There was a low (median of 8.2 %) overlap in the isotopic niches of sympatric species; in addition, host identity accounted for over 75% of the dissimilarity in both δ13C and δ15N values and microbiome community structure among individual samples within a site. There was also a strong phylogenetic signal in both δ15N values and microbial community diversity across host phylogeny, as well as a correlation between microbial community structure and variation in δ13C and δ15N values across samples. Together, this evidence supports a hypothesis of strong evolutionary selection for ecological divergence across sponge lineages and suggests that this divergence is at least partially mediated by associations with microbial symbionts.Marine sponges host diverse communities of microbial symbionts that expand the metabolic capabilities of their host, but the abundance and structure of these communities is highly variable across sponge species. Specificity in these interactions may fuel host niche partitioning on crowded coral reefs by allowing individual sponge species to exploit unique sources of carbon and nitrogen, but this hypothesis is yet to be tested. Given the presence of high sponge biomass and the coexistence of diverse sponge species, the Caribbean Sea provides a unique system in which to investigate this hypothesis. To test for ecological divergence among sympatric Caribbean sponges and investigate whether these trends are mediated by microbial symbionts, we measured stable isotope ( Associations with microbial symbionts allow their hosts to exploit novel pools of nutrients and expand into ecological niches that would otherwise be inhospitable , 2. The The expansion of host metabolic capacity is likely a crucial feature of these interactions , but thesensu ) among Caribbean sponges that have variable associations with microbial symbionts, these studies are limited to specific locations or to only a few species, and have neither quantitatively tested for metabolic divergence across host phylogeny nor determined the relationship between microbial community structure and δ13C and δ15N values  concentration) was quantified in sponges from sites in Honduras, Panama, and the Florida Keys as in [a [g dry sponge tissue]−1. Scopalina ruetzleri samples were not analyzed for chl a because they were too small to provide tissue for both isotope and chl a analyses.Stable isotope values  that was attributed to host identity, collection site, and microbial abundance.Statistical analyses and visualizations used the R packages  picante picante , vegan [e [vegan , RVAidMedMemoire , and ggp[ggplot2 . To test[ggplot2  between in vegan . We inclin vegan . The PERδ13C and δ15N values to measure and visualize the isotopic “niche” of each species and calculate the overlap of this isotopic “niche” with those of other sympatric species within that site [δ13C and δ15N) plots with isotopic niches represented as standard ellipse area (SEAc) according to [Ecological divergence within a site was also calculated by using intraspecific dispersion in hat site , 53. Thirding to . The mearding to , 54 thesvegan package [The alpha diversity of the microbial community within each sample was calculated as the observed richness (S), Shannon index (H′), and inverse Simpson’s index (D) using the  package .δ13C and δ15N values, chl a concentration, ASV richness (S), Shannon index (H′), inverse Simpson’s index (D), and elemental composition . To do this, we used mean values of species that were well represented in sampling across sites within at least two regions of the Caribbean. Monanchora arbuscula was not included in analyses for S, H′, and D due to low microbiome sample size. Bayesian phylogeny of sponge species was constructed using sequences from the small ribosomal subunit (18S) and the large ribosomal subunit (28S) downloaded from Genbank to assess genetic relatedness using methods similar to previous research [picante. This analysis assesses whether more closely related organisms possess more similar traits; higher values for K (Blomberg’s K) indicate trait patterns that are strongly linked to the evolutionary history of the organisms and low K values indicate trait patterns shaped by stochastic changes over evolutionary time [To investigate ecological divergence across host phylogeny, we tested for a phylogenetic signal in research ; see Supary time .δ13C and δ15N) of sponge tissue, as well as between the mean δ13C and δ15N values of sponge tissue and microbiome community richness and diversity, we used linear regressions. In addition, we used an analysis of variance (ANOVA) to test for differences in the δ13C and δ15N values of sponge tissue between HMA and LMA groups. Both of these analyses were carried out in JMP (Ver 14).Mantel tests were used to identify correlations between dissimilarity patterns in microbial community structure (Bray–Curtis dissimilarity) and isotope values (Euclidean distance). To test for relationships between elemental composition  and isotope values . Although δ13C and δ15N values varied between HMA and LMA groups, overall microbial abundance (HMA vs. LMA) accounted for only 20% of the dissimilarity among individual samples from across the Caribbean  and from 5 to 64% of the dissimilarity in δ13C and δ15N values at individual sites ; the reC. vaginalis) to 449 (in M. laevis). Only ten phyla had an average relative abundance of at least 1%  and diversity (Shannon index: range of 1.03 for I. birotulata to 4.72 for A. cauliformis and Inverse Simpson’s Index: range of 1.42 for I. birotulata to 65.36 for A. cauliformis) among these sponge species  by a single ASV . Collection site was significantly but weakly related to community structure , and host species and site exhibited a significant interaction . Microbial abundance classification (HMA or LMA) accounted for ~19% of the dissimilarity in microbial community structure across all samples . There was also a significant effect of host identity within individual sites or geographic regions, with a range of 79–88% (PERMANOVA) of the dissimilarity in microbial community structure across samples being driven by host identity (Table P ≤ 0.05 with FDR correction) except for C. vaginalis and E. ferox in Belize (P = 0.10) and A. cauliformis and A. crassa at Isla Pastores in Panama (P = 0.06). In contrast to host identity, microbial abundance (HMA vs. LMA) accounted for less of the dissimilarity in microbial community structure within sites  at the level of the Caribbean, with 67% of the dissimilarity in microbial community structure across all samples explained by host identity , with evidence of divergence in δ15N values across lineages of the subclasses Verongimorpha and Keratosa  and species in the subclass Heteroscleromorpha  , with generally elevated chl a values in members of the subclasses Verongimorpha and Keratosa and lower values in the subclass Heteroscleromorpha , but there was only a weak phylogenetic signal for C:N  compared with those of microbiome diversity  Fig. . In contδ15N and δ13C values together , as well as with dissimilarity patterns in δ15N values  and, to a lesser extent, δ13C values . Mean δ13C and δ15N values of LMA sponges were higher than HMA sponges , but δ15N values were not influenced by microbiome richness   for C:N  of all species within a site were widespread across the δ15N and δ13C space, individual sponge species generally had narrow isotopic niches, with a median of <10% overlap between coexisting species [δ15N and δ13C values provides evidence of broad-scale partitioning of resources [Our results show evidence of ecological divergence among coexisting sponge species on Caribbean reefs. On individual reefs and across the Caribbean, host sponge identity was the strongest determinant of dissimilarity in  species , 53. Theesources , 40. Ouresources , 30, 57,esources , 33. In esources , 40 thatesources –60.V. rigida, A. crassa, and Aplysina spp.), but at the other end of this continuum were I. birotulata, C. vaginalis, and A. compressa. In all three of these species, a single, unique ASV dominated the microbial community at all collection sites in the Caribbean. Striking host specificity in microbial community structure and diversity in Caribbean sponges [Coexisting species also had distinct microbiomes , 26, witδ13C values are not tightly constrained across host phylogeny may therefore be driven by variation in the dependence of sponge species on spatially and temporally variable sources of organic carbon , with more closely related species having more similar patterns of diversity than would be expected under a Brownian motion model of evolution . Although photosymbiont abundance was also linked to the phylogenetic history of the sponge host, this trait is not always a reliable proxy for host sponge reliance on photosynthate , 31. Insand DOC; , 40, 61)δ13C values and the phylogenetic history of host sponges makes the trends in δ15N values from these same sponge samples even more striking. Microbial symbionts mediate the nitrogen cycle within sponges, allowing transformations like N-fixation, nitrification, denitrification, and anaerobic ammonium oxidation [δ15N values across host phylogeny within this study, but higher δ15N values are generally associated with trophic enrichment from heterotrophic feeding and consistently depleted δ15N (−2 ‰ to ~0 ‰) values in some species (members of the genus Ircinia in the current study) are indicative of biological nitrogen fixation by diazotrophic bacteria [δ15N trends across host species. For instance, nitrate release (as a proxy for nitrification) has been reported from in situ or laboratory-based experiments for some , but not all  sponges from this study [The lack of a strong correlation between xidation , 17. It bacteria . Nitrifibacteria , 62, 63,is study , 64.