Grapes are categorized as a non-climacteric type of fruit which its ripening is not associated to important rises in respiration and ethylene synthesis. The starch metabolism shares a certain role in the carbohydrate metabolic pathways during grape berry development, and is regarded as an important transient pool in the pathway of sugar accumulation. However, the comprehensive role of starch and its contribution to the quality and flavor of grape berry have not been explored thoroughly. In this study, the expression levels of genes enzyme activities and carbohydrate concentrations related to starch metabolism, were analyzed to understand the molecular mechanism of starch accumulation during grape berry development. The results indicated that starch granules in grape berry were located at the chloroplast in the sub-epidermal tissues, acting as the temporary reserves of photosynthetic products to meet the needs for berry development, and relatively high starch contents could be detected at véraison stage. Moreover, both ADP-glucose pyrophosphorylase (EC 2.7.7.27) and sucrose phosphate synthase (EC 2.3.1.14) involved in starch synthesis displayed elevated gene expression and enzymes activities in the sub-epidermal tissue, while α- and β-amylases involved in its degradation were highly transcribed and active in the central flesh, explaining the absence of starch in this last tissue. Change in the gene expression and activities of ADP-glucose pyrophosphorylase, β-amylase and sucrose phosphate synthase revealed that they were regulated by the circadian rhythms in the fruitlets compared with those in the leaves. Both the morphological, enzymological and transcriptional data in this study provide advanced understandings on the function of starch during berry development and ripening that are so important for berry quality. This study will further facilitate our understanding of the sugar metabolism in grape berry as well as in other plant species. Starch is pivotal for the plant growth and development, and its accumulation may influence the final soluble sugar concentration at ripe stage, in many fruits like apple and banana. Even grape berry contained no significant amounts of starch compared the leaves and roots,5 Grape berries accumulate different kinds of soluble sugar, mainly sucrose, glucose and fructose during ripening. Glucose and fructose are the two predominant sugars of the grape berry at maturity. Whereas, the grape starch metabolism is a highly complex, dynamic process, which is represented by the contemporaneous synthesis and degradation of starch.6–8 Many fruit crops such as apple, banana and kiwifruit contain large contents of starch before the onset of ripening, but grape was reported to have a low quantity of starch granules present in the fruitlets at anthesis period,6 then the starch presented only in the epidermis and a few layers of sub-epidermal cells at maturity period, and finally disappeared in most tissues.1 Many studies have already been conducted on the developmental changes of sugar metabolism in grapevine (Vitis vinifera).9–11 These studies focused on the metabolic changes of glucose, fructose and even sucrose which total account for about 99 percent of the entire carbohydrate content of grape berry, regulated both the sugariness of the berry and the ethanol content of the wine produced through the fermentation of grapes. Starch may represent an important intermediate in the global pathway of sugar accumulation in grape berry. Some reports on the role of starch during the grape berry development have indicated that the starch reserves in plastid are transported to produce new hexose (glucose and fructose).9 However, it is still necessary to have more insights into the regulation network about starch metabolism pathways at different periods of grape development, and in the circadian rhythm, which will provide profound understandings in the developmental regulatory mechanism of starch is coordinated with other sugars, and the information will benefit both scientific motivation and economic interest.Carbohydrates are biosynthesized in the source tissues (leaves) and translocated to the sink tissues  in most plant species in the form of soluble sugars to sustain heterotrophic metabolism, including pineapple, cherry, fig, strawberry, citrus fruits and grape. Growth and development of plants is accompanied by changes in the relations of source/sink tissues.12–15 It was accepted that ADP-glucose pyrophosphorylase (AGPase), granule bounded starch syntheses (GBSS), alpha-amylase (AMY) and beta-amylase (BMY) played a crucial role in controlling starch synthesis and degradation in the tubers, germinating cereal seeds and roots.16–20 It is often suggested that AGPase, GBSS and BMY played a similar role in the degradation of transitory starch in chloroplasts.21–23 John et al.24 demonstrated that the low activities of starch synthase and adenosine diphosphate glucose pyrophosphorylase may result in the shortage of starch in grape berries. Xie et al.25 found that during the first rapid growth phase of the grape berry, cells had more abundant mitochondria, endoplasmic reticulum, multivesicular bodies, vesicles, and plastids than in control fruit under root restriction. However, the role that starch played in grape berry still need further study.The circadian fluctuation of transient accumulation and degradation of starch in source leaves has been studied extensively in many plants.V. vitifera, which may be beneficial for our understanding the process of quality formation and accelerating the breeding program in the future.In this study, transmission electron microscopy was used to locate where the starch granule in grape berry tissues resides. The carbohydrate contents, the activities of key enzymes and the expression patterns of genes related to starch-sucrose metabolism in grape berry were also examined to investigate the changes of starch metabolism occurring in the developmental process of grape berry. The results may provide a background of relationship between starch metabolism and berry quality of Vitis vinifera×Vitis labrusca) (red cultivar) have been grown for 8 years from the grapevine germplasm repository . Vines were grown with 3.0×1.5 m spacing, east-west direction and suitable plant protection measures. To select representative samples in the vineyard, we sampled based on the method of Boulton et al.26 The vineyard was divided into three plots from which three grapevine trees per plot were selected for tissue collection.Field-grown grapevines cv. ‘Yongyou 1 Hao’  from the outer southern canopies at 9, 13, 17, 21, 1 and 5 O’clock in the daytime. The anthesis (flowering) was described as the time when about 50% flowers are fully open. For exploring the difference between the skin and flesh on starch metabolism during fruit development, three berries from top, middle and bottom of the berry bunch were sampled at eight stages  and immediately divided into skin and flesh employing a fine edged scalpel. These seeds were discarded, and berries mixed to one sample at per point.While, the fruitlets at 7 and 14 DAA were too small to separate the skin and flesh, so the fruitlets at 7 and 14 DAA can be considered respectively as both the skin and flesh at 7 and 14 DAA, that is, in the subsequent results, the enzyme activities and gene expression between the skin and flesh at 7 and 14 DAA were the same. For carbohydrate analysis as well as enzyme and RNA extractions, each tissue from individual sampling points and dates was immediately frozen using the liquid nitrogen and then stored in −70 °C freezer until needed. Each data presented here was the mean value of three replicate samples and each sample was measured three times.27 with modifications. The tissues were cut into small blocks (about 4–6 mm3), and immediately fixed in 100 mM phosphate-buffered saline  with 2.5% (V/V) glutaraldehyde for 4 h at 4 °C with thorough degassing. After wash for three times with PBS, the tissue blocks were fixed with 1% (W/V) OsO4 in PBS for 2 h at 4 °C. Following another extensive rinse in PBS, the tissue was dehydrated in a graded ethanol series (30–100%) and 100% acetone. The tissue was infiltrated with the Spurr Embedding Kit for 24 h at 4 °C. Polymerization was conducted at 70 °C for 8 h. The specimen was first semi-thin sectioned to localize the epidermal cells under the light microscope before the proceeding of the ultrathin (~60–90 nm) section preparation. The ultrathin section was put on the copper-grids of 100 meshes covered with 0.5% formvar films which were stained first with 2% uranyl acetate in 60% ethanol for 15 min at 25 °C and then with alkaline lead citrate for 10 min. The ultrathin sections were investigated with a Hitachi H-7650 electron microscopy.To investigate the changes of starch granules in the skin and flesh cells during grape berry development, the skin and flesh tissues were prepared for electron microscopy. Tissue preparation was done following the method that Diakou and Carde reportedet al.28 with slight modifications. The Samples (1 g fresh weight (FW) berry and 0.5 g FW leaf) were ground into powder in liquid nitrogen, and then were extracted in 5 ml or 2.5 ml 80% (V/V) ethanol at 70 °C for 30 min. The homogenate was centrifuged at 12,000 g for 15 min at 4 °C, and then the supernatant was removed and centrifuged at the same condition. The precipitate was re-extracted three times with 25 ml aliquots 80% (V/V) Ca(NO3)2 for 10 min at 100 °C, and the three extracts were made up to 25 ml in a volumetric flask. The 2 ml extract solution and 2 ml the standard starch solution was added respectively 0.01 mol L−1 I2-KI solution. The starch content was then measured at optical density 620 nm.Starch was extracted according to the method in Xu et al.29 with slight modifications. Triplicate tissues were prepared for fruits and leaves at each stage of development. Frozen samples (about 3 g FW) were ground in liquid nitrogen and extracted in 70% (V/V) ethanol of 6 ml and was incubated in water at 35 °C for 20 min, then it was centrifuged , and the supernatant was transferred to a 25 ml flask, the volume was topped up with 80% ethanol. The ethanolic extract was concentrated by nitrogen gas and the concentrate was reconstituted with 1 ml of ultrapure water. The soluble carbohydrates were separated by HPLC using a CARBOSep CHO-620 CA carbohydrate column  dissolved in double-distilled H2O, following the manufacturer's instructions, and a refractive index detector . The column temperature was 80 °C and the flow rate was 0.6 ml min−1 with water as the eluent. Sucrose, glucose, and fructose were determined through their retention times and quantified based on standards.Soluble carbohydrate of fruits and leaves were measured based on the method used by Niu 2, 2.5 mM DTT, 1.0 mM EDTA, 0.05% (V/V) Triton X-100, 0.1% (W/V) BSA, 0.1% β-mercaptoethanol and 2% (W/V) in soluble PVPP. PVPP was needed in the extraction method of V. vinifera. L since extracts quickly browned in its absence.30 The homogenate was centrifuged at 12,000 g for 15 min, crude supernatant was dialyzed for 16 h with 25 mM Hepes-NaOH (pH 7.5) and 0.25 mM EDTA-Na2. The insoluble pellet was homogenized in two times in 10 ml extraction buffer and then suspended in 3 ml of 50 mM Hepes-NaOH (pH 7.5) and 0.5 mM EDTA-Na2. Cell wall composition used to assay the activity of insoluble invertase was washed in 200 ml scattered (1:40 v/v) extraction buffer minus PVPP. Extracts were transferred into pre-chilled vials and examined immediately. All the process mentioned above were implemented at 0–4 °C. All activities were proportional to the amount of extracts and to reaction