Genomic analysis of the first european bacteriophages with depolymerase activity and biocontrol efficacy against the phytopathogen Ralstonia solanacearum

Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plants were isolated from river water in Europe but not fully analysed, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum

Persistent warm Mediterranean surface waters during the Roman period

Reconstruction of last millennia Sea Surface Temperature (SST) evolution is challenging due to the difficulty retrieving good resolution marine records and to the several uncertainties in the available proxy tools. In this regard, the Roman Period (1 CE to 500 CE) was particularly relevant in the socio-cultural development of the Mediterranean region while its climatic characteristics remain uncertain. Here we present a new SST reconstruction from the Sicily Channel based in Mg/Ca ratios measured on the planktonic foraminifer Globigerinoides ruber. This new record is framed in the context of other previously published Mediterranean SST records from the Alboran Sea, Minorca Basin and Aegean Sea and also compared to a north Hemisphere temperature reconstruction. The most solid image that emerges of this trans-Mediterranean comparison is the persistent regional occurrence of a distinct warm phase during the Roman Period. This record comparison consistently shows the Roman as the warmest period of the last 2 kyr, about 2 °C warmer than average values for the late centuries for the Sicily and Western Mediterranean regions. After the Roman Period a general cooling trend developed in the region with several minor oscillations. We hypothesis the potential link between this Roman Climatic Optimum and the expansion and subsequent decline of the Roman Empire

An insight into curcumin-based photosensitization as a promising and green food preservation technology

Consumer awareness on the side effects of chemical preservatives has increased the demand for natural preservation technologies. An efficient and sustainable alternative to current conventional preservation techniques should guarantee food safety and retain its quality with minimal side effects. Photosensitization, utilizing light and a natural photosensitizer, has been postulated as a viable and green alternative to the current conventional preservation techniques. The potential of curcumin as a natural photosensitizer is reviewed in this paper as a practical guide to develop a safe and effective decontamination tool for industrial use. The fundamentals of the photosensitization mechanism are discussed, with the main emphasis on the natural photosensitizer, curcumin, and its application to inactivate microorganisms as well as to enhance the shelf life of foods. Photosensitization has shown promising results in inactivating a wide spectrum of microorganisms with no reported microbial resistance due to its particular lethal mode of targeting nucleic acids. Curcumin as a natural photosensitizer has recently been investigated and demonstrated efficacy in decontamination and delaying spoilage. Moreover, studies have shown the beneficial impact of an appropriate encapsulation technique to enhance the cellular uptake of photosensitizers, and therefore, the phototoxicity. Further studies relating to improved delivery of natural photosensitizers with inherent poor solubility should be conducted. Also, detailed studies on various food products are warranted to better understand the impact of encapsulation on curcumin photophysical properties, photo-driven release mechanism, and nutritional and organoleptic properties of treated foods

(Des)encuentros : las retóricas delirantes y las cosas

Este (Des)encuentros pretende pensar las condiciones de aparición de las narrativas delirantes en nuestra cultura visual hoy. Concretamente, en el cine, la dislocación formal que antes definía un gesto de vanguardia ha sido asimilada e integrada en el cine mainstream contemporáneo. El debate también busca explorar las consecuencias de este fenómeno nuevo y complejo mediante el cruce poco habitual de distintos discursos (filosofía, psicoanálisis, física, neurofisiología, teoría cinematográfica y cultura visual)

Fish meal-free diets supplemented with health promoters support optimal growth in gilthead sea bream, with benefitial changes in gene expression, intestinal microbiota and improved intestinal disease recovery