δ13C and δ15N values across host phylogeny are especially interesting considering pioneering and recent work focused on organic carbon as the limiting nutrient on Caribbean reefs [Our trends in an reefs , 37, 65.an reefs . This, aan reefs , 37. Unlan reefs , 67. Unian reefs . This, cδ13C and δ15N values differed between HMA and LMA groups, supporting the contention that sponges hosting abundant communities of microbial symbionts can more efficiently exploit and transform nutrients on these reefs [δ13C and δ15N values compared with host identity . Therefore, it is unlikely that microbial biomass had a substantial influence on the isotope values of bulk sponge tissue. Instead, we identified a relationship between δ15N values and broad trends in microbiome diversity, with the inverse Simpson’s index explaining about 30% of the variation in mean δ15N values across host species. In addition, at an even finer scale, microbiome dissimilarity was strongly correlated with δ15N and δ13C dissimilarity across host sponges. Associations with specific symbionts can drive divergence in host resource use in terrestrial [δ15N values across species were stronger than between δ13C alone or δ15N and δ13C together, supporting trends from our phylogenetic signal analysis that show divergence in nitrogen metabolism due to associations with microbial symbionts. Thus, although δ15N values of bulk sponge tissue are at least partially influenced by microbial biomass, our data also provide evidence that different sponge lineages are obtaining nitrogen in fundamentally different ways and that this variation is coupled to microbiome community structure. These results act as an important reminder that carbon is not the only nutrient shaping sponge holobiont evolution, and as our understanding of the complex nutrient cycling  within and across sponge species increases [se reefs , 10, 24.restrial , deep-serestrial , and correstrial . On corarestrial , 29, 59,ncreases , it is lδ13C and, to a lesser extent, δ15N can be influenced by variation in the biochemical  or structural  composition of tissue [δ13C and δ15N values . Thus, it is unlikely that the interspecific trends we observe in our δ15N and δ13C values are being strongly influenced by variation in the composition of sponge tissue [The values of f tissue . Althouge tissue –74.The high biomass and successful coexistence of diverse sponge species with different life history traits  has been cited as evidence of a lack of resource limitation on Caribbean reefs, as no single sponge type has gained a competitive advantage , 37, 65.Based on our observations, we posit that, over evolutionary time, the acquisition of novel symbiont taxa (or perhaps even shifts in the abundance of specific taxa shared across particular sponge species) allowed Caribbean sponges to exploit novel resources and expand into available niche space afforded by a combination of diverse sources of organic carbon and reduced competition with reef-building corals compared with other ocean basins , 61. ThiASV AbundanceSupplementary Material


DNA originating from degenerate tumour cells can be detected in the circulation in many tumour types, where it can be used as a marker of disease burden as well as to monitor treatment response. Although circulating tumour DNA (ctDNA) measurement has prognostic/predictive value in metastatic prostate cancer, its utility in localised disease is unknown.n = 189), we identified the presence of ctDNA TP53 mutations in preoperative plasma in a retrospective cohort and determined its association with biochemical- and metastasis-free survival.We performed whole-genome sequencing of tumour-normal pairs in eight patients with clinically localised disease undergoing prostatectomy, identifying high confidence genomic aberrations. A bespoke DNA capture and amplification panel against the highest prevalence, highest confidence aberrations for each individual was designed and used to interrogate ctDNA isolated from plasma prospectively obtained pre- and post- (24 h and 6 weeks) surgery. In a separate cohort .Tumour variants in ctDNA were positively identified pre-treatment in two of eight patients, which in both cases remained detectable postoperatively. Patients with tumour variants in ctDNA had extremely rapid disease recurrence and progression compared to those where variants could not be detected. In terms of aberrations targeted, single nucleotide and structural variants outperformed indels and copy number aberrations. Detection of ctDNA CtDNA is uncommonly detected in localised prostate cancer, but its presence portends more rapidly progressive disease. The majority of men diagnosed with localised prostate cancer are effectively cured with surgery and/or radiation therapy ; howeverA number of molecular biomarkers for use on primary prostate cancer tissue are already in clinical use  across the  disease . We founn = 189), patients were selected from a prospectively collected and clinically annotated institutional bio-repository [Two separate patient cohorts were used for this study. For tumour-based WGS analysis (WGS cohort), we prospectively enrolled 10 consecutive patients presenting to a single surgeon with clinically localised prostate cancer suitable for prostatectomy with curative intent. For the TAm-Seq analysis  for probe design. A single probe was selected for small variants, while SVs were targeted using a probe from each side of the rearrangement junction. For indels, a probe adjacent to the variant was selected  and stored at − 80 °C until analysis. Circulating DNA was extracted using the QIAamp Circulating Nucleic Acid Kit , and DNA quality was assessed using an Agilent BioAnalyzer and quantified using the Qubit method .For each patient, blood was collected from up to three timepoints in EDTA tubes for ctDNA analysis; sample (a) was taken pre-surgery, sample (b) 24 h post-surgery and sample (c) 6 weeks post-surgery. Immediately after collection, plasma was collected following a double spin . Adapters containing UMIs enable detection of genuine, very low-frequency variants through the reduction of background noise in sequencing data. Due to the small fragment length of the starting material, the DNA shearing step of the TST170 protocol was omitted. Libraries were enriched with the custom panel then sequenced as paired-ends (2 × 150 bp) plus indexing reads on NextSeq High output. Five or six samples were loaded per flow cell to achieve ~ 40,000x raw depth per sample.Samples were processed using an Illumina R&D analysis pipeline to take advantage of the UMIs incorporated during library preparation. Reads were grouped into read families by alignment to the hg19 human reference sequence followed by collapsing and stitching into unique fragments utilising start and stop positions, UMI barcodes and the paired read (where present) to generate collapsed BAM files. These collapsed families of reads are low noise and high confidence. Positions targeted by the panel were reviewed using Integrative Genomics Viewer  to confirm the presence or absence of the targeted variants.TP53 gene were combined for use in eight separate PCR reactions per patient. Input was 2 ng of DNA, which was added to a PCR reaction mix composed of 2 μl of primer (0.5 μl ea. @ 10 μM) and 10 μl Q5 DNA pol 2X master mix made up to 20 μl with MQ water. The reaction was run on a ViiA 7 PCR machine with the following settings: initial denaturation at 95 °C × 1 min, then cycle denaturation at 95 °C × 10s following by annealing at 58 °C × 1 s and then extension at 72 °C × 18 s for 38 cycles. Amplicons were then combined into two pools of non-overlapping products and cleaned with the Qiagen QIAquick PCR purification kit following the manufacturer’s instructions. DNA was quantified using a nanodrop and 100 ng of DNA of each pool end repaired and adaptor ligated using NEBNext Adaptors for Illumina according to the manufacturer’s instructions. Following a AMPure XP bead clean-up, a second round of PCR amplification was performed in a total reaction volume of 50 μl consisting of 20 μl of adaptor ligated DNA fragments, 25 μl Q5 2X master mix, 2.5 μl index primer (10 μM) and 2.5 μl universal PCR primer (10 μM). PCR amplification was then performed in a thermocycler under the following conditions: initial denaturation at 98 °C × 30 s, then cycle denaturation at 98 °C × 10 s following by annealing/extension at 65 °C × 75 s for 8 cycles, with a final extension at 65 °C for 5 min. The reaction mixture was then cleaned again using AMPure XP beads following the manufacturer’s instructions at a ratio of 0.9:1 beads to ligation mixture. The pool was then quantified and quality checked on a Bioanalyzer  before sequencing on a MiSeq (Illumina Inc.). A detailed description of the experimental procedure is provided as supplementary information . For amplicon production, forward and reverse primers  population frequency greater than 1%, a set of high-confidence variants was selected based on having a minimum filtered depth of coverage of 500 reads, a minimum log Bayes factor of 6  and a cohort frequency of less than four samples. The predicted pathogenicity of detected variants was assessed using Varsome [For each sample in the study, paired-end DNA sequencing reads (length 151 bp) were aligned to the human genome reference hg19 using BWA mem (version 0.7.17). Reads were assigned to their corresponding PCR amplicons based on their alignment coordinates. To be considered for further analysis, reads were required to have a minimum alignment length of 75 bp, a minimum overlap of at least 75% of their target amplicon and no more than 2 bp edit distance from the reference genome. Using the filtered set of reads, the frequency of each DNA base was counted at each genomic locus within the target PCR amplicon regions for each sample. Using the control samples, at each locus, a null model was computed to represent the expected distribution of allele counts when no variant