time.Flesh, skin (~1 g) and leaf samples (~0.5 g) were ground with a buffer at 1:8 (W/V) ratio of buffer. The extract buffer was composed of 50 mM Hepes-NaOH (PH 7.5), 10 mM MgClet al.31 using 0.25 ml of 100 mM phosphate buffer (pH 6.0), 0.25 ml enzyme extract and 0.5 ml of 1% (g mL−1) starch solution. The reaction mixtures were pre-incubated for 5 min at 40 °C and terminated by adding 1 ml NaOH (0.4 M). The produced reducing sugars concentration was determined using DNS reagent based on the method of Miller.32 Activity of α-amylase was determined by following the same procedure as for total amylase. The enzyme extract was first pre-incubated for 15 min at 70 °C to deactivate as to β-amylase. Activity of β-amylase was calculated by the difference between the total amylase activity and the α-amylase activity. The activity of amylase was defined as the amount of maltose produced per hour per kg fresh weight of berry and leaves tissues under the experiment conditions.Activity of amylase was assayed according to Hagenimana et al.33 with slight modification. The 0.3 ml enzyme extract was incubated for 40 min at 37 °C with pH 4.5 80 mM acetate-K3PO4, and 500 mM sucrose, or pH 7.5 80 mM acetate-K3PO4 and 500 mM sucrose in a total volume of 1 ml for acid or neutral invertase activity assays, respectively. Reaction was stopped at 30 min by adding 600 μL 1% (w/v) 3, 5-Dinitrosalicylic acid (DNS) at 100 °C for 5 min and then read absorbance displayed at 540 nm. The activity of invertase was defined as the amount of glucose produced per h per kg fresh weight of grape berry tissue in the experiment conditions.The soluble acid invertase (AI) and neutral invertase (NI) activity were determined by the method of Lowell et al.34 involving 55 μl of reaction solution and 85 μl crude enzyme extract sample. The reaction solutions were composed of 0.5 M Hepes-NaOH (pH 7.5), 0.14 M MgCl2, 0.028 M EDTA-Na2, 0.112 M F-6-P (fructose-6-phosphate), and 0.042 M uridine diphosphate glucose. The mixture was incubated for 40 min at 37 °C, following then the reaction was terminated by adding 70 μl of 1.0 M NaOH. Non-reacted F-6-P was degraded by placing the tubes in 100 °C water for 10 min. After cooling, 0.25 ml of 0.1% resorcinol solution  and 0.75 mL of 35% HCl (V/V) were added into the mixture and then the tubes were incubated at 80 °C water bath for 8 min. The procedure of the sucrose synthase (SUS) assay was completely same to that of SPS except the replacement of F-6-P with 0.084 M fructose in the reaction mixtures. The amount of sucrose produced from F-6-P was calculated finally under the utilization of the standard curve made using sucrose and absorbance value at A520. Enzymes activity was described as micromoles of sucrose or glucose generated per h per g fresh weight of samples.Activity of sucrose phosphate synthase (SPS) was determined by the methods of Zhang et al.,35 with slight modification, the crude enzymatic extract and reaction buffer were conducted at 30 °C in totally volume of 450 μl including 50 mM Hepes-NaOH, pH 7.5, 6 mM MgCl2, 10 μLM Glc-1,6-bisP, 6 mM NADP+, 5 mM Na-PPi, 0.08 unit per mL phosphoglucomutase (from rabbit muscle), 0.07 unit per mL G-6-P dehydrogenase (from yeast), and 1.2 mM ADPG with 3 mM DTT. Parallel assays were conducted lacking either Na-PPi or ADPG. Blank activity was insignificant for at least 30 min and was reduced when detected.For the assay of ADP-Glucose pyrophosphorylase based on the method of Tiessen 36 To isolate APS, APL, GBSS and AMY homolog genes from grape fruits, the corresponding full-length cDNA sequenced of APS, APL, GBSS1 and AMY in Arabidopsis thaliana  as querying probes, a blast search performed on the National Center for Biotechnology Information  database and further search against the Grape genome browser database Genoscope website were done. The results showed that the predicted CDS of these genes in grape  were highly homologous to their genes in Arabidopsis thaliana. On Based on the predicted CDS sequences of these genes in grape, specific primers .PCR products of genes were ligated into pMD19-T vector and then transformed into http://expasy.org/tools/protparam.html) to predict their characters such as molecular weight and theoretical pI (Isoelectric point). The catalytic domain of these four amino acid sequence were identified using the InterProScan program (http://www.ebi.ac.uk/interpro/). The phylogenetic tree was constructed through the neighbor-joining method using the MEGA version 6.0.The similarity between starch metabolism genes in grape and those in other plants was determined using NCBI BLAST. The obtained cDNA sequences were further blasted to verify the open reading frame (ORF) of the predicted cDNA sequences and BioXM (Version 2.6) was used to deduce the amino acid sequences of the four genes. Amino acid sequence of each gene was submited to ExPASy server , and that of VvBMY, VvSuS1, VvSPS were based on the partial cDNA using Primer Premier 5.0 and Oligo 7 Software, VvNIN, VvcwIN, VvAIN1 and VvAIN2 were in according to the study of Hayes et al.37 All amplified production of primer pairs (see actin (LOC100232968), ubiquitin (LOC100253716), and β-tubulin (LOC100247828) as appropriate reference genes, and then actin was used for normalization in all experiments. Samples were collected in technical triplicates and the mean values of cycle threshold (Ct) were used to calculate the mean normalized expression level (and the Standard Error) of each gene in each cDNA tested relative to the reference gene by Q-Gene software.38The gene-specific primers of airs see  was a si40 At the same time, the tangential cell walls of the skins became thickened, and the flesh cell walls remained very thin, during which some profound changes in chemical composition occurred.42The grape berries from three different developmental stages were sampled and observed via transmission electron microscopy . These t43 Compared with the stage I and stage II, the electron density both of the berry flesh and skin cell walls during the ripening stage  became lower and the space between cells changed to be larger, which might be associated with berry softening. In addition, the flesh cell cytoplasm was so thin that the chloroplasts were hard to observe at this stage.Transmission electron microscopy (TEM) micrographs showed clear difference between the structures of the skin and the flesh cells. At stage I (28 DAA), the young berries were firm, both the flesh and skin cells were arranged closely, enriched in chloroplasts, and had a dense cytoplasm. In skin cells, most of the chloroplasts were distributed along the inner cell walls, presenting large starch granules that occupied 50% of chloroplast volume. The size of starch granules was about 1.5 μm at 14 DAA. At stage II (56 DAA), there appeared some osmiophilic granules in the flesh cells. The starch granules in the flesh cells disappeared soon and the cytoplasm became thinner. Meanwhile, the starch granules in the skin were hardly observed during the appearance of these osmiophilic granules possibly corresponding to tannins formed in tannosomes.VvAPL, VvAPS, VvGBSS1 and VvAMY genes in grape. Their GenBank accession numbers, predicted molecular weights and theoretical pIs are shown in http://www.ebi.ac.uk/Tools/msa/clustalo/) was performed to study the amino acid sequences’ similarity between the genes of APL, APS, GBSS1 and AMY in grape and those in other plant species. The analysis revealed that the similarities of amino acid sequences between the four genes in grape and those in other plant species were high (70–94%). It was found that VvAPL, VvAPS, VvGBSS1, and VvAMY shared the highest identities of 87, 94, 78, and 74% with ADP-glucose pyrophosphorylase large subunit of Fragaria×ananassa (Accession no. AY518345), ADP-glucose pyrophosphorylase of Diospyros kaki (Accession no. AB514525), granule-bound starch synthase of Nelumbo nucifera (Accession no. EU938541), secreted α-amylase in Malus×domestica (Accession no. AY939870), respectively. InterProScan analysis showed that the proteins deduced from VvAPL and VvAPS belonged to the same family, the Glucose-1-phosphate adenylyltransferase (InterPro acc. no. IPR011831) family also called ADP-glucose pyrophosphorylase, indicating that this enzyme was involved in the glycogen synthesis pathways in grape.24VvGBSS1 protein had a catalytic domain of starch synthase (InterPro acc. no. IPR013534) that used ADP-glucose (EC: 2.4.1.21), as in animals, as the glucose donor for starch synthesis. The analysis result revealed that VvAMY belonged to alpha-amylase family in plant (InterPro acc. no. IPR013775), and it hydrolyzed the glycosidic bond between two or more carbohydrates, or between carbohydrate and non-carbohydrate moiety  at around 14 DAA before gradually decreasing until the day of collection. However, the significant differences between the starch contents of flesh and skin were found from 21 DAA. The berry flesh established a remarkable decrease in the starch content and the concentration remained at a low level (<0.077 mg/g FW) until fruit ripening. Meanwhile, the skin always presented higher starch content than did flesh. From stage I to stage II, the starch content in the skin remained at a low level (0.184–0.217 mg/g), and then the starch content slightly increased to the second peak (0.317 mg/g FW) at véraison stage (56 DAA), followed by a significant decrease during fruit ripening. As shown in There was an obviously distinct variation of the carbohydrate content in flesh and skin throughout the grape berry development  and 3. TAnalysis of the activities of selected enzymes  in flesh and skin showed that they followed a similar pattern, where they all decreased slightly at the initial stage of berry growth and development, and then increased significantly around from 42 DAA until collecting time, with an exception concerning the activity variation profiles of AGPase and of the SPS in skin . It was VvAPS) had an obvious increase before 14 DAA and then decreased to a relatively lower level at 21 DAA that was kept constant until collection. However, the expression level of VvAPS in the skin keep steady during berry development, suggesting the more active role of VvAPS in starch accumulation there. In most of the time, the expression level of AGPase large subunit gene (VvAPL) was kept at low level in both the flesh and the skin compared to VvAPL. VvAPL was highly expressed in the flesh at 14 DAA, 28 DAA and reached the highest level at 56 DAA, but it was only highly expressed in the skin at 14 DAA. Another expression mode was those of granule-bound starch synthase 1 gene (VvGBSS1) and α-amylase gene (VvAMY). It was the situation that there were sharp increases to the detectable levels at 28 and 56 DAA in the flesh tissue, and then their expressions were almost undetectable in both the flesh and skin. The expression patterns of VvBMY, VvSPS, VvSuS1 and VvAIN2 were of the third situation. Their expressions seemed to coincide with the increasing contents of sucrose and hexose in both the flesh and the skin during the late development period. The expression of β-amylase gene (VvBMY) remained at a constant level up to 42 DAA followed by a significant increase, and then slightly decreased in both the flesh and the skin. The expression levels of VvSPS and VvAIN2 in the two tissues showed similar pattern as that of VvBMY throughout the fruit development. The expression of VvSuS1 both in the flesh and the skin increased until 56 DAA and its expression in the flesh kept at the constant high level with minor decrease before collection stage, while its expression level in the skin had a decrease after 56 DAA. The acid invertase