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021.[Introduction]: The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. Tolerance to fish meal (FM) and fish oil replacement in the economically important gilthead sea bream (Sparus aurata) is being extensively studied with many products emerging as alternative feed ingredients. It has been demonstrated that alternative diets influence the composition of intestinal adherent microbial populations, which have a key role on host metabolism, health and disease resistance. In addition, low fish meal diets showed an increased susceptibility to enteric parasites (Piazzon et al., 2017). Clearly, differences in diet have an impact on the overall health and metabolism of the fish and many parameters have to be taken into account when studying alternative diets for their use in aquaculture. In this study we evaluated the effect of a novel feed formulation (NoPAP SANA) with total replacement of FM by insect meal and bacterial fermentation biomass, and supplemented with the health-promoter additive SANACORE®GM (Palenzuela et al., 2020), on growth performance, gene expression, intestinal microbiota and disease resistance in gilthead sea bream.[Methods]: Tagged gilthead sea bream of mean weight 21.3 g were distributed in two open-flow tanks (160 fish/tank) and fed ad libitum during 34 days with control or NoPAP SANA diets. Twelve fish/diet were sacrificed and head kidney (HK), liver (L) and posterior intestine (PI) were taken for RNA extraction. From the same fish, the adherent bacteria of PI were collected and immediately used for DNA extraction. RNA from HK, L and PI was used to run three customized PCR-arrays including genes of interest for each tissue, with markers of performance and metabolism (L), immune system (HK and PI), epithelial integrity, nutrient transport and mucins (PI). Using the bacterial DNA, the V3-V4 region of the 16S rRNA of each individual sample was amplified and sequenced by Illumina MiSeq. After quality filtering, taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq and beta diversity using PERMANOVA and PLS-DA models. Metagenome prediction and pathway analysis were performed using Piphillin. Differential gene expression and OTU presence and abundance correlations were studied using the corrplot R package. From the remaining fish, 70 fish/group were challenged with the intestinal parasite Enteromyxum leei by effluent exposure and the remaining fish were used as controls. The challenge lasted 78 days, including a non-lethal diagnosis sampling at day 40. At the end of the challenge all fish were sampled for histological and molecular diagnosis. Biometric values from all fish were taken in all sampling points.[Results]: A slight decrease in condition factor and specific growth rate was detected in the NoPAP SANA group. However, all fish grew efficiently considering gilthead sea bream standards. NoPAP SANA group showed differential expression of 17 out of 44 genes in L, two out of 29 in HK, and 4 out of 44 in PI. The bacterial composition at the PI showed no major differences in diversity or at the phylum level. However, 29 abundant (>1%) OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differential expression of genes in the different tissues, highlighting Pseudoxanthomonas which was positively correlated with the expression of seven L genes, or Actinomyces, significantly correlated with the expression of L and HK genes (Fig. 1). Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge was not significantly different between diets. In fact, infected fish from both groups showed similar recovery rates.[Conclusions]: NoPAP SANA promoted good growth parameters and efficient conversions arising as a good alternative for a FMbased diet in gilthead sea bream diets. This diet modulated the expression of several genes in L showing the capacity to reduce lipogenesis, mitochondrial activity and the risk of oxidative stress and, at the same time, promoting an antiinflammatory gene expression profile in HK and PI. Changes were also detected in the adherent bacterial populations of PI, with significant changes of OTUs that could potentially account for significant metabolic alterations. The correlations between presence and abundance of intestinal bacteria with changes in gene expression of different tissues, together with the pathway analysis results, show that microbiota changes can have an impact on host metabolism at a systemic level, and vice versa. Clearly, the changes induced by this novel FM-free diet supported an accelerated growth with an overall feed conversion ratio close to 1 and no increased susceptibility against this intestinal parasite, as often observed in studies when replacing a FM-based diet.GAIN (EU-H2020 #773330); RYC2018-024049-I/AEI/10.13039/50110001103

A novel fish meal-free diet formulation supports proper growth and does not impair intestinal parasite susceptibility in gilthead sea bream (Sparus aurata) with a reshape of gut microbiota and tissue-specific gene expression patterns