is present. Allele count data was modelled (with add-one smoothing) using the Dirichlet Multinomial distribution with best hyperparameter computed by maximising the log-likelihood function using the Newton-Raphson method. For each locus in the case samples, the allele count was compared with the null model. The weight of evidence against the null was measured by the Bayes factor B. The prior distribution of the count data of the case samples was given by a Dirichlet distribution on a 3-simplex, with all pseudocounts set to ½ . Variant allele frequencies (VAF) were computed using the maximum a posteriori (MAP) estimate on the posterior distribution marginalised to the component representing the non-reference allele. After filtering out any variants with gnomAD  as appropriate. No patient had evidence of metastatic disease at the time of surgery.Paired tumour and germline WGS was completed on eight patients, the clinical and pathological characteristics of whom are shown in Table The number of genomic aberrations identified by tumour WGS per patient is summarised in Additional file Changes in specific variants identified in the ctDNA in patients 11196_3 and 11201_4 over time are shown in Figs. n = 189), focussing on TP53, one of the most commonly mutated genes in prostate cancer, and one that is positively enriched for in metastatic disease (including in patient 11196_3) [TP53 ctDNA variants was identified in 22 cases (12%), with no variants detected in the remainder . In contrast, metastasis-free survival (MFS) was significantly shorter in patients positive for a variant . Similar results were observed using a contingency analysis  , 30. As 1196_3 , ant Fig. b, with mAlthough ctDNA is reported to be relatively abundant in metastatic prostate cancer, at the time of initiation of this study, it was unclear if ctDNA could be detected in patients with localised disease, as well as the clinical implications of its presence. A number of early studies identified an association between total cell-free plasma DNA concentration and the presence of cancer and biochemical recurrence post prostatectomy –33; howeHere we have been able to identify ctDNA in two of eight patients with localised disease using a personalised approach based on individual whole-genome sequencing followed by deep-targeted sequencing of selected variants in plasma cell-free DNA. Furthermore, in a retrospective analysis of patient samples (enriched for high-risk patients) using the previously described TAm-Seq method to tile across a single gene, we have been able to detect ctDNA in 22 out of a further 189 patients. Importantly, we were able to demonstrate that detection of ctDNA in the pre-prostatectomy setting has prognostic implications and is associated with more rapid progression to metastatic disease in both analyses. This is of clear clinical relevance, given that MFS is a strong surrogate of overall survival in localised prostate cancer .Despite differences in sequencing and analysis methods between the studies, at least for the individual targeted analysis, it is likely that clinical differences between the cohorts are the basis of the observed discrepancy. For instance in the Hennigan study, although 8/9 patients who underwent tissue and plasma cell-free DNA sequencing had locally advanced and/or high grade tumours, only one patient experienced a biochemical recurrence with 24 months of surgery, and no patients were reported to progress to metastasis. In contrast, from our cohort, three patients had primary PSA persistence, and a further two patients experienced biochemical recurrence within 24 months. Of these, ctDNA was only detectable in 2 patients, both of whom had primary PSA persistence and very rapid disease trajectories, characterised by early progression to overt metastatic disease and death. Patient 11196_3 had a moderately sized tumour, with only ISUP grade group 3 disease at prostatectomy, but was radiorecurrent, with extensive extracapsular extension and invasion into the seminal vesicle. Prominent lymphovascular invasion, a pathological feature associated with aggressive disease , was alsTP53, mutations of which are enriched in patients with metastatic disease, we were able to identify ctDNA in just under 12% of patients, where it again was associated with the development of metastatic disease. Clearly, all patients with potentially detectable ctDNA levels were not identified, as many patients who were TP53 ctDNA negative also experienced metastatic progression post-operatively. Extending the number of variants assessed will likely increase test sensitivity [A key finding in this study is the consistent association between pre-treatment ctDNA detection and clinical outcomes, which may serve as a useful adjunct to select patients for treatment intensification at the time of diagnosis. Creating a custom panel based on sequencing of the primary tumour and identifying individual-specific variants may be the most sensitive way of positively identifying ctDNA in patients, but outside of particularly invested centres, its use as a clinical strategy is limited by the cost and turn-around time. An alternative approach is to target common variants identified in a landscape-type analysis . We weresitivity , but forTP53 mutation is a relatively frequent driver, has been shown to be a common source of variants detected in deep cell-free DNA analyses and ideally should be controlled for by concomitant sequencing of white blood cell DNA which we have not performed [TP53 mutations in localised prostate cancer [There are a number of limitations to our study that warrant some consideration. Undiagnosed clonal haematopoiesis of indeterminate potential (CHIP), of which erformed , 39. Howerformed . Enrichme cancer , 21. Howe cancer , such imIn summary, using two separate cohorts and two different approaches, we have found that ctDNA can be identified in patients with clinically localised prostate cancer and is associated with a significantly shorter time to metastatic progression. Efforts to improve levels of detection, specificity and universal applicability of ctDNA detection in this setting are warranted.Additional file 1: Supplementary Tables S1, S3–5. DNA samples analysed and variants identified by WGS; TP53 variants identified by TAm-Seq and distribution of variants by clinical outcome.Additional file 2: Supplementary Table S2. Genomic variants targeted for deep sequencing.Additional file 3: Supplementary Figure S1-S3. Probe design strategy for panel sequencing and primer/amplicon map for TAm-Seq; clinical outcomes of additional patients.Additional file 4: Supplementary Methods. Detailed description of TAm-Seq methodology.


Slc6a8 gene showing reduced brain creatine and cognitive deficiencies or impaired motor function. We present a new knock-in (KI) rat model of CTD holding an identical point mutation found in patients with reported lack of transporter activity. KI males showed brain creatine deficiency, increased urinary creatine/creatinine ratio, cognitive deficits and autistic-like traits. The Slc6a8Y389C KI rat fairly enriches the spectrum of CTD models and provides new data about the pathology, being the first animal model of CTD carrying a point mutation.Creatine is an organic compound used as fast phosphate energy buffer to recycle ATP, important in tissues with high energy demand such as muscle or brain. Creatine is taken from the diet or endogenously synthetized by the enzymes AGAT and GAMT, and specifically taken up by the transporter SLC6A8. Deficit in the endogenous synthesis or in the transport leads to Cerebral Creatine Deficiency Syndromes (CCDS). CCDS are characterized by brain creatine deficiency, intellectual disability with severe speech delay, behavioral troubles such as attention deficits and/or autistic features, and epilepsy. Among CCDS, the X-linked creatine transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different mouse models of CTD were generated by doing long deletions in the  Cr is taken from the diet or endogenously synthetized in a two-step pathway by the enzymes AGAT (arginine-glycine amidinotransferase) and GAMT (guanidinoacetate methyltransferase), and specifically transported into the cells by SLC6A8 2. Guanidinoacetate (GAA) is synthetized from arginine (Arg) and glycine (Gly) in the first step, and it is methylated generating Cr in the second step. Cr breaks down spontaneously in creatinine (Crn).Creatine (Cr) is a nitrogenous organic acid used as phosphate energy buffer to recycle ATP by the Cr-PCr system; Cr is also one of the main osmolytes of the brain, and suggested to play a role as neuromodulator or neurotransmitter6. CCDS are characterized by brain Cr deficiency, intellectual disability with severe speech delay, behavioral abnormalities such as attention deficits and/or autistic features, and seizures for GAMT and SLC6A8 deficiencies7. Among CCDS, Cr transporter deficiency (CTD) is caused by loss-of-function mutations in the SLC6A8 gene .Deficits in endogenous synthesis or transport of Cr cause Cerebral Creatine Deficiency Syndromes (CCDS)16. In particular, CrT knock-out (KO) mouse males with ubiquitous deletions of the exons 2–416 or 5–714 showed brain creatine deficiency and decreased body weight, features present in CTD patients17. Ubiquitous CrT KO males with exons 5–7 deleted also showed impaired motor function12. Cognitive deficits with normal sociability were demonstrated in the ubiquitous KO males with deletions in exons 2–416 and 5–714. Deletion in exons 2–4 also caused some changes in monoamine neurotransmitters correlating with higher resilience to stress, while deletion in exons 5–7 leaded to more stereotypies, impaired working memory performance and some brain alterations associated with aging. Interestingly, while ubiquitous deletion of the exons 2–4 after tamoxifen induction in neonatal and adult mice led to