genes  seemed to have similar functions, but their expression profiles were different from each other. VvAIN1 was expressed at the highest level around 14 DAA, showed the profile as the second situation of VvGBSS1 and VvAMY, while VvAIN2 did the same as the gene group of VvBMY and VvSuS11. The cell wall invertase gene (VvcwIN) and neutral invertase gene (VvNIN1) were found highly expressed both in the flesh and the skin during the first phase and in flesh and/or the skin at the second rapid period of berry growth and development. Their expression patterns could suggest that they might play role for sugar accumulation at the early developmental stage.In order to know the transcription profiles of genes related to starch and sucrose metabolisms in flesh and skin of grape berry and their roles in berry development, the transcript levels of the 11 genes were surveyed using the qRT-PCR technology. These 11 genes showed four different expression patterns  and 6. IVvAPS and VvAIN1, but a negative correlation with the expression levels of VvSuS1 , although carbohydrate metabolism in the sink tissues  also affect transportation. rhythms . In the To explore the effect of photoperiod on the activity of starch and sucrose metabolism enzymes, we measured the changes in enzyme activities during the day and night time. As shown in As a whole, the changes of AGPase and SPS activity displayed a clear circadian rhythm both in grape leaves and berry, which increased in daytime and declined at night. In addition, the changes of AI activity also displayed the circadian rhythm in the leaves and the berries, the highest level at night and the lowest level at daytime. In contrast, the starch degradation enzyme BMY not showed a clear circadian pattern.To research the effect of photoperiod on the expression patterns of sugar metabolism related genes, we assayed the relative expression levels of these genes by quantitative real-time PCR (RT–PCR), of which the variation could imply the roles of the genes.VvAPS, VvAPL, VvAMY, VvBMY and VvGBSS1 obviously shared the same expression patterns between the leaves and fruitlets tissues, indicating the starch metabolism happened both in grape leaves and berry . Likewise, the starch grains gradually disappeared in the amyloplast with simultaneous appearance of osmiophilic globules filling up the amyloplast gradually.Starch is a good storage of carbohydrates widely synthesized in photosynthetic tissues of plant, such as leaf and skin, and in non-photosynthetic tissues, such as seed, root and tuber. Like melon and apple, starch accumulates during the initial stage of grape berry development, and then degraded mainly during ripening. With the berry development, starch is rapidly metabolized and is converted into soluble sugars, making it almost undetectable on grape berry at collection. Starch is one of the primary productions of photosynthesis occurred in the chloroplast and serves to buffer the changing availability of photosynthesis resulting from the light and dark cycle in a day. It plays a key role in the daily carbohydrate metabolism of the leaf. Accordingly, starch in leaf is regarded as a short-time carbohydrate pool and therefore is often called ‘transitory starch’.VvAPL and VvGBSS1 in the leaf and fruit were regulated by light-dark cycles, indicating that these genes played the essential roles in starch biosynthesis during the grape berry development, and their expression levels could also suggest the ongoing of photosynthesis in grape fruitlets, more specifically, in the skin. Hunter et al.1 found that the starch content of leaves increased from the morning to the afternoon during the grape growth before veraison, indicating a percentage change in storage or transportation of carbohydrates between day and night. At later developmental stages the starch levels slightly declined or remained the same under the diurnal cycle. With fruit development and ripening, the ultrastructural changes in the skin and the flesh cells occurred and they were clearly observed with the transmission electron microscope. The microscope window showed that the chloroplasts kept degrading, the thylakoids disintegrating, and the starch granules disappearing gradually with the appearance of the cluster-like osmiophilic globules that was in larger size  in sink organs, which is then changed into hexose-phosphate and ADP-glucose (ADPG) involved in the synthesis of starch.48 The high expression level of VvSuS1 in our work may indicate a potential important role of VvSuS in cell wall synthesis and starch synthesis during the initial development phase of berry. Sarry et al.49 also proposed a futile sucrose recycling in the cytoplasm of grape berry, involving UDP glucose pyrophosphorylase and SuS. In the skin, the VvAPS and VvAIN2 also showed high expression levels. The dramatic drop of starch in the flesh of grape berry was consistent with the previously described degradation in the starch during the early stage of strawberry fruit,51 together with the steady increase of the content glucose and fructose, the result may imply that the trait can be the signal for the beginning of sugar accumulation.At the stage I (from 7 DAA to 28 DAA) of grape berry development, the berry cells underwent rapid division and expansion.52 The variation in the activities of enzymes and expression patterns of genes involved in sugar metabolism were accompanied by the accumulation of sugar in both flesh and skin during this stage.53 The activity of AGPase, AMY, SPS, SuS and the expression of VvAPS and VvSuS1 increased significantly in skin, together with the activity of AMY and the expression of VvBMY and VvSuS1 in flesh. The starch content in the skin during the lag phase started to increase and finally reached a maximum at véraison, at least partially due to the role of AGPase and SuS. Moreover, both the expression and activities of acidic and neutral invertases (VvAI and VvNI) increased near the start of ripening and reached a peak in the later stage. This also was described by Zhang et al.34 After véraison, the development of grape berry enters the stage III that was represented by the initiation of coloring process, and accumulation continuously of sugar. During this period, the berry usually doubles its size. In addition to AGPase and SPS, all the enzyme activities increased rapidly both in the flesh and the skin. Previous studies indicated that the primary role of SuS was to degrade sucrose and supply simplicial glucose and fructose for many important metabolic processes.54 This could explain why in this study the expression of VvSuS1 and SuS activity increased in the flesh with the fruit development, and meanwhile the amount of glucose and fructose in the berry continuously increase. This result was also consistent with some previous report on Arabidopsis sus1/sus2/sus3/sus4 mutant,48 suggesting an important function of sucrose synthase in the normal cellulose and starch synthesis.9 In contrast, genes related to starch metabolism in the flesh, such as VvAPL, VvGBSS1, VvAMY, VvBMY, were expressed at high levels at véraison and finally caused the low content of starch in the flesh. This revealed that the low content of starch in the grape flesh was from the combined functions of the genes related to starch synthesis and degradation.The stage II (from 28 DAA to 56 DAA) of grape berry development was the lag phase, during which berry size did not increase. It is well known that grape berry started to accumulate sugar around 45 DAA just after softening but possibly before color change.55 Changes in gene expression generally led to strong driving or damped alters of enzyme activities. In this study, starch content displayed a similar pattern both in grape leaves and fruitlets with a peak at 17:00 and the lowest content at 9:00. Here, it was shown that the high expressions of VvAPL and VvGBSS1 both in leaves and fruitlets were more likely to contribute to starch accumulation in the early morning from 5:00 to 9:00 .58 The APL1 mutations resulted in a significant reduction in the AGPase activity and starch content in the leaves of the plants (adg2 mutants).60 Scheidig et al.61 studied the starch-overexpression phenotype in potato (Solanum tuberosum) leaves by the antisense method. The ability to degrade starch significantly decreased in potato leaves due to the lacking of the chloroplastic BMY, especially in the dark, suggesting that hydrolytic cleavage was the predominate pathway instead of the phosphory-lytic cleavage within the degradation process of temporary starch. In Arabidopsis leaves, there were nine AtBMY genes that showed different diurnal pattern at transcript levels.21 The expression pattern of VvBMY in this study was similar to that of the AtBMY. The AMY3 protein was the only known chloroplast-targeted AMY in Arabidopsis leaf, and the expression of AtAMY3 gene increased in the daytime and decreased in the dark. However, it had been demonstrated that AMY3 is not essential for starch breakdown and other enzymes can compensate its absence.62 The diurnal patterns of VvAMY expression both in leaves and fruitlets were different from those of AtAMY3, but the AMY activity showed a similar pattern between grape and Arabidopsis, suggesting that the VvAMY cloned in this study might be not located in the chloroplast. In other plants, such as the sweet potato, the GBSS1 gene was expressed well in the tuberous roots, leaves and stems, and the expression profile in leaves exhibited a circadian-regulated pattern which was dramatically expressed after 2 h treatment of light and almost completely disappeared at 12 h dark condition during the light/dark period.64 Compared to the expression pattern in the grape leaves, the expression of VvGBSS1 in grape fruitlets was more similar with that of GBSS1 in sweet potato leaves, which implied that VvGBSS1 could affect the carbon allocation under the circadian regulation in grape berry.The carbon metabolism including starch metabolism during day and night time shows a circadian rhythm phenomenon. In many plants, starch accumulates in the daytime and degrades in the night almost linearly, with the starch accumulation can adaptively change which supply the sugar continuously even in long-dark circumstances. to 9:00 . The expVvAPS and VvSPS) regulated the starch synthesis in the skin, while the genes (VvAMY and VvBMY) and enzymes (AMY and BMY) regulated starch degradation in the flesh. During the circadian cycle, the expressions of genes  and the activities of enzymes  were regulated by the circadian rhythms in the fruitlets.In this study, we found that ta relatively high starch contents could be detected in the skin at véraison stage. The enzymes  and genes (


