstrate and to better understand how spatial and temporal variation in water column carbonate chemistry are influenced by the different physical and biogeochemical attributes of a reef system acting in combination, not just by rates of benthic metabolism alone. Our results combined with data in the literature allow us to conclude the following: Firstly, most of the changes in carbonate chemistry occur during the transit of water across the reef flat, although these changes can be further augmented by particularly high rates of metabolism in shallow lagoons. Secondly, changes in carbonate chemistry are as sensitive to the combined length and depth of the reef flat as they are on the ratio of metabolic to wave forcing; however, they are much less sensitive to variations in channel morphology and to the overall frictional resistance of the reef system. Thirdly, the long-term (weeks to months) net offset in water column Appendix S1Derivation of wave setup formulation.(DOC)Click here for additional data file.Appendix S2Derivation of cross-reef transport formulation.(DOC)Click here for additional data file.Appendix S3Bottom current drag formulations.(DOC)Click here for additional data file.Appendix S4Net production and net calcification parameterizations.(DOC)Click here for additional data file.Movie S1Time-dependent changes in the depth-averaged Total Alkalinity (TA) of reef waters relative to offshore waters over a 24-hour period.(WMV)Click here for additional data file.Movie S2Time-dependent changes in the depth-averaged Dissolved Inorganic Carbon (DIC) of reef waters relative to offshore waters over a 24-hour period.(WMV)Click here for additional data file.Movie S3Time-dependent changes in the depth-averaged p2 of reef waters relative to offshore waters over a 24-hour period.CO(WMV)Click here for additional data file.Movie S4Time-dependent changes in the depth-averaged pH of reef waters relative to offshore waters over a 24-hour period.(WMV)Click here for additional data file.Movie S5Time-dependent changes in the depth-averaged aragonite saturation state of reef waters relative to offshore waters over a 24-hour period.(WMV)Click here for additional data file.Table S1Variation amplitude and time-average difference in depth-averaged Total Alkalinity (TA) between reef waters and offshore waters over a 24-hour period.(DOC)Click here for additional data file.Table S2Variation amplitude and time-average difference in depth-averaged Dissolved Inorganic Carbon (DIC) between reef waters and offshore waters over a 24-hour period.(DOC)Click here for additional data file.Table S3Variation amplitude and time-average difference in depth-averaged p2 between reef waters and offshore waters over a 24-hour period.CO(DOC)Click here for additional data file.Table S4Variation amplitude and time-average difference in depth-averaged pH between reef waters and offshore waters over a 24-hour period.(DOC)Click here for additional data file.Table S5Variation amplitude and time-average difference in depth-averaged aragonite saturation state between reef waters and offshore waters over a 24-hour period.(DOC)Click here for additional data file.