brain Cr deficiency and lower body weight, cognitive deficits were only present if the induction was during the neonatal period highlighting the important role of Cr during brain development10.To better understand the pathology and with the ultimate goal of finding a treatment, several mouse models for CTD have been generated11 or 5–718 in CNS nestin-positive cells exhibited brain Cr deficiency but normal Cr levels in periphery. Brain CrT KO of exons 2–4 mice maintained the same learning and memory deficits as the corresponding ubiquitous KO, plus hyperactivity and normal body weight11. In contrast, brain CrT KO of exons 5–7 mice showed a rescue in the general phenotype  but declined at early age, correlating with some brain alterations related with aging18. Deletion of CrT exons 2–4 in CaMKII-positive cells (excitatory neurons)15 revealed similar phenotype as the nestin-targeted KO mouse, while targeting only DAT-positive cells (dopaminergic neurons) exhibited hyperactivity19.Brain-specific KO mice of CrT have shown additional and somehow different phenotypes. Deletion of exons 2–417. Further studies are thus needed to fully understand CTD pathology, and to allow, through the use of various complementary animal models, the best translation to human pathology20.All the accumulating knowledge using these various mouse models highlights the importance of the Cr transporter in brain function and physiology, and allows a better understanding of SLC6A8 deficiency. However, CTD patients do not always present the same phenotype22. In order to increase the fundamental knowledge and to increase the relevance of preclinical studies with complementary preclinical models, we generated a novel rat model of CTD: the Slc6a8Y389C knock-in (KI) rat line. Slc6a8Y389C KI rat holds a point mutation found in CTD patients which was reported to completely abolish Cr transporter activity7. Here we show the first analysis of Slc6a8xY389C/y rat KI males as model of CTD, being the first KI animal model of CTD.As an alternative to mouse models, rats have a number of potential advantages over mice. In particular, they are closer to human physiology, biochemistry and behavior in comparison with miceSLC6A8 c.1166A > G; p.(Tyr389Cys) point missense mutation reported as completely abolishing Cr transporter activity17  rat line  and 3 heterozygous Slc6a8xY389C/x female founders were generated from the Sprague–Dawley (OFA-SD) rat strain using CRISPR/Cas9 technology  or KI males and wild type females or heterozygous females generated litters with the expected Mendelian ratios and with the 5 possible genotypes: . DNA was obtained from ear punch using the KAPA BIOSYSTEMS kit (ref. KK7352) and finally diluted in nuclease-free water. Amplification primers were: for WT fragment (250 bp) F-WT/5′-GGTCTAGCCTTCATTGCCTA-3′ and R-WT/5′-GCCCTCCACACCTACAAACT-3′; for KI fragment (211 bp) F-KI/5′-ACTCTGGCATGAGACCCTGT-3′ and R-KI/5′-CAGTGTGACAGCCCGTGGGC-3′. PCR conditions were as follows: 10 min 94 °C, 2x, 36x, 10 min 72 °C, store at 4 °C. A representative PCR of the 5 possible genotypes is shown in Fig. Animals were maintained under a 12 h/12 h light–dark cycle. Food and water were available ad libitum. All experiments were performed with the approval of the veterinary authorities of the Canton de Vaud  and in accordance with the regulations of the Swiss Academy of Medical Science, as well as followed the ARRIVE Guidelines 2.0. Efforts were made to minimize stress and number of animals used.1H-MRS 3 months-old rats were anesthetized with isoflurane  using a nose mask under 70% compressed air and 30% O2. Body temperature (kept at 37.5 ± 1.0 °C using circulating warm water) and respiration were monitored continuously by a small-animal monitor system . In vivo 1H-MRS was performed on a horizontal actively shielded 9.4 T system  interfaced to a Varian Direct Drive console . A home built 1H-quadrature surface coil (14 mm diameter) was used as transceiver and placed over the head of the animal. The volumes of interest (VOI) were positioned in dorsal hippocampus , striatum  and cerebellum  using axial and sagittal anatomical T2 weighted images . The static magnetic field homogeneity was adjusted using first and second order shims by FAST(EST)MAP leading to water linewidths of 10–12 Hz for hippocampus and striatum and 14–16 Hz for cerebellum23. Localized spectra were acquired with SPECIAL spectroscopy sequence  to obtain the specific neurochemical profile in the 3 mentioned VOIs25. Metabolite concentrations were estimated using LCModel software26 and water as internal reference. The LCModel basis-set for spectral fitting contained a spectrum of macromolecules acquired in vivo27 and 17 individual metabolites measured in vitro: alanine (Ala), ascorbate (Asc), aspartate (Asp), glycerophosphocholine (GPC), phosphocholine (PCho), creatine (Cr), phosphocreatine (PCr), γ-aminobutyrate (GABA), glutamine (Gln), glutamate (Glu), glutathione (GSH), myo-inositol (Ins), lactate (Lac), N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), phosphoethanolamine (PE) and taurine (Tau). PCho and GPC were expressed as tCho (PCho + GPC) due to better accuracy in the estimation of their concentration as a sum. The ultra-short echo-time 1H-MRS allowed the detection of the abovementioned 17 metabolites24.For g for 7 min. Supernatant (plasma) was transferred in a new tube. Liquids  were stored at – 80 °C for further analysis.The same day of the sacrifices, 6 WT and 7 KI rats aged of 3 months were anesthetized with 4% isoflurane under 70% compressed air and 30% O2. Fur around the base of the head and the dorsal part of the neck was shaved with an electric shaver. A little incision was made in medial position and longitudinally through the skin and muscles at the base of the skull. The cavity was maintained opened using little separators and cleaned of blood using cotton swabs. Carefully, puncture in cisterna magna was made to collect CSF. After CSF collection, the rat was positioned in supine position and the abdominal cavity was opened to collect urine from the bladder. Finally, the thoracic cavity was opened and blood was collected transcardially with heparin (5000 u/ml) and centrifuged at 2710 × 3-Creatine, Sigma-Aldrich and 13C2-Guanidino acetic acid, VU Medical Center Amsterdam) containing 2.2% formic acid (v/v) was added to 20 µl of centrifuged plasma or urine. For CSF, 100 µl of 0.5 µM internal standard solution was mixed with 100 µl of CSF sample. The samples were loaded on a MCX µSPE plate  previously activated with MeOH and water, and then washed with formic acid 2% (v/v) and MeOH. Elution was obtained with 0.67 mol/l NH4OH solution on collection wells prefilled with acetonitrile. After centrifugation, samples were directly injected onto a LC–MS system (Waters I-Class UPLC system coupled with a Waters XEVO TQS micro mass spectrometer). Separation of the analytes was performed in a 4.5 min gradient at a flow rate of 0.5 ml/min on a HILIC column  maintained at 30 °C, with a 0.2% formic acid containing ammonium formiate (10 mmol/l):acetonitrile mobile phase gradient. Mass spectrometry was performed by electrospray ionization in positive mode using two SRM (Selected Reaction Monitoring) transitions  for each analyte. The m/z values of the selected quantitative and qualitative transitions, respectively, were the following: 118 → 100.85 and 118 → 30 for GAA, 120 → 103 and 120 → 31 for 13C2-GAA, 132.1 → 43.82 and 132.1 → 90 for Cr, 135 → 46.88 and 135 → 93 for d3-Cr. Quantified concentrations were calculated with TargetLynx (Waters Corp.). Creatinine (Crn) was measured in urine by a COBAS 8000 automate .Cr and GAA were quantified by liquid chromatography coupled to mass spectrometry (LC–MS) following a µSPE sample preparation protocol. For plasma and urine, 180 µl of a 0.28 µM aqueous solution of internal standards , Y-maze for spontaneous alternation and Social preference/Social memory (SP-SM) tests for the other one. Each cohort contained 3 to 4 litters in which WT and KI were littermates between them. The set of tests began at 12 weeks of age.Rats were introduced in a round (1 m diameter) open arena with black walls and floor made of black PVC for 10 min while spontaneous behavior was recorded with a videocamera. Grooming behavior was hand-scored blindly with the Observer XT software and cumulative duration and frequency of such behavior were analyzed. 10 WT and 10 KI males were used.A Y-shape maze of 50 cm arm length, grey walls and floor made of grey PVC in conditions of 4 lx in each arm was used. For the learning phase, the rat was introduced in the Y-maze for 10 min with one arm closed. After 4 h, the rat was located in the same starting arm and was allowed to explore all the arms for 5 min. EthoVision tracking software was used to track the time spent in each arm. Exploration time  and Recognition Index ((tN—tF)/(tN + tF)) were calculated from the cumulative time spent in the familiar (tF) and the new (tN) arms. 9 animals per group were used.An arena of 57 × 36 cm with grey walls and floor made of grey PVC in 4 lx conditions was used. For habituation, the rat explored the empty arena for 5 min. On next day, the rat was introduced in the arena with two identical objects for 5 min in the learning phase. After 4 h, the rat was allowed to explore again the arena for 5 min but one of the objects was changed for a different one. Both, new and familiar objects and the right and the left sides were randomized. All phases were recorded with a videocamera and exploratory behavior was hand-scored blindly with the Observer XT software. The cumulative duration times for exploring familiar (tF) and new object (tN) in testing phase were used to calculate Exploration Time  and Recognition Index ((tN − tF)/(tN + tF)). 