Compliance mismatch between stent graft and host artery may induce complications and blood flow disorders. However, few studies have been reported on stent graft compliance. This study aims to explore the deformation and compliance of stent graft in stented and unstented zones under three pressure ranges. Compliance of two stent grafts incorporated with polyurethane graft (nitinol-PU) and polyester graft (nitinol-PET) materials respectively were tested; the stents used in the two stent grafts were identical. For the circumferential deformation of the stent grafts under each pressure range, the nitinol-PET stent graft was uniform in both zones. The nitinol-PU stent graft was circumferentially uniform in the stented zone, however, it was nonuniform in the unstented zone. The compliance of the PU graft material was 15 times higher than that of the PET graft. No significant difference in compliance was observed between stented and unstented zones of the nitinol-PET stent graft regardless of the applied pressure range. However, for the nitinol-PU stent graft, compliance of the unstented PU region was approximately twice that of the stented region; thus, compliance along the length of the nitinol-PU stent graft was not constant and different from that of the nitinol-PET stent graft. The use of endovascular stent grafts for endovascular aneurysm repair (EVAR) has been accepted widely since the first application of an endovascular repair technique to treat an abdominal aortic aneurysm (AAA) reported in 1991 by Parodi . HoweverThe change in aneurysm neck diameter (8.4%–13.3%) during cardiac cycle can exceed the maximum diameter of a non-compliant stent graft; thus, stent-graft migration may occur ,16. At tSome studies have been conducted to investigate the blood flow features in vitro or angiography, hemodynamic, scanning-electron-microscopic, and histological analyses in vivo with compliance matched and compliance mismatched stents. Similarly, finite element analysis was used to evaluate the wall stress and compliance of the stent/artery hybrid structure. The results confirmed that compliance mismatch induced blood flow disturbances; restenosis developed outside the compliance-mismatched stent, whereas compliance-matched stents elicited no or focalized cell accumulations at endings, thereby demonstrating the importance of mechanical properties for stent or other interventional therapies ,23. The The PET woven graft a was preThe nitinol stent from Micro-Tech Co. Ltd.  consisted of three Z-wires with five apexes, with an angle of 35° for each Z-wire, and a longitudinal bar parallel to the stent-graft axis to provide columnar support. The nitinol filament diameter was 0.4 mm. The structural parameters of the Z-stents are shown in The two stent grafts shown in As shown in p1 (mmHg) is the lower pressure value, p2 (mmHg) is the higher pressure value, pS1 (mm2) is the cross-sectional area of lower pressure, and pS2 (mm2) is the cross-sectional area of higher pressure.The compliance (C) calculation formula is shown as follows:The outer diameter value along the time of the test was recorded by measuring the sheltered beam width illustrated in To fully explore the compliance of the stented and unstented zones of the stent graft, the compliance of two positions along the Z-stent-supported stent-graft axis was tested. The compliance of S3, which was the middle point of the stented zone of the stent graft and representing the compliance of the stented zone, was tested under three pressure ranges. Similarly, the compliance of the middle point of the unstented zone, denoted as G3, representing the compliance of the unstented zone under the three pressure ranges was also tested, as shown in The stent used in this study was special because it had a longitudinal bar parallel to its axis, which made it similar to the commercial stent graft Talent. The longitudinal bar was designed to provide columnar support and help to stabilize relative position between two Z-wires. The longitudinal bar has been avoided in later generations of stent grafts because it may decrease flexibility of the device; whether it would make a difference on the compliance is still unknown. In this study, both stent grafts were kept straight along their axis when testing. If the compliance property of a device with the longitudinal bar was studied, the compliance property of devices without the longitudinal bar could also be easily understood. However, the test instrument could only measure the diameter change of one position of the sample at a time. To fully explore the circumferential deformation behavior of the stent, the diameter changes of the following three circumferential bar positions were tested separately: when the vertical bar is in front (0°), on top (90°), and between those two positions (45°), as illustrated in p was less than 0.05.All the data were expressed as mean ± SD. The compliance comparison between and within groups was assessed with one-way ANOVA using SPSS 19 . A statistically significant difference was considered when In order to compare the compliance behavior of both materials, it is useful to relate the compliance behavior to the mechanical properties of both materials. For that purpose, we performed circumferential extension tests on PET tubular graft samples according to the standard method of ISO 7198. The length of the tubular specimen tested was 20 mm. The stretching velocity was 100 mm/min. This test was repeated 3 times. Thus, its modulus and ultimate tensile strength can be calculated.For the PET graft, there was no significant difference of the diameter changes among the three circumferential positions  regardless of the pressure range. Meanwhile, no significant difference of the diameter changes was detected among the three pressure ranges  regardless of the circumferential positions.P was applied, according to the force analysis of the graft wall, it can be decomposed into circumferential stress P is the inner pressure (MPa), R is the radius of the tubular graft (mm), L is the length of the tubular graft (mm), and T is the wall thickness of the tubular graft (mm).The tubular graft could be regarded as a hollow cylinder when considering its circumferential force. If the inner pressure R is 5 mm for PET graft, and T is 0.127 mm.Circumferential stresses PET graft’s circumferential tensile strength was tested; its tensile strain under each stress value was obtained as well as the modulus of the graft. A representation of the PET tubular graft stress-strain curve is shown in However, the circumferential stresses imposed on the PET graft were low when compared with PET graft’s ultimate tensile stress, so the particular stress-strain curve under low stress is depicted in The diameter changes of the PU graft showed no significant differences among the three circumferential bar positions  under each pressure range . HoweverAs shown in For both the PET and PU grafts, the diameter change showed no significant difference among the circumferential positions under a certain pressure range due to their homogeneous wall structure, indicating that the grafts experienced uniform deformation circumferentially. The shape of the cross section of the grafts remained circular under each pressure range.The cross-section area change under particular pressure ranges can be calculated according to its diameter, as well as its compliance, as shown in The diameter change of both stent grafts were evaluated in the middle point of stented zone S3 and unstented zone G3, as shown in p value was 0.014 and 0.021 under 50–90 mmHg, 0.026 and 0.043 under 80–120 mmHg, 0.044 and 0.031 under 110–150 mmHg, respectively). Meanwhile, no significant difference in diameter change was detected between the circumferential bar positions at 0° and 45° . The existence of the vertical bar should be the main factor that caused the non-uniform diameter change circumferentially in the unstented zone of the nitinol-PU stent graft which greatly inhibited the graft’s diameter expansion compared with the diameter change results of the PU graft alone. When the circumferential bar position was at 90°, the diameter change value under the pressure range of 50–90 mmHg showed a significantly lower result than that of the pressure ranges of 80–120 mmHg and 110–150 mmHg, respectively, in both stented zone S3  and unstented zone G3 . No other significant difference in diameter change among the three pressure ranges was observed except in the circumferential bar position at 0° in the stented zone S3 between 80–120 mmHg and 110–150 mmHg .For the nitinol-PU stent graft shown in The diameter change ratios between 0° and 90° and between 45° and 90° were calculated and graphed in The diameter change in the nitinol-PU stent graft was at least 8 times higher than that in the nitinol-PET stent graft in the stented zone S3 and 12 times higher than that in the unstented zone G3 , which sFor the stented zone of the nitinol-PU stent graft, its diameter changed with the pressure wave so stent deformation must have occurred simultaneously. A possible deformation pattern is the apex angle changing rather than the nitinol stent filament elongating due to its high modulus . The nitinol stent consisted of three Z-wires with five apexes whose initial angle (α) with no internal pressure was 35° for each Z-wire. The apex angle α could be increased slightly with increased pressure. According to the diameter of the stent graft, each apex angle can be calculated; it increased to 37.47° at 150 mmHg, and the increase amounts were 0.59°, 0.72°, and 0.76° under pressure ranges of 50–90 mmHg, 80–120 mmHg, and 110–150 mmHg, respectively.The compliance of the nitinol-PET and nitinol-PU stent grafts are shown in The radial deformation in the stented zone of the nitinol-PU stent graft was limited by the stent. Along the length of the stent graft, the amount of compliance of the unstented zone G3 was approximately twice that of the stented region S3 ; thus, iA less compliant stent graft could cause biomechanical issues, mainly changes in the flow pattern, relative pulsatility, and pulsatile diameter after device implantation. This kind of complication may be remitted by introducing a stent graft incorporated with a more compliant tubular graft than the PET graft. However, the compliance of the stent graft with a more compliant graft are not uniform longitudinally. There is a big difference between stented zone S3 and unstented zone G3 in our research; this could lead to new blood flow disturbances. In this study, only one kind of stent structure was studied. Different stent structural parameters, such as apex angle, apex number circumferentially, and the length of stent region, may correspond to different radical deformation characteristics and compliance properties. More types of stents with various structures need to be evaluated in a further study to determine the superior stent structure which makes the stent graft have uniform compliance between stented zone S3 and unstented zone G3 longitudinally.(1)The PU graft material showed significantly greater compliance than the PET graft material. The difference was at least 17 times higher.(2)Analysis of the nitinol-PET stent graft showed no difference in the amount of compliance between the stented and unstented zones regardless of the applied pressure range.(3)For the nitinol-PU stent graft, significant differences were observed in the amount of compliance in the stented and unstented regions. Thus, the compliance of the unstented PU region was approximately twice that of the stented region along the length of the stent graft. In addition, the pressure range was observed to influence the compliance of the stented region, with hypertensive pressures being associated with the highest compliance values in the stented zone S3 of the nitinol-PU stent graft.(4)Furthermore, because the stent graft was constructed with a longitudinal bar connecting the stents, the nitinol-PU stent graft was associated with a different kind of non-uniformity along its length, different from that of nitinol-PET stent graft. The shape of the unstented PU cross-section was found to be non-circular and rather “heart-shaped”, which was significantly different from the circular cross-sectional shape observed in the PU-stented region. Further work needs to be undertaken so as to translate these cross-sectional size and shape changes into disruptions of laminar flow through the stent graft.In this study, the variation in compliance was measured along the length of stent grafts with distinct stented and unstented regions. Stent grafts incorporating either PET or PU graft materials were included.