8 WT and 9 KI males were used.The rat was introduced in the abovementioned Y-maze and allowed to explore the three arms opened for 8 min. EthoVision tracking software was used to track rat position and calculate the number of total entries in all the arms (nº entries in arm A + arm B + arm C) as well as the percentage of alternation ). 8 WT and 10 KI males were used.A three-chamber arena 80 × 34 cm with grey walls and floor made of grey PVC with 10 lx in the center was used for SP-SM test. A transparent cylinder made of PVC, 13 cm in diameter and with openings, was positioned in each lateral chamber. During habituation, a juvenile rat was located in one cylinder while an object was positioned in the other one, and the experimental male rat was allowed to explore only the center chamber for 5 min. During the social preference phase, doors were opened and the rat could explore all the chambers (with the juvenile rat or the object) for 10 min. After 2 h, the object was changed for another juvenile, and the experimental rat was allowed to explore both new and familiar juveniles for 10 min. Relative positions of the items (object and juveniles) were randomized. All phases were recorded with a videocamera and exploration behavior towards the object or the juveniles was hand-scored blindly with the Observer XT. Cumulative duration time exploring each item, the number of visits (frequency) per each item, and the first item to be explored in each phase were taken into account for the analysis. 9 WT and 10 KI were used.28. A Shapiro test was used to assess the normality of each sample. To address the significance between WT and KI groups or between each group with respect to the value expected by chance, Mann–Whitney tests were performed when normality was rejected while, for normal distributions, we used Bartlett tests to evaluate equality of variances and t-tests for equality of the means. Potential influence of litter variability was considered and discarded after conducting linear mixed-effect tests in which litter factor was modelled as a random effect (package lme429).All statistical analysis and graphs were conducted with R-3.5.110 of the ratio KI/WT concentrations of different metabolites in brain, a pseudocount (+ 0.001) was added to all concentrations before doing the ratio. Graphs were done using ggplot2 package30, while heatmaps were generated with gplots31.To calculate the log32).In the analysis of first decision for the SP-SM experiment, we evaluated different questions. First, we asked if there was a significant bias in the decision of exploring first one of the two possibilities. We modelled this question considering a Bernouilli variable with probability q = 0.5 (no preference), and estimating the probability of selecting one of the two choices with a binomial distribution of parameters q, N, k, where N is the number of animals tested and k the number of visits of one of the items. We tested if the preferred choice was significantly different than random performing a two-tail exact binomial test, or a one-tail exact binomial test when prior information existed . Finally, we tested if the preferences of KI and WT were significantly different between them using a Barnard’s CSM Test  missense mutation found in human patients and reported with no functional activity of the transporter17  rat was generated with the 1H-MRS. High magnetic field 9.4 T 1H-MRS spectra in hippocampus  and a significant decrease of Crn . We also measured GAA in urine, and show, very interestingly, that GAA/Crn ratio was also strongly and significantly elevated in Slc6a8xY389C/y KI males .CTD patients show an increased Cr/Crn ratio in urine due to the deficiency in functional Cr transporter present on kidney proximal tubular cells . We thus measured Cr and Crn in the urine of our Slc6a8xY389C/y KI males at around 3 months of age to see whether Cr and GAA concentrations were altered. We show a significant decrease of Cr and a significant increase of GAA concentrations in plasma of KI males in comparison to those of WT males   presented a significant reduced body weight gain just after weaning, as compared to WT males, showing for instance 56% of the body weight of their WT littermates at 14 weeks of age   concentrations were significantly increased in all the brain regions analyzed while other metabolites changed their levels only in specific brain regions . Similar results were obtained when analyzing cumulative exploration times and recognition index every 30 s from the beginning  test measures declarative memory. In Y-maze for spatial memory, both recognition index and total exploration time of the arms were similar between genotypes Fig. A. In NOR33. Additionally, to assess attention deficit in rodents, a task evaluating working memory is typically used. Thus, we carried out Y-maze for spontaneous alternation test in a different cohort of animals. Percentage of alternation index was used to compare working memory performance between genotypes and each genotype with the value of a random exploration . While there were no differences in the number of entries between genotypes, the percentage of alternation for Slc6a8xY389C/y KI males was significantly reduced in comparison with that of WT males, having similar value than the expected by chance  to evaluate the general sociability and Social Memory test (SM) to evaluate the interest in social novelty. For this analysis, we used the cumulative duration of the rat exploring an item  but not the object (+ 7.6%). These little changes of KI males resulted in a nearly significant reduction of the social exploration time along the two phases . Furthermore, the number of visits of KI rats towards the juveniles across the two phases was in general significantly lower than those of WT littermates but similar towards the object . These data may point towards an aversion of KI males in establishing the first contact with a social novelty.Regarding the social novelty paradigm, WT and Slc6a8Y389C knock-in rat holding an identical patient’s missense mutation abolishing CrT activity17. Slc6a8xY389C/y KI rat males are brain creatine deficient, and exhibit increased Cr/Crn urine ratio as well as neurological symptoms similar to those of CTD patients, such as cognitive deficits and some autistic-like traits.We present here the first analysis of a new in vivo model of CTD, the Slc6a8xY389C/y KI rat males by 1H-MRS to check whether they present the same pathognomonic signature as CTD patients. All the Slc6a8xY389C/y KI rat males analyzed showed the specific markers of CCDS and CTD: a strong reduction of Cr levels in the brain and a strong increase in urinary Cr/Crn ratio17. These results validate the Slc6a8Y389C rat line as a plausible model of CTD, being the first model of ubiquitous CrT deficiency confirming the two known biochemical signatures of CTD.Using similar techniques as in the clinic, we first analyzed the brain metabolic profile of 34. Given that plasma Cr is being filtered and lost in the urine due to CrT deficiency, we could expect that Cr levels in plasma were diminished and those of GAA, the precursor of Cr, were increased in an attempt to compensate Cr levels by AGAT feedback loop of up-regulation due to Cr deficiency (for this mechanism in periphery36 or for CNS37). Indeed, a tendency to decreased Cr levels and significant increased GAA were observed in the plasma of Slc6a8xY389C/y KI males. This finding is in agreement with the decreased levels of Cr also observed in the ubiquitous KO mouse males with exons 2–4 deleted16. These data contrast however with the low number of patients with available measurements of Cr and GAA in plasma showing normal or mildly elevated Cr levels as well as normal GAA17. Further studies are needed to unravel the possible causes of these differences between human and rodents.The increase in urine Cr levels reveals a functional deficiency in the reuptake of Cr from the primary urine, which is normally done at the renal proximal tubular cells by CrTSlc6a8xY389C/y KI males, suggesting that GAA/Crn ratio in urine may also be a valid biochemical marker for CTD when is found together with increased Cr/Crn ratio. This result might help to bring attention towards a CTD diagnosis in patients with suspected GAMT-D . New data from patients will help to confirm whether this double marker would be useful for CTD diagnosis. Whether GAA is also re-absorbed from primary urine in proximal tubular cells of kidney through transport by CrT (as we have shown in brain cells38) remains to be determined. Indeed, while Cr was decreased in plasma and increased in urine of Slc6a8xY389C/y KI males demonstrating CrT deficiency, GAA was increased in plasma but normal with a tendency to increase in urine.Furthermore, we present the first measurement of the GAA/Crn ratio in urine of an in vivo CTD model. This ratio was strongly and significantly elevated in 17 and all the ubiquitous CrT KO mouse models16, Slc6a8xY389C/y KI males presented lower body weight. We observed this difference already after weaning, but taking into account the degree of significance, it is likely that it extends from previous time points. This body weight gain issue is in agreement with reported early signs of CTD39.Consistent with CTD patientsSlc6a8xY389C/y KI males than in their WT littermates, Cr levels in the CSF were mildly higher or similar to those of WT ones. This is consistent with what was observed in CTD patients7, confirming that CSF does not always reflect what is happening in brain parenchyma, and that CSF can also be a useful diagnostic sample for the differential diagnosis between CTD and GAMT deficiency, in which Cr is decreased7. Interestingly, while WT males showed higher levels of Cr in plasma than in CSF, Slc6a8xY389C/y KI males exhibited similar levels in both liquids (60–70 µmol/l). Altogether, these data support the fact that the Cr pool in CNS does not fully depend on the peripheral one, owing to the low permeability of blood–brain barrier for