International Journal of Environmental Research and Public Health [http://www.mdpi.com/1660-4601/11/9/9897.The authors wish to update the Acknowledgments Section in their paper published in the c Health , doi:10.


Acinetobacter dijkshoorniae, a novel human pathogen within the Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex. Strain JVAP01T has an estimated genome size of 3.9 Mb, exhibits a 38.8% G+C content, and carries a plasmid with the blaNDM-1 carbapenemase gene.Here, we report the draft genome sequence of the type strain of  Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex currently comprises six different Acinetobacter species, the environmental A. calcoaceticus and five Acinetobacter species that are potential human pathogens, that is, A. baumannii, A. pittii, A. nosocomialis, A. seifertii, and A. dijkshoorniae, the latter two only recently discovered , the type strain of Acinetobacter dijkshoorniae that was recovered in 2009 from a urine sample in Turkey. JVAP01T produces the NDM-1 metallo-β-lactamase and is resistant to β-lactam antibiotics and kanamycin , ABySS v1.5.2 and Spades v3.5.0  manufacturer’s protocol with paired-end libraries (2 × 150). Sequencing was performed in an Illumina MiSeq system. s v3.5.0 –10. Cont v3.5.0 –. CISA coblaNDM-1 gene and a type VI secretion system was previously published (The sequence of the 47 kilobase plasmid (pNDM-JVAP01) containing the blaNDM-1 and aphA6 genes, described previously and conferring resistance to most β-lactams and aminoglycosides, respectively (blaADC- and blaOXA-213-family genes, respectively encoding an Acinetobacter-derived cephalosporinase and a class D oxacillinase, both of chromosomal location and conferring resistance to β-lactams. PathogenFinder v1.1 was used for the prediction of bacterial pathogenicity (A. baumannii ATCC 17978, AB0057, ACICU, and AYE strains.All 92 contigs and the plasmid were further annotated using the RAST server , which pectively , but alsgenicity . ResultsLJPG00000000 and KM923969 for its associated plasmid. The version described in this paper is LJPG00000000.1.This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession no.


Ten years ago, a few ACMP members began discussing how the new communication medium, the internet, could be used to publish a peer‐reviewed clinical medical physics journal. Acting with a lot of faith, the ACMP Board of Chancellors pledged the resources of the ACMP on the promise the journal would be successful; publication began just two years later. Although a few internet journals were already flourishing by 2000, the JACMP was one of the very first open‐access journals in Medicine and Radiology. Despite some stumbling, we have completed 8 years of publication and the journal now has an international presence. This is my last Editorial as Editor‐in‐Chief, and with it, I complete four rewarding years and sixteen issues of service.The internet was an exciting new frontier 10 years ago, and it represented a new way of thinking about the communication of scholarly ideas. I see another such frontier just emerging, and I want to be a part of it. Today, encyclopedias, operating systems, news stories and many other items are produced through mass collaboration. I believe that the medical physics community needs a Wikipedia‐type solution to the problem of expanding knowledge and information. It seems to me that the JACMP could be a large part of that effort since the ownership of the articles in the JACMP remains with the author, and the information in those articles could be contributed to such a collaborative effort. Maybe ten years from now, we will have such a resource thanks to the generosity and integrity of the medical physics community.I would like to welcome several new Associate Editors to the JACMP: Patrick Cadman, Marco Carlone, Nathan Childress, Lawrence Court, and John Gibbons. I also want to welcome the new Editor‐in‐Chief of the JACMP, my very good friend and colleague, George Starkschall. Let me offer my very best wishes and congratulations to George as he takes the JACMP to the next level. Finally, I want to thank you, the readers of the JACMP for making us part of your professional life and community.The Journal of Applied Clinical Medical PhysicsMichael D. Mills, PhD


We report the case of a 69-year-old female with Fuchs endothelial dystrophy and posterior chamber in-the-bag intraocular lens, whom we treated with DMEK surgical technique. We encountered difficulties both during obtaining the endothelium from the young donor and during the intraocular unrolling and its application on the stroma. We evaluated both preoperative and postoperative the following parameters: visual acuity, slit lamp, and optical coherence tomography appearance of the cornea, corneal thickness, intraocular pressure, endothelial cell density. In all postoperative assessments, the endothelium was attached to the host's stroma, with small peripheral, inferior folding area. Short-term results were favorable with a clear cornea and significant improvement in visual acuity obtained in the first 3 months of postoperative follow-up.Abbreviation: BCVA = best-corrected visual acuity, RE = right eye, LE = left eye, OCT = optical coherence tomography, DMEK = Descemet membrane endothelial keratoplasty  DMEK is a partial-thickness corneal graft surgery in which only Descemet’s membrane and endothelium are replaced, unlike DSAEK and UT-DSAEK, in which the donor tissue includes a fine lamella of corneal stroma.Great progress has been achieved lately in corneal surgery, especially in corneal grafting for endothelial failure. The once gold standard penetrating keratoplasty has been replaced by more advanced and superior techniques such as DSAEK , UT-DSAEK (ultra-thin DSAEK) and, most recently, DMEK , corneal topography , choice of patient [6]. Although still very used (for their more rapid learning curve), DSAEK and UT-DSAEK have lost a great part of their indications in the advantage of DMEK [7].Today, numerous articles and studies show the superiority of DMEK versus DSAEK and UT-DSAEK in terms of BCVA, graft rejection [8] but also a challenge, because of both donor tissue preparation and its manipulation for positioning on the recipient's stroma.In the majority of cases, endothelial keratoplasty by DMEK is a method of choice for corneal pathologies with endothelial dysfunction [A 65-year-old woman presented in our clinic with gradual decreased vision for the past two years, more significant in the RE. The patient was diagnosed with Fuchs’ dystrophy 3 years prior to presentation and she had cataract surgery of the RE two years earlier. She was also complaining of foreign body sensation and, sometimes, eye pain. Past medical history of the patient was insignificant with no family history of eye diseases. General review of systems was negative.At presentation, BCVA was: RE = 0.1, LE = 0.7; manifest refraction was RE: +1,5 dcyl 160, LE: +3 dsf +1,25 dcyl 176; intraocular pressure on non contact tonometry was RE: 26 mmHg, LE: 23 mmHg; pachymetry was RE: 675 µm, LE: 631 µm. Slit-lamp examination of the RE revealed corneal beaten metal aspect, confluent guttae, descemets folds, significant stromal edema, epithelial edema with small defect areas, posterior chamber intraocular lens implanted in the bag. LE presented endothelial beaten metal appearance, confluent guttae, stromal edema, cortical opacities, and mild nuclear sclerosis of the lens. Dilated fundus examination was impossible to perform at RE, retinal angiosclerosis being shown at LE.Specular microscopy of the corneal endothelium of the LE revealed decreased cell density (1100/ mm²) with abnormal cell morphology (polymegathism and pleomorphism) and the presence of endothelial guttae. Specular microscopy was not possible to perform at the RE. The anterior segment OCT showed increased central corneal thickness in both eyes, posterior corneal surface irregular and highly reflective, prominent formations in anterior chamber, small, hyporeflective intrastromal spaces. The patient’s symptoms and examination were consistent with the diagnosis of Fuchs’ endothelial dystrophy in both eyes, complicated with bullous keratopathy in RE, cataract in LE, posterior chamber pseudophakia in RE.Treatment options including medical therapy and surgical intervention were discussed with the patient and she elected surgery by Descemet’s membrane endothelial keratoplasty.Fig. 1 A-D). We have centered the donor cornea with the endothelial side up on the punch block, we scored the peripheral cornea at the Schlemm’s canal level with a blunt instrument, we have gently stripped the endothelial tissue leaving a small adhesion in center, we have punched the donor with 8,5 mm trephine, lifted it from the stromal adhesion carefully using the “one touch” technique and stained it with trypan blue. The donor tissue, double rolled, was loaded into a glass injector by aspiration. The difficulty of the preparation was the younger age of the donor: the endothelial tissue was thin, prone to tears, and adherent to the corneal stroma.For the preparation of the donor tissue, we used the SCUBA (Submerged Cornea Using Backgrounds Away) technique : performing retrobulbar anesthesia, desepitheliation for better visualization, marking the peripheral cornea at 9 mm diameter, creating 3 paracentesis, filling the anterior chamber with air, scoring and stripping Descemet’s membrane with reverse Sinskey hook following the peripheral markings, completing a descemetorhexis, keratotomy and removal of the Descemet membrane through the main wound, injecting acetylcholine intraocular solution into the anterior chamber to constrict the pupil, creating an inferior peripheral iridotomy, removing air and injecting BSS leaving a shallow anterior chamber, injecting the endothelium very carefully into the anterior chamber with double-roll up, suturing the wound with 10-0 nylon. Unscrolling and orientating the DMEK-graft using different “no-touch” techniques were very difficult due to the young donor’s age. The graft was properly positioned and centered on the recipient’s stroma but the persistence of peripheral folds could not be avoided. The graft position was secured by filling the anterior chamber with air and positioning the patient supine as much as possible until complete air resorption. The surgical technique we used for this patient was Descemet Membrane Endothelial Keratoplasty, with the following main steps .On postoperative week 1, the graft was attached but the corneal edema was significant and the visual acuity was decreased. Anterior segment OCT showed the attached endothelium graft with inferior peripheral folds .On postoperative week 2, the corneal edema decreased and BCVA was 0.4, dilated fundus exam showed retinal angiosclerosis, and a good number of endothelial cells was found at cellular microscopy .1 month postoperatively, BCVA was 0,6 and increased at 0,7 at 3 months after the surgical procedure. Good endothelial cells number and improved OCT aspects were observed: attached graft, decreasing central corneal thickness, stable peripheral folds  is encouraging for resolving similar cases with this technique. There are some disadvantages though: the poor availability of donors and the necessity of selecting the donor for an optimal result.