Cr and to brain endogenous synthesis of Cr41.Although Cr levels in plasma and in brain were lower in Slc6a8xY389C/y KI males were accompanied by significant increases in Gln, NAA and NAA + NAAG. Given that these molecules are important osmolytes in CNS44, and that the magnitudes of both changes tend to be similar, it might hint at an osmolyte compensation mechanism by Gln, NAA and/or NAAG for the brain Cr deficiency. Other metabolites with a role in osmoregulation are taurine, myo-inositol (Ins) or choline47. Interestingly, the concentration of these metabolites change in some of the regions analyzed as well (see below). We have recently shown a similar potential mechanism of osmolytes compensation involving the same molecules in another pathological condition of CNS . As Gln, NAA and NAAG play other important roles in CNS, a line for future research is to establish whether their changes only compensate Cr deficiency osmotically, or are truly pathological.In every brain region analyzed, the strongly decreased Cr levels in brain parenchyma from S-adenosylmethionine which is involved in the synthesis of Cr from GAA48. Gln is a precursor for Glu, which is a precursor for GABA. Transporters for GABA and Tau can transport Cr or GAA40. It would thus be possible that alterations in Cr and/or GAA levels might affect GABA and Tau homeostasis and their concentrations. As described above Tau and total choline in hippocampus might also participate to an osmolyte compensation mechanism under Cr deficiency. Overall, these metabolic changes may suggest that endogenous Cr synthesis may also be affected, together with disturbances in neurotransmission in the hippocampus of Slc6a8xY389C/y KI males. Hippocampus is involved in motivation, emotion, learning and memory, and these changes could affect its functions and also promote epilepsy in CTD patients. In striatum, Slc6a8xY389C/y KI males showed an increase in Ins, osmolyte also involved in many other cellular processes. The decrease in lactate might reflect a change in striatal energy metabolism.Other metabolites changed differentially depending on the brain region analyzed. In hippocampus, neurotransmitters such as GABA, Glu and Tau increased together with total choline, a precursor for another neurotransmitter (acetylcholine), membrane phospholipids and, (via betaine) the methyl group donor Slc6a8xY389C/y KI males showed similar learning and memory performance in simple tasks about recognition and spatial memory  but failed in a working memory task . Higher exploration times of objects in both phases of NOR test without any advantage in declarative memory might reflect a deficit in cognition related with attention and working memory as well. These negative results in learning and memory performance contrast with the data from the ubiquitous CrT KO mice with exons 2–4 and 5–7 deleted, where deficits were present although with milder phenotype in NOR test around the same age in the last one16. Interestingly, these results showing normal recognition of an object or a location in KI males are aligned with data from CTD patients, as they are able to remember people, objects or locations but fail in the performance of more complex daily tasks and learning new abilities39. Performance of tasks with little or high level of complexity/difficulty can also be influenced by emotional learning and memory, and by other cognitive processes such as language or attention. Whether all these cognitive processes are affected and how, and whether they are playing a role in the cognitive features of CTD pathology are still open questions to be addressed in the future.These alterations in brain metabolites correlate with changes in behavior and cognition. In comparison with their WT littermates, 13 and with the presence of attention deficits observed in patients17. However, not all CTD patients, including the one sharing the c.1166A < G; p.(Tyr389Cys) mutation with KI males, are diagnosed with attention deficits17 . Interestingly, similar variability was present in the cohort of KI males in relation with working memory assessment, where although the average of the group showed significant reduction in the percentage of alternation, few animals presented values higher than the random expectation overlapping with the smallest values of WT males  are in line with the autistic phenotype of the CTD patient sharing the same missense mutation as Slc6a8xY389C/y KI males17 . Importantly, the Slc6a8Y389C rat line is the first animal model of CTD showing a difference in sociability. Further studies will be needed to unravel the nature of this difference.Besides intellectual disability and attention deficits, autistic-like behavior is another common symptom in CTD patients. We evaluated the presence of two core features of autism spectrum disorder: repetitive self-stimulating behaviors (like grooming for rats) and sociability. Slc6a8Y389C rat line is the first CTD model generated so far with one of the single nucleotide mutations described in CTD patients. Slc6a8xY389C/y male rats present characteristic features of CTD pathology and reveal the Slc6a8Y389C rat line as a valuable model of CTD, fairly enriching the spectrum of in vivo CTD models. Additionally, we have shown new data confirming what has been shown in patients and highlighting common aspects of CTD pathology. Future work will aim at further characterizing the molecular mechanism and the consequences of CTD in the Slc6a8Y389C rat line both in periphery and in CNS. Recent work demonstrated that several variants of CrT are expressed although misfolded, some of them being rescued with chemical chaperones thus opening new avenues for the treatment of CTD patients54. Using the Slc6a8Y389C rat line to develop new strategies of treatment for CTD, this chaperone approach will also be tested in this new in vivo model.In summary, this new Supplementary Information


Symbiotic dinoflagellate density and algal pigment concentration, photosynthetic yields, lipid concentrations and ratios and growth varied among the turbidity treatments, with corals exhibiting photoacclimation within low turbidity treatments. A range of physiological responses were observed within the high turbidity treatments (low light), including bleaching and changes in lipid levels and ratios. Most corals, except P. damicornis, were capable of adjusting to a turbidity treatment involving a mean light level of 2.3 mol photons m−2 d−1 in conjunction with a SSC of 10 mg L−1 over the 7 week period.Dredging increases suspended sediment concentrations (SSCs), causing elevated water turbidity (cloudiness) and light attenuation. Close to dredging, low light periods can extend over many days, affecting phototrophic epibenthic organisms like corals. To improve the ability to predict and manage dredging impacts, we tested the response of corals to an extended period of elevated turbidity using an automated sediment dosing system that precisely controlled SSCs and adjusted light availability accordingly. Replicates of four common species of corals encompassing different morphologies were exposed to turbidity treatments of 0–100 mg L Knowing when the hazard becomes a risk to sensitive organisms such as corals depends on understanding tolerance limits. For dredging, this knowledge can be used for management purposes and alert dredging proponents of water quality conditions when effects may occur allowing them to make changes to dredging schedules as required (i.e. reactive management10). The same understanding can be used with hydrodynamic and sediment transport models11 to predict the possible areal extent of impacts before dredging at the environmental impact assessment stage13.Elevated turbidity (water cloudiness) from natural resuspension events17. The key stressors or pressures associated with elevated turbidity include elevated suspended sediment concentrations (SSCs), sediment deposition as the sediments fall back out of suspension, and a decrease in the quantity and quality of benthic light through scattering and attenuation. These pressures can act either alone and/or in combination7.There are several different mechanisms (cause-effect pathways) whereby the increased turbidity from suspended sediments could affect the health of the underlying communities; for recent reviews for tropical organisms see18. These studies of several large-scale capital dredging projects in tropical Western Australia showed that within a few hundred metres of dredging the intensity, duration and frequency of turbidity events increased by 10–, 5– and 3–fold respectively compared to baseline (pre-dredging) levels. SSCs varied 2–3 orders of magnitude over a day and although occasionally exceeding hundreds of mg L−1, over longer periods (i.e. weeks) the near worst case scenario (95th percentile) was typically a few tens of mg L−1. The high turbidity levels profoundly affected underwater light levels with sites close to dredging frequently experiencing days in darkness and extended periods of very low light. The water quality data also indicated a strong power-decay relationship, with conditions improving with increasing distance from dredging19.Temporal and spatial changes in pressure fields associated with dredging activities have only recently been described in detailin situ water quality measurements of SSC, benthic light availability and estimates of sediment deposition were conducted in conjunction with observations of the health of hundreds of individually marked corals at 5–10 m depth. The corals were examined at roughly fortnightly intervals over an extended (200 d) dredging period. Threshold values for the probability of non-zero mortality of shallow water corals  over different running mean intervals were derived, suggesting strict thresholds  of ∼1 mol quanta m2 d−1 of photosynthetically active radiation (PAR) and SSCs of ~10 mg L−1 over a 14 day period. Although elevated turbidity and light reduction could be detected many kilometres away from the dredging (see21), biological effects occurred considerably closer to the dredging activities22.In one of these studies the ex situ laboratory-(aquarium) based studies that allow careful manipulation and even isolation of individual variables and more detailed testing of cause-effect pathways. To be relevant, such studies need to use environmentally realistic exposure conditions7, which is now possible using water quality information described above. Using this approach, Bessell-Browne, et al.23 examined the effects of three SSCs crossed with three different light levels on a number of coral species. The study emphasized the significance of the light attenuation associated with high turbidity. Exposure to low light for extended periods resulted in the dissociation of the symbiosis i.e. bleaching, but if light levels were manipulated to compensate for the light reduction, then corals could survive extremely high SSCs (i.e. 100 mg L−1) over extended periods. Sediment deposition and smothering of corals was prevented in the study, which effectively suggested it was the light attenuating properties of the suspended sediments that was the primary concern for corals, rather than the suspended sediments themselves. Following from this experiment, Bessell-Browne et al. (2017b) tested the physiological responses of light limitation alone on corals and found the EC10 threshold for bleaching over 30 days was 1.2–1.9 mol photons m−2 d−1.Another approach to developing guidelines is by 24. Although some capital dredging projects can last for several years, in practice spatial separation of excavation activities  often means that reefs may only be exposed to plumes for shorter periods. Throughout the study we use the term turbidity to refer to the various treatments which are effectively combinations of elevated SSCs with associated light reduction. The light levels tested and the SSCs (as mg L−1) associated with the light level were empirically derived (see7), and based on a shallow water tropical reef. The study was intended to experimentally support the initial guideline values developed from in situ studies of22, but also to quantify the broader physiological consequences, survivorship and energetic status of corals, including lipid status, growth, zooxanthellae densities and chlorophyll concentrations over extended exposure to turbidity. Smothering of corals by sediment was prevented in the study, thereby ensuring the results are specific to turbidity tolerance.In this study, we examine the chronic effects of turbidity on a range of corals and morphologies using a suite of sub–lethal indicators to quantify the detailed physiological effects of six different turbidity levels over an extended (7 week) period. The exposure period was chosen as it is the typical duration of maintenance dredging activities in coastal waters of the Great Barrier Reef−1 with the light levels in each treatment  adjusted to approximate light levels that would occur at 5–6 m depth at the SSC, based on light profiles collected during a dredging program and described in7 , and lower for treatments with ≤2.3 mol quanta m−2 d−1  treatments than the highest turbidity (lowest light) treatments with these two groups clearly delineated statistically  treatments, peaking at 6.3 mol quanta m−2 d−1/5 mg L−1, although there was substantial overlap among treatments  varied somewhat among treatments across all species and was generally lowest for the higher turbidity/low light treatments Fig. . There wDLI Fig. . For P. d−1 Fig. . For Porlly Fig. . For T. nts Fig. .Table 1RFv/Fm) of the algal symbionts decreased from the start to the end of the 7 week exposure period for all species  that even the 0.25 mol quanta m−2 d−1 treatment was lower than that with the highest efficiency , all coral species were much lighter  and was highest in the 2.3 mol quanta m−2 d−1/10 mg L−1 treatment  treatments having the lowest values across all species  complex dominating all coral species , but for Porites lobata/lutea and T. reniformis the concentrations consistently increased with increasing turbidity to 2 and 1.6 × the zero turbidity (12.6 mol quanta m−2 d−1) values respectively in the highest turbidity treatment  and 2 mg L−1 treatment (10.1 mol quanta m−2 d−1), and then systematically decreased with increasing turbidity  and storage lipids  were identified during the lipid analyses Fig. . Total lra Table . In all ent Fig. . The grenis Fig. . For allnis Fig. .Figure 622. During the dredging period, the 42-d running mean light and turbidity levels and at two sites at 7–9 m depth and few hundred metres from the dredging were calculated. The upper percentile (P) values18 were determined for turbidity and the lower percentiles determined for light. Similar analyses were conducted at reference sites 20–30 km north of the dredging at similar depths but which were not influenced by dredging-related sediment plumes26. The P95 of the sites a few hundred of metres away were 12–15 NTU, nearly an order of magnitude higher than at distantly located references >20 km away from dredging . Underwm−2 Fig. .Figure 727. This transition point occurs between the 2.3 mol quanta m2 day−1 (in combination with 10 mg L−1) and 0.25 mol quanta m2 day-(in combination with 30 mg L−1) treatment and provides experimental support for guidelines derived from combined in situ water quality sampling and coral health monitoring on a shallow, clear water reef system during a large scale capital dredging project22.Seven weeks of exposure to a range of elevated turbidity (i.e. a combination of elevated SSCs and associated light reduction) resulted in a range of physiological and biochemical shifts in the health of corals, across all the species examined. The most conspicuous effects of the turbidity treatments were the darker pigmentation of the corals in the low to middle turbidity treatments and the lightening and bleaching of corals in the highest turbidity (near zero light) treatment. When considered collectively, and despite some differences between species and growth forms, the suite of sublethal indices suggest acclimation and tolerance in the low to mid turbidity treatments and then a transition to light deprivation at the highest turbidity (zero light) treatment. Bleaching of corals under conditions of low light involved the dissociation of the coral-algal symbiosis which is a well-known stress response of coralsSymbiodiniaceae, LaJeunesse, et al.28) and concentration of their primary photosynthetic and accessory pigments. The majority of the pigments are associated with the peridinin-chlorophyll a-protein (PCP) complex30 including chlorophyll a (with absorption greatest at 435–440 nm and 670–680 nm), chlorophyll c2 (450–460 nm) and the carotenoid, peridinin (478–500 nm)31. For all species the deep brown pigmentation was caused by high pigment concentration per algal cell and for A. millepora, P. damicornis and Porites lobata/lutea by higher symbiont density. The higher algal pigment concentration is consistent with the photoacclimatory responses described previously for dinoflagellates33 and in symbiotic dinoflagellates in symbiosis with corals, is considered to be a mechanism to increase light absorptivity under progressive light limitation39.The caramel brown colour of corals, in the absence of strong host pigments, epilithic algae and colour in the skeleton, is a product of both the density of the symbiotic dinoflagellates (family T. mesenterina (see below). Some studies have reported no change in algal density under light reduction i.e.41, whereas others have clearly shown an increase in areal specific symbiont density in more turbid, lower light environments42. The discrepancy might be due to different methodologies used i.e. in situ sampling of colonies down light gradients versus ex-situ laboratory-based manipulative studies, or sampling techniques using whole colonies versus exterior branches. It could also be due to differences in experimental durations as increases in algal density takes several weeks to months43 as opposed to algal pigment changes that can occur within days39.The dark brown colouration was also caused by higher algal density in all species except T. reniformis explants still retained ~50% of their algal symbionts. Despite the heavy bleaching there was no whole colony mortality and partial mortality only occurred in the low light (high turbidity) treatments, and even then, only for P. damicornis and to a lesser extent A. millepora. The maximum quantum yield or photochemical efficiency (Fv/Fm) of the algal symbionts decreased within 2–4 weeks for all species at higher turbidity treatments. However, the Fv/Fm values in the remnant algal population at the end of the experiment were nevertheless quite high (i.e. >0.6), even in the heavily bleached corals. These photochemical efficiency values are much higher than the rapid decreases that have been reported in previous studies when corals have been exposed to sediments45. As discussed in Bessell-Browne et al. (2017b), those exposures involved smothering of corals by organic rich sediments which results in rapid reduction in tissue O2 and pH, anoxia and hydrogen sulphide formation46. It follows that if experiments showing pronounced decreases in dark-adapted Fv/Fm involved smothering, then inferences of ‘photo-physiological’ stress may be inappropriate if the measurements are made of algal symbionts that are trapped in a milieu of dead and decaying host tissue. The slight decreases in the quantum yield at very high turbidity (zero light) could have been due to unstacking and structural changes of the thylakoid membrane, leading to reduced electron transport48. Considering that the corals had been held in near darkness for 7 weeks (and at a SSC of 100 mg L−1), the relatively