Rhinosporidium seeberi. Approximately 15% of cases of rhinosporidiosis are ocular, occurring mainly in the tarsal conjunctiva. There are only 11 cases of scleral melt with staphyloma formation associated with bulbar conjuctival oculosporidiosis and none of them was associated with partial regression of the scleral ectasia after a corneoscleral tectonic graft.Rhinosporidiosis is a rare chronic infection of the mucous membranes caused by the Rinosporidium seeberi. Treatment was conducted by conjunctival resection and tectonic corneoscleral graft (13x13mm) over the staphyloma. Within 1 year of follow-up the patient presented a partial staphyloma reduction, 9x9x2.5 mm, and the patch detached from the lesion. A novel surgical approach was done reducing the corneal patch and no recurrence was seen after 9 months.a 13-year-old girl with a progressively increasing black mass in the upper nasal part above the cornea of the left eye. The biomicroscopy revealed an oval, bluish mass measuring 10x10x5 mm with congestion of the overlying conjunctiva. Conjunctival biopsy showed sporoblasts of This case is one of the largest anterior scleral staphylomas secondary to rhinosporidiosis described in the literature. Scleral anterior staphyloma partial regression is an unusual outcome after a tectonic corneoscleral graft. Infection resolution and graft covering of thinned area contributed to scleral reepithelization. Rhinosporidium seeberi, a microorganism of Mesomycetozoa clade. The common sites affected are nose and nasopharynx, although it may also be present on the eye, ear and even genitalia in both sexes. Approximately 15% of cases of rhinosporidiosis are ocular, and the tarsal conjunctiva is the most common site of infection followed by bulbar conjunctiva, lid, the lacrimal gland, and sclera. [Rhinosporidiosis is a rare chronic infection of the mucous membrane that is caused by the  sclera.  However, sclera. , 4Bulbar oculosporidiosis represents about 12.4% of the cases and usually presents as polypoidal and vascular masses that bleed even upon touch. The definitive diagnosis is made by microbiological scrapings and histopathological examination of resected tissues. Scleral melting associated with bulbar conjunctival rhinosporidiosis is rare, there have been only 11 cases of scleral melt and staphyloma formation reported in the literature. –10 This A 13-year-old girl from Presidente Juscelino, Maranhão, Brazil with recurrent episodes of irritation, bleeding and watering of the OS for the past 3 years (Fig. On examination, uncorrected Snellen visual acuity was 20/20 in OD and the best-corrected visual acuity was 20/20 in OS with a − 4.75-diopter cylinder at 120 degrees. In OD, slit-lamp biomicroscopy, intraocular pressure and fundus examination were normal. In OS, an oval, bluish swelling measuring 10 × 10 mm and 5 mm in height was seen in the equatorial area of the upper nasal quadrant. There was congestion of the overlying conjunctival and episcleral vessels. Slit-lamp biomicroscopy revealed scattered white corpuscular bodies with less than 1 mm in diameter at the posterior margin of the hyperemic conjunctiva (Fig. Rinosporidium seeberi (Fig. A complete rheumatologic evaluation of overlying diseases was performed with no positive results. Therefore, an incisional biopsy of the hyperemic conjunctiva was performed and revealed scattered inflammatory cells and multiple double-walled sporangia containing sporoblasts of eri Fig. . A conjuAt surgery, the involved conjunctiva was excised with a 1 mm cauterized margin. Both superior and medial rectus muscles were preserved even though they were less than 1 mm from the ectasic area. A 13 × 13 mm corneoscleral tectonic graft was applied over the staphyloma wall and secured in place with interrupted 9–0 nylon sutures. Conjunctiva and tenon were mobilized to cover the scleral portion of the graft. Postoperatively, the patient was given topical antibiotic for 15 days and dexamethasone 0,1% eye drops for 2 months. No oral medications were prescribed. She was monitored weekly in the first month, then every 3 months until 1 year of the surgery.After 1 year, the staphyloma showed signs of reduction in height; a space was visualized between the lesion and the patch Fig. . The cylRhinosporidium seeberi thrives in a hot tropical climate. [The climate. , 12 In BThe ocular structures are the second most commonly involved after the nasal mucosa. Although quite rare, when present, scleral ectasia is associated with scattered grey–white subepithelial conjunctival granules along the posterior margin of the defect.  Common dThe treatment of this condition is essentially surgical and during surgery it is important to completely excise the involved conjunctiva. The staphyloma repair of the scleral thinning over a large area did not allow a direct approximation of the edges. Therefore, a patch graft over the area was necessary. Castelino et al.  describeCovering the exposed sclera by either a conjunctival flap or a properly secured single/multilayered amniotic membrane is important to achieve a viable graft. There were at least two important challenges to tackle in this particular case. One was removing the tissue above the thinned area without perforate the eye, and the other was performing a conjunctival flap to cover a scleral graft because the conjunctiva over the lesion must be well resected to prevent recurrence. Jacob et al.  describeIn the early postoperative period, the patient showed recovery without signs of infection recurrence or graft failure. Additionally, the corneal transparency of the patch was essential to observe the ectasia during follow-up. After 1 year, it was possible to visualize an enlarged space between the corneal graft and the staphyloma. During the second surgical intervention was possible to visualize a thicker sclera and a staphyloma height of 2,5 mm. This reduction was attributed to scleral fiber rearrangement, reepithelization and child eye growth.This case is one of the largest anterior scleral staphylomas described in the literature. Ocularsporidiosis scleral involvement was probably secondary to underlying chronic conjunctivitis. Treating large staphylomas is challenging, especially after infection. The use of corneascleral graft was crucial to observe the eventual recurrence of rhinosporidiosis and the partial regression of staphyloma.


The purpose of this study was to evaluate the effect of glazing on flexural strength of highly translucent zirconia materials. N = 10): control, heat-treated, and glazed. Heat-treated specimens were fired without the application of the glaze material. The glaze material was applied to the glazed specimens before being fired. A three-point bending test (15 mm span) was performed in an Instron universal testing machine (ISO 6872). Data were analyzed by ANOVA and Tukey's HSD post hoc test (α = 0.05).  Specimens of three brands of zirconia bars  were prepared and polished according to manufacturers' instructions. Final specimen dimensions were 20 × 4 × 2 mm. The specimens from each brand were divided into 3 groups (P ≤ 0.05). There was no significant difference in flexural strength among the different brands of highly translucent zirconia (P ≥ 0.05). Tukey's HSD post hoc test showed that specimens in the “glazed” group had significantly lower flexural strength than the control and heat-treated groups (P ≤ 0.05).  Two-way ANOVA showed a significant influence of surface treatments on flexural strength of zirconia materials ( Within the limitations of the study, external glazing decreased the flexural strength of highly translucent zirconia. The glazing of porcelain dental restorations is a routine procedure designed to provide esthetic and hygienic glass-coated surfaces on the finished restoration , 2. ApplHowever, many studies have shown that glazing does not increase flexural strength , 4, 6. IThe introduction of highly translucent yttrium oxide-stabilized tetragonal zirconia polycrystals (Y-TZPs) enabled the esthetics of monolithic zirconia restorations to be sufficiently improved. The high translucency of the Y-TZP was achievable by decreased alumina content , which iAlthough many clinicians prefer to glaze monolithic zirconia restorations, data regarding the effect of glazing on the mechanical properties of these materials are still lacking. The purpose of this study was to evaluate the effect of glazing on the flexural strength of highly translucent zirconia materials. The study examined the flexural strength of three different brands of translucent zirconia with different surface treatments.Three different brands of zirconia and two different glaze materials  were tesEach specimen was polished and beveled  in a Buehler Ecomet 250 grinder/polisher . The polishing was then done with a 15-micron grit diamond polishing pad at 30 rpm with water irrigation for 90 s for each side, after which the specimen was rinsed thoroughly. For beveling, the specimen was placed at 45° on a 15-micron grit diamond polishing pad at 30 rpm with water irrigation. The specimen was inspected for the presence of chamfer edge after the beveling. All specimens were air-dried at room temperature for at least 24 h to minimize the possibility of water being trapped in the zirconia structure. The sectioned bars were sintered with the parameters as in μm polycrystalline diamond suspension with a polishing pad, respectively, at 30 rpm with water irrigation (90 s for each side), and then thoroughly rinsed. The dimensions of the bars after being sintered and polished were approximately 20 × 4 × 2 mm according to ISO 6872:2015.After being sintered, the specimens were polished again in a Buehler Ecomet 250 grinder/polisher . The polishing was accomplished with a 15-micron grit diamond polishing pad, followed by a 6 Specimens were divided into different groups according to surface treatments: (a) no treatment, (b) heat-treated, and (c) glazed.Zirconia specimens were air-dried and placed on a firing tray. They were then fired with the glaze firing cycle but without glaze materials. The firing parameters are shown in Zirconia specimens were air-dried. The overglaze pastes were mixed with glaze liquids and applied in a thin coat on the entire surface of each zirconia bar using a ceramic brush. For Prettau Zirconia and inCoris TZI specimens, Zirkonzahn glaze paste and Zirkonzahn ICE stain liquid were used. For Zirlux FC specimens, Zirlux FC glaze paste and universal liquid were used. The glazing group specimens were then placed on a firing tray. Glaze firing was done according to manufacturers' instructions .F is the failure load (force) at the fracture point (N), L is the length of the support span, b is the width, and d is the thickness.All specimens were then stored in 37°C deionized water for 24 h before testing began. Specimens were tested for flexural strength on a three-point bending test conducted on an Instron 5566A universal testing machine  with a 1 kN load cell at a crosshead speed of 0.5 mm/min. Each specimen was loaded to failure and failure load data was obtained. The data were then calculated into flexural strength according to the following formula:P values less than 0.05 were considered statistically significant in all tests. Data from all zirconia brands were pooled, and multiple comparisons between and among different surface treatments were evaluated by Tukey's HSD test.Statistical analysis was performed by means of SPSS System 23 for Windows. The means of each group were analyzed by two-way ANOVA, with flexural strength as the dependent variable and the zirconia systems and surface treatments as the independent factors. P < 0.05). The Tukey HSD post hoc test showed that specimens in the glazed group had significantly lower flexural strength than did those in the control and heat-treated groups (P < 0.05). There was no significant difference in