high quantum yields of the remnant algal populations is emphasized here, as opposed to the slight reductions.The bleaching of the tissues in the high turbidity (very low light) treatments was consistent across all species and involved the loss of the algal symbionts. Some of the corals turned bone-white although the 50, and in conjunction with the results of recent experiments51 it seems reasonable to attribute the dissociation of the symbiosis to the low light component of the elevated turbidity as opposed to the elevated suspended sediments. The bleaching of the corals to the low light was very uniform and without the variegated patterns often on upper sunlight exposed surfaces seen with elevated water temperatures55 and herbicides56. A reduction in translocation of algal-derived photosynthate to the host could be the cue to initiate the dissociation, as has been suggested for warm bleaching59.Bleaching has been reported many times previously following exposure to darkness62 can be quickly fixed into structural phospholipid (i.e. polar lipids and sterols) and acts as a precursor pool for storage lipids, principally as non-polar wax esters and triglycerides68. Lipids can represent up to 40−50% of the dry weight of corals64 and once photoautotrophy has been reduced under prolonged energy deficits and negative energy balances, lipids are likely to be one of the biochemical pools corals could draw on for energy69. Changes in lipid class composition  can provide insights into how corals are consuming their lipid reserves70. The corals did not lose all lipid classes at similar rates, instead free fatty acid and triacylglycerol which consist of many lipid types that contribute greatly to cellular structure and integrity72 showed the greatest decreases. The lowering of the lipid ratios for all species exposed to SSC ≥ 10 mg L−1 (≤2.3 mol quanta m−2 d−1) is likely due to a gradual decreasing of reserves of lipids under sub-optimal light conditions. The results are consistent with previous studies examining effects of experimentally reduced light levels on lipid concentrations and branch growth64.The most conspicuous trend in the lipid analysis was a decrease in lipid content and change in lipid class composition with increasing turbidity. Excess fixed carbon translocated to the host from the algal symbionts−2 d−1 and 2 mg L−1 SSC) compared to the zero-turbidity treatment (12. 6 mol photons m−2 d−1 and ∼0 mg L−1) and higher turbidity treatments. Without follow up studies replicating the effect, it is not clear whether this trend indicates an optimal light level (10.1 mol photons m−2 d−1 versus 12.6 mol photons m−2 d−1) or possibly an interactive effect with a low level of sediment. However, it is noticeable that it was consistent in all four species and also largely reflected in the growth trends.A notable secondary trend was that lipid concentrations were highest in the lowest turbidity/high light treatment  reduction in light from dredging was first identified in the 1970sAcropora millepora (Ehrenberg 1834) and Pocillopora damicornis (Linnaeus 1758), a foliose species, Turbinaria reniformis (Bernard 1896) and the massive species, Porites lobata (Dana 1846) and/or Porites lutea . P. lobata and P. lutea are morphologically very similar and difficult to distinguish in situ due to their small and variable corallites (Veron 2000), and therefore a mixture of species was probably used and are referred to here as Porites lobata/luteaExperiments were conducted on two branching species, Porites lobata/lutea were cut from large colonies using a pneumatic drill. Corals were transported to the SeaSim aquarium facilities at the Australian Institute of Marine Science  where each fragment was glued onto numbered aragonite coral plugs. Corals were left to recover from the handling and preparation procedures for several weeks at 7.2 mol photons m−2 d−1 and fed using enriched Artemia spp.All coral species were collected from ∼5 m depth at Davies and Broadhurst reefs which are mid-shelf reefs (~18 °S) in the central Great Barrier Reef. Fragments from up to 10 colonies each of the branching and foliose species were collected while 30 mm diameter cores of −1 . Experiments were conducted at a water temperature of 27 °C achieved by mixing two streams of FSW (22 °C and 36 °C) through manipulating valves regulated through a Siemens PCS7 Supervisory Control and Data Acquisition (SCADA) system (see below) and with feedback from temperature thermistor in a mixing chamber  each with an associated light intensity .The sediments used were collected from Davies Reef and were biogenic and predominantly calcium carbonate based. After collection, sediments were screened to 2 mm and ground with a rod mill grinder until the mean grain size was ~30 µm ). Sediments were thus predominately silt-sized, and typical for sediments in dredge plumes−1) stock solution recirculating at high velocity (>3 m s−1) in the ‘sediment delivery loop’ , based on filtering water samples through pre-weighed 0.4 µm polycarbonate filters, washing in deionized water, drying the samples (60 °C for 24 h) and then re-weighing the filters. All nephelometers were connected to the SCADA system, which recorded turbidity and episodically controlled the opening of the solenoid valves to inject sediment into each of the independent tanks from the sediment delivery loop  aliquots of a highly concentrated  digitally converted each pixel to grayscale. For each coral, the polygon selection tool in ImageJ was used to draw around the replicate, digitised to grayscale to calculate a colour mean, which was then compared to the means of the colour squares. Initial coral colour was statistically similar (ANOVA: P > 0.05) between SSC for all species.To examine colour changes, the E1–E6 colour squares on the colour reference chartFv to maximal fluorescence Fm of the symbiotic dinoflagellates) was measured at the start and end of the experiments using a Mini-PAM fluorometer .Dark-adapted (for ≥1 h), quantum yield , symbiotic dinoflagellate density , and pigment (1 mL) analyses. The lipid aliquots were temporarily stored at −20 °C, while the other two samples were stored at −80 °C. The surface area of the corals was determined using the wax dipping technique−1 and a column temperature of 60 °C. Finally, the various pigments in each sample were identified by retention time and PDA spectral confirmation before being quantified against calibration curves that were established under the same running conditions using certified reference pigments .For symbiotic dinoflagellate density, each aliquot was counted 8 times using a Neubauer haemocytometer containing 8 µL of homogenised solution. For pigment extraction each sample was centrifuged at 1500 × g for 1 min at 4 °C to obtain an algal pellet, which was then suspended in 0.9 mL of pre-chilled buffered extraction solvent , sonicated for 10 s and stored in the dark on ice for 30 mins. The sample was centrifuged again, with the supernatant subsequently transferred to a volumetric flask. This extraction process was repeated, with the combined supernatant made up with the extraction solvent to the prescribed volume and then passed through a 0.2 µm filter before chromatography. All samples and extracts were kept on ice throughout the extraction process which was performed under dimmed light. The extracts were analysed by chromatography on a Waters Acquity UPLC  system coupled to photo-diode array (PDA) detector. The extracted pigments were separated on an Acquity UPLC BEH C8 column  over a 20 min run using a binary gradient of methanol-tetrabutylammonium acetate (70:30) and methanol-acetonitrile (50:50) at a constant flow rate of 0.45 mL min2O (3:1) and left to separate overnight. The next day, the bottom lipid layer was carefully removed using a glass syringe, placed into a pre-weighed vial, dried-down under nitrogen, and finally weighed. For samples with sufficient lipid content (>1 mg), the amount of storage lipids  and structural lipids  were extracted following82, and analysed using a thin layer chromatography-flame ionization detector . The lipid ratio of storage to structural lipids was then calculated according to83.For lipid analysis, each sample was first freeze-dried for 48 h, placed in a 10 mL test tube together with 2 mL of dichloromethane-methanol (2:1). The sample was sonicated for 10 min, extracted 3 times with 7 mL  of dichloromethane-methanol using a solvent rinsed cotton-stuffed Pasteur pipette, washed with 3.5 mL of 0.44% KCl in methanol-Ha, chl c and peridinin cm−2), photochemical quantum yield (dark-adapted Fv/Fm), total lipids, and storage to structural lipid ratio) a Gamma distribution was used as these were continuous on the scale of >0. Models were fit using the glmer function in the R package lme484 using tank as a random effect. Relative model weights85 were calculated using AICc values for the model including the treatment effect, and a null model including only the random tank effect. We used a Bayesian approach for comparing individual treatment effects for each species, based on posterior contrasts. Bayesian models of the equivalent GLMM design were fit using an integrated nested Laplace approximation (INLA) for approximating posterior distributions . Statistically similar groupings were based on a posterior probability of similarity of 0.95% or greater.A generalised linear mixed modelling (GLMM) framework was used to assess the strength of the treatment effect and examine the differences among individual treatments. GLMMs were fit individually for each species, for each of the response variables of interest. For growth  we used a Gaussian distribution, as this can theoretically take positive and negative values  and appeared to be normally distributed. For the remaining variables , pigment concentration (µg chl Supplementary Information.Supplementary Information2.