flexural strength between specimens in the control and heat-treated groups (P > 0.05). There was no significant influence of zirconia brands on flexural strength (P > 0.05).The flexural strength data obtained from the three-point bending tests are presented in Glazing after grinding is believed to increase the strength of a ceramic restoration because it decreases the depths of surface cracks . HoweverIn this study, for investigation of the effect of glaze on the flexural strength of three different highly translucent zirconia systems, Prettau Zirconia, inCoris TZI, and Zirlux FC were used. Glazes were applied according to the manufacturer's recommendations for each system. It has been reported that 0.05 mm glaze thickness is sufficient to prolong its integrity . TherefoStatistical analysis showed that glazing decreased the flexural strength of the highly translucent zirconia. This finding was supported by the results from another study , reportiWe conducted this study on heat-treated groups to determine whether the glaze-firing cycle had an effect on the flexural strength of zirconia. We found that there was no significant reduction in the flexural strength of zirconia after being fired with a glazing cycle without glazing materials. The only difference between specimens in this group and those in the glazed group was the absence of glaze materials (a mixture of glaze powder and liquid). Therefore, the strength reduction in glazed zirconia found in this study might be due to the glazing.Residual stresses played an important role in determining the strength of ceramic materials. In cases of residual tensile stress, preexisting stress will amplify the applied cycling stress and induce cracks in that region. Compressive global residual stresses within the ceramic surface can somehow strengthen the material; however, excessive compressive residual stresses can cause lateral cracks to grow and propagate on the surface, and these will eventually cause the material to fail. According to Swain , residuat→m transformation with the associated volume increase [t→m transformation during this procedure. The coefficient of thermal expansion (CTE) of tetragonal zirconia is approximately 10.5 × 10−6·K−1, while the CTE of monoclinic zirconia is only 7.5 × 10−6·K−1. Therefore, the CTE of polished zirconia might depend on the degree of phase transformation brought about by polishing. The CTE of porcelain was also believed to be changed during firing. Since glazed materials consist of porcelain powder (∼60%), the CTE change in porcelain might occur in glazed materials during porcelain firing.Due to the metastability of tetragonal zirconia, stress-generating surface treatments such as grinding or sandblasting are also capable of triggering the increase . In thisTempering stresses associated with temperature gradients during cooling have also been reported to cause residual stress on ceramics. The poor thermal conductivity of porcelain and zirconia, which is much lower than that of metal alloys, combined with poor thermal diffusivity , resultsThe composition of the highly translucent zirconia materials used in this study was different from that of the conventional zirconia designed for coping or a fixed dental prosthesis (FDP) framework. The most notable difference is the reduction or absence of alumina, which is reported to play a significant role in phase stabilization. The effect of residual stresses on the mechanical properties of highly translucent zirconia can be more than that of conventional zirconia. Crown geometry, as is commonly understood, affects residual stress. The scenario of crowns may never be captured in its entirety with the use of simple bar models as a result of their multifaceted geometric properties.It would be particularly useful in understanding the stress mechanic of this study. Fractographic analysis can give useful information regarding failure origin, failure stress, fracture toughness, and residual stress. Further investigations including fractographic analysis and effect of crown geometry are recommended to complement the present study.P < 0.05).Glazing decreased the flexural strength of the highly translucent zirconia tested (P < 0.05).Glaze firing without the glaze material had no effect on the flexural strength of the highly translucent zirconia tested (P > 0.05).There was no significant difference in the flexural strengths of different brands of the highly translucent zirconia tested (Within the limitations of the study, the following conclusions can be drawn:


Three different bar-shaped zirconia/porcelain bilayer specimens were prepared for a three-point flexural strength (TPFS) test (n = 15): no-liner (NL), commercial liner (CL), and EL3. Specimens were tested for TPFS with the porcelain under tension and the maximum load was measured at the first sign of fracture. The strength data were analyzed using one-way ANOVA and Tukey’s test (α = 0.05) as well as Weibull distribution. When compared to NL, the CL application had no effect, while the EL3 application had a significant positive effect (p < 0.001) on the flexural strength. Weibull analysis also revealed the highest shape and scale parameters for group EL3. Within the limitations of this study, the novel ceramic liner containing 3.0 wt % zirconia (EL3) significantly enhanced the zirconia/porcelain interfacial bonding.The adhesion of porcelain to zirconia is a key factor in the success of bilayered restorations. In this study, the efficacy of a novel experimental liner (EL) containing zirconia for improved bonding between zirconia and veneering porcelain was tested. Four ELs containing various concentrations  of zirconia were prepared. Testing determined the most effective EL (EL3 containing 3.0 wt % zirconia) in terms of shear bond strength value ( When highly esthetic restorations are required in clinical situations, all-ceramic restorations are often preferred over metal-ceramic restorations ,2. Pure Although zirconia (Y-TZP) materials are more translucent than metal alloys ,9, they Failures in zirconia-based restorations either originate from the veneer and propagate to the interface or originate from the zirconia/veneer interface ,18. Void2), indicating a similar composition to veneering porcelain . SBThe CTEs of the zirconia, veneering ceramic, CL, and EL3 used in this study were measured using a thermomechanical analyzer . For each material, five rod-shaped specimens  were prepared. The ends of the specimens were ground with abrasive papers so that they were flat, parallel and perpendicular to the axis of the specimens. After each specimen was placed in the TMA oven, the length was measured using the software’s “read length” function. An expansion measurement of the test specimen was performed at 10 °C/min between 25 °C and 500 °C. For each specimen, the CTE between 25 °C and 500 °C was determined by referring to plotted curves indicating the expansion in relation to the temperature.A total of 45 bar-shaped zirconia specimens were prepared in the same way as the SBS test specimens. The dimensions (20 mm × 4.0 mm × 0.7 mm) of each specimen were checked with a Vernier caliper . All zirThe bilayer ceramic specimens were then subjected to a TPFS test, with the porcelain layer under tension , using tm) and scale parameters .The data  were examined for the normality of distribution with the Shapiro-Wilk test and the equality of variances with the Levene test. The data set, which met both assumptions, was then subjected to a one-way ANOVA followed by Tukey’s post hoc test (α = 0.05) using SPSS 18.0 for Windows software . The SBS and TPFS results were also analyzed using Weibull distribution to determine the shape parameters (Weibull moduli, Within the limitations of this in vitro study, the novel ceramic liner containing 3.0 wt % zirconia significantly enhanced the zirconia/porcelain interfacial bonding. On the contrary, the commercial liner application did not show any positive effect on the bond strength nor flexural strength values. Considering the multifactorial and complex nature of the fracture behavior of veneered zirconia restorations, in vivo studies are still required to better understand the clinical significance of the findings of this in vitro study.


Parvilucifera parasitoids infect the toxic dinoflagellate Alexandrium minutum during bloom conditions but their prevalence and impact remain unexplored. In this study, we evaluated the in situ occurrence, prevalence, and dynamics of Parvilucifera parasitoids during recurrent blooms of A. minutum in a confined site in the NW Mediterranean Sea as well as the contribution of parasitism to bloom termination. Parvilucifera parasitoids were recurrently detected from 2009 to 2013, during seasonal outbreaks of A. minutum. Parasitic infections in surface waters occurred after the abundance of A. minutum reached 104–105 cells L−1, suggesting a density threshold beyond which Parvilucifera transmission is enhanced and the number of infected cells increases. Moreover, host and parasitoid abundances were not in phase. Instead, there was a lag between maximum A. minutum and Parvilucifera densities, indicative of a delayed density-dependent response of the parasitoid to host abundances, similar to the temporal dynamics of predator-prey interactions. The highest parasitoid prevalence was reached after a peak in host abundance and coincided with the decay phase of the bloom, when a maximum of 38% of the A. minutum population was infected. According to our estimates, Parvilucifera infections accounted for 5–18% of the total observed A. minutum mortality, which suggested that the contribution of parasitism to bloom termination is similar to that of other biological factors, such as encystment and grazing.Dinoflagellate blooms are natural phenomena that often occur in coastal areas, which in addition to their large number of nutrient-rich sites are characterized by highly restricted hydrodynamics within bays, marinas, enclosed beaches, and harbors. In these areas, massive proliferations of dinoflagellates have harmful effects on humans and the ecosystem. However, the high cell density reached during blooms make them vulnerable to parasitic infections. Under laboratory conditions parasitoids are able to exterminate an entire host population. In nature,  In the last decades, toxic and harmful planktonic protist species have been the focus of scientific and public attention due to their environmental, economic, and public health impacts in coastal areas, which are of major importance for food production  commonly occur in areas with restricted hydrodynamics, such as bays, lagoons, harbors, beaches, and estuaries. These coastal proliferations are an emerging problem whose frequency has increased in response to the intensive urbanization and recreational use of the Mediterranean shoreline, which has resulted in nutrient-rich (semi-) confined areas with low turbulence levels. These areas constitute a unique environment that favors HAB formation by several planktonic protist species   species are mostly detected in the marine sediment, with a much lower abundance in the water column  to assess host-parasitoid dynamics during a bloom of the toxic dinoflagellate A. minutum; and (iii) to quantify the impact and contribution of Parvilucifera spp. parasitism to bloom termination. The present work constitutes the first record of the impact of Parvilucifera parasitoids in the field.The main goals of this study were: (i) to determine the timing of 2, has a depth ranging from 1 m at confined sites to 6 m at the entrance, and receives large freshwater inputs rich in nutrients. Intense and recurrent A. minutum blooms between December and August have been recorded every year since 1999  is located on the coast of Catalonia (NE Spain), in the NW Mediterranean Sea , pre-filtered through a mesh with a 60-μm pore size to discard possible predators, and then incubated for 4–5 days at room temperature (20°C) under natural light. The sample was concentrated by the inverse filtration (10-μm pore size) of a 4-L water subsample and the presence of infected cells, defined as the detection of sporangia, was determined using light microscopy. The detection of <10 and >10 sporangia per concentrated sample was defined as the low and high presence of Parvilucifera spp., respectively. Samples with no sporangia were considered to be non-infected.Parvilucifera infection during a bloom of A. minutum was quantified using only the data obtained during the bloom of 2013. Because the dinoflagellate host sinks after Parvilucifera infection  was 6–7 days and was estimated using the Q10 temperature corrector . Based on this estimation, samples were collected from the sediment traps with a frequency of 5–7 days throughout the 2013 bloom period (from January to April). The same sampling frequency was used for host sampling (surface water samples). All settled material was fixed with formaldehyde  and stored for 1 h in the dark at 4°C. Subsamples (100 mL) were filtered through a mesh of 10-μm, rinsed with 250 mL of autoclaved seawater to remove small sediment particles, and then concentrated in 10 mL of autoclaved seawater in 15-mL Falcon tubes . From the latter, 1–5 mL were filtered onto 0.8-μm polycarbonate filters (25-mm diameter) using a vacuum pump at 150 mbar at room temperature. Cellulose-acetate support filters were used during filtration to promote the homogeneous distribution of the cells. The filters were cut into pieces with a razor blade, dipped in low-gelling-point agarose (0.1%) to avoid cell loss, and then dried face-down on Parafilm. The filter sections were then mounted on a microscope slide, placed in a mixture consisting of four parts Citifluor and one part Vecta Shield containing 4′-6′-diamidino-2-phelylindole , and stored at 4°C in the dark until they were observed at 400x using an Olympus BX61 epifluorescence microscope. Ultraviolet excitation allowed the detection of the DAPI signal of the host nuclei and of the parasitoid nuclei in the sporangium stage. Blue-light excitation was used to detect the green autofluorescence of Parvilucifera parasitoids in the sporangium stage vs. the red autofluorescence of host chlorophyll. Parvilucifera infections were confirmed within the whole dinoflagellate community. Parasitoid sporangia were classified into morphotypes based on the size and disposition of the nuclei inside the sporangia and on sporangial morphology. Micrographs were taken using an Olympus DP72 camera (Olympus America Inc.) attached to the microscope. Parvilucifera sporangia were counted in 3–4 transects (~11 × 0.5 mm2 each) across the filter piece to analyze a representative area of the whole filter. The detection limit under these conditions was ~75 cells L−1.The impact of B Figure  to quant2 day−1) during the bloom. Infection fluxes were obtained by multiplying the abundance of infected cells (infected cells L−1) by the trap volume (L−1) and dividing first by the trap aperture (cm2) and then by the corresponding time interval (days) between the deployment and removal of the traps. To estimate parasitoid prevalence within the host population, the number of infected cells accumulated during the deployment of the sediment traps was divided by the corresponding duration of their deployment to obtain the number of infected cells per day. Parasitoid prevalence within an A. minutum population was calculated as the number of infected cells per day as a percentage of the total A. minutum population according to:Parvilucifera species infect a wide range of dinoflagellate species  was used to determine the daily infection fluxes  recorded during the A. minutum bloom in 2013, and the ~24 h needed for sporangia to release their zoospores at 20°C  at a specific temperature (T); i.e., R1 is the per day rate at temperature T1, and R2 the per day rate at temperature T2.Host mortality induced by kstahler  for Amoed et al.  and at 1d et al.  under cuin situ net mortality rate of A. minutum was calculated as the decrease in host cell abundance during the decay phase, in this case from March 26th to April 23rd, following the method of Guillard  and caused high-biomass blooms. Other peaks in the abundance of this species occurred in June and July, although the abundances were lower than those reached in winter.Between 2009 and 2012, dinoflagellates caused annually recurrent high-biomass blooms at location A. Between June 2010 and 2011, host concentrations at this site were as high as 10Parvilucifera parasitoids were detected every year, from 2009 to 2012, when A. minutum reached bloom abundances as high as 104–105 cells L−1 . Exponential growth stopped when A. minutum reached a peak abundance of up to 105 cells L−1 at location A and 106 cells L−1 at location B. Abundances of 105 cells L−1 were sustained for 10 days at location A and for 15 days at location B; in the latter, abundances increased to as high as 106 cells L−1 during a 1-week period. After the peak of the bloom, during the decline in A. minutum, the dinoflagellate community again became more diverse, with an increased dominance of Scrippsiella spp. and P. micans, whereas A. minutum abundances reached their lowest values.During the sampling period, from February to April 2013 and at both sampling sites, total dinoflagellate abundance increased two-fold from January to late March, reaching a peak on March 26th of up to 10 Figures . Total dParvilucifera spp. infections were restricted to dinoflagellates, but from a total of 17 recorded dinoflagellate taxa (data not shown) only three of them were infected: A. minutum, Scrippsiella spp., and P. micans. However, while infected cells of A. minutum were observed throughout the bloom at both locations, infection of only a few cells of Scrippsiella spp. was observed at location A on only two sampling dates, February 19th and March 4th, and of only a single infected cell of P. micans on March 4th, also at location A. The detection of infected Scrippsiella spp. cells coincided with the maximum abundance of this dinoflagellate (104 cells L−1) and with a decrease of A. minutum.Parvilucifera spp. parasites followed dynamics similar to those of their hosts but with a one-period phase lag between A. minutum and Parvilucifera densities  on March 26th. After the peak, the abundance of A. minutum gradually decreased, to 103 cells L−1, although the total dinoflagellate concentration was 104 cells L−1. The slight decrease in the infection flux coincided with the decrease in A. minutum abundance. At location B , achieved immediately after the peak of the bloom, was the same at the two sites. Following the dramatic decline of the A. minutum population, by more than two orders of magnitude, the infection flux declined by one order of magnitude.The infection flux caused by  Figures . The num Figures , during B Figure , the iniA. minutum. Maximum prevalences, reached between April 3rd and 15th, were much higher at location A than at location B, evidenced by maximum values of 38 and 12% of the A. minutum population, respectively. The impact of the parasite prevalence on the total dinoflagellate community was very low at both locations over the entire course of the bloom, with a maximum of 7% at location A and 4% at location B.The parasitoid prevalences at locations A and B followed a similar pattern Figures , with veA. minutum density, with a higher abundance occurring close to the A. minutum (host) peak and then diminishing as the bloom declined . It was recurrently present at the late phase of the bloom, from April 3rd to 23rd and at a higher concentration (~103 cells m−2). At location B  and 0.7% (−0.007day−1) of the host population per day at locations A and B, respectively. Host mortality increased after the peak of Parvilucifera abundance, with the parasites killing, on average, 10% (−0.1 day−1) of the A. minutum population every day. The estimated mortality rate during the termination phase of the bloom was lower at location B than at location A, with 2.3% (−0.023 day−1) of the host population killed. Maximum host mortalities of −0.21 day−1 and −0.053 day−1 at locations A and B, respectively, were reached immediately after the maximum in Parvilucifera abundances. On average, at location A, Parvilucifera parasites killed 3.4% of the A. minutum population (−0.034 day−1) each day. Considering a decrease of −0.2 day−1 in A. minutum abundance at the end of the bloom, parasitism due to Parvilucifera was estimated to account for 18% of the total A. minutum mortality at location A between March 26th and April 23rd, and for 5% at location B during the same period.The highest  Figures  and afteA. minutum is a potentially toxic dinoflagellate and a common species in the Mediterranean. In the NW Mediterranean, it is present at low abundances throughout the year in the studied location, but once or twice per year, mainly in winter, it proliferates to form high-biomass blooms. As demonstrated in this study, Parvilucifera infections accompany these outbreaks, becoming prominent when the dinoflagellate community is almost mono-specific (>90%) for A. minutum. Algal blooms, with their low species diversity and very high abundances  during the bloom agreed with the results reported by Turon et al. (Parvilucifera isolates from A. minutum blooms from the Atlantic and Mediterranean coast (most of them in Arenys de Mar harbor) were P. sinerae. Although in host-range laboratory experiments using monospecific cultures Parvilucifera species were shown to be generalist pathogens of dinoflagellates  and thus alternate between the water column and the sediment. Taken together, our results explain why, in discrete environmental samples, Parvilucifera parasitoids are more abundant and active in the marine sediment   of the A. minutum planktonic population per day. It was only at the end of the bloom, coinciding with the sharp decrease in A. minutum abundance, that Parvilucifera reached prevalences high enough to cause maximum host mortalities. Similar patterns were observed in field studies of Amoebophrya infection dynamics (Alves-de-Souza et al., Amoebophrya infection may be so extreme that the bloom collapses (Chambouvet et al., A. minutum population attributable to Parvilucifera, it can be concluded that parasitism strongly influences bloom dynamics, as it leads to losses that are of the same order of magnitude as those due to other biological factors, such as encystment (Anglès et al., Parvilucifera parasitoids and dinoflagellates, since their interaction is host-density dependent, the natural course of the infection will lower the density of the host and thus its contact rate with the parasitoid. Fewer host individuals and lower infection rates will lead to the establishment of a population equilibrium between dinoflagellate cells and their parasitoids.In the present study, during the whole bloom period Parvilucifera are well adapted to those of their blooming hosts and therefore that eukaryotic parasitism is an important factor accounting for biological loss during dinoflagellates massive proliferations. In addition, parasitism exhibits both temporal and spatial heterogeneity during high-biomass blooms. Further investigations of the effects of abiotic and biotic factors on the ecology of these parasitoids are needed to understand parasitoid abundance, host interactions, and the link with other trophic levels of the marine food web.Our study demonstrates that the dynamics of EA, JC, and EG designed the study. EA, AR, and EG contributed to data acquisition. EA analyzed the results. EA, AR, JC, and EG contributed to interpretation and discussion of the results. EA prepared the manuscript with contributions from all-coauthors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.