The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. The maximized replacement of fish meal (FM) and fish oil (FO) with plant ingredients has been extensively studied in the economically important species gilthead sea bream (Sparus aurata). Recently, major progress has been done with other alternative raw materials, though some non-pathological inflammatory response persisted with feed formulations that increased the circularity of resource utilization. In the present study, we evaluated the effects on growth performance, gene expression, intestinal microbiota and disease resistance of a FM-free diet (NoPAP SANA), based on plant ingredients, aquaculture by-products, algae oil, insect meal and bacterial fermentation biomasses as main dietary oil and protein sources, and supplemented with a commercially available health-promoting feed additive (SANACORE®GM). Juveniles of 21 g initial body weight were fed control or NoPAP SANA diets for 34 days, and head kidney, liver and posterior intestine were collected for gene expression analyses using customized PCR-arrays. Each tissue-specific PCR covered 96 genes in total and included markers of growth performance, lipid and energy metabolism, antioxidant defence, immune system, and intestinal function and integrity. From the same fish, the adherent bacteria of the posterior intestine were studied by Illumina sequencing of the V3-V4 region of the 16S rRNA. The remaining fish were challenged with the intestinal parasite Enteromyxum leei for 78 days and sampled for parasite diagnosis. Both control and NoPAP SANA fish grew efficiently considering gilthead sea bream standards. Before parasite challenge, the NoPAP SANA group showed differential expression of 17, 2 and 4 genes in liver, head kidney and posterior intestine, respectively. The intestinal bacterial composition showed no major differences in diversity or at the phylum level. However, 29 abundant OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differentially expressed genes in the different target tissues. Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge did not significantly vary between dietary treatments, and infected fish from both groups showed similar disease outcome. Altogether, these results indicate that the NoPAP SANA diet promoted optimal growth and a healthy condition in gilthead sea bream without affecting susceptibility against the tested intestinal parasite, as often observed with alternative diets following current industry formulations.This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 773330 (GAIN, Green Aquaculture Intensification). This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH, RTI2018–094128-B-I00, AEI/FEDER, UE). M.C·P was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I, co-funded by the European Social Fund & ACOND/2020 Generalitat Valenciana)

Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction

Limfòcits b; Metilació de l'ADN; GenomaLinfocitos b; Metilación de ADN; GenomaB-lymphocytes; DNA methylation; GenomeActivation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.Spanish Ministry of Science, Innovation and Universities [SAF2017-88086-R to E.B.]; cofunded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe. E.B is supported by Instituto de Salud Carlos III (ISCIII), Ref. AC18/00057, associated with i-PAD project (ERARE European Union program); P.L. and C.P. are supported by the German Cancer Aid project CO-CLL [70113869]; B.G. is funded by the Deutsche Forschungsgemeinschaft [GR1617/14-1/iPAD, SFB1160/2_B5, RESIST–EXC 2155–Project ID 390874280, CIBSS–EXC-2189–Project ID 390939984]; BMBF [GAIN 01GM1910A]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities [SAF2017-88086-R]

Understanding how high stocking densities and concurrent limited oxygen availability drive social cohesion and adaptive features in regulatory growth, antioxidant defense and lipid metabolism in farmed gilthead sea bream (Sparus aurata)

The study combined the use of biometric, behavioral, physiological and external tissue damage scoring systems to better understand how high stocking densities drive schooling behavior and other adaptive features during the finishing growing phase of farmed gilthead sea bream in the Western Mediterranean. Fish were grown at three different final stocking densities (LD, 8.5 kg/m3; MD, 17 kg/m3; HD, 25 kg/m3). Water oxygen concentration varied between 5 and 6 ppm in LD fish to 3–4 ppm in HD fish with the summer rise of water temperature from 19°C to 26°C (May–July). HD fish showed a reduction of feed intake and growth rates, but they also showed a reinforced social cohesion with a well-defined endogenous swimming activity rhythm with feeding time as a main synchronization factor. The monitored decrease of the breathing/swimming activity ratio by means of the AEFishBIT data-logger also indicated a decreased energy partitioning for growth in the HD environment with a limited oxygen availability. Plasma glucose and cortisol levels increased with the rise of stocking density, and the close association of glycaemia with the expression level of antioxidant enzymes (mn-sod, gpx4, prdx5) in liver and molecular chaperones (grp170, grp75) in skeletal muscle highlighted the involvement of glucose in redox processes via rerouting in the pentose-phosphate-pathway. Other adaptive features included the depletion of oxidative metabolism that favored lipid storage rather than fatty acid oxidation to decrease the oxygen demand as last electron acceptor in the mitochondrial respiratory chain. This was coincident with the metabolic readjustment of the Gh/Igf endocrine-growth cascade that promoted the regulation of muscle growth at the local level rather than a systemic action via the liver Gh/Igf axis. Moreover, correlation analyses within HD fish displayed negative correlations of hepatic transcripts of igf1 and igf2 with the data-logger measurements of activity and respiration, whereas the opposite was found for muscle igf2, ghr1 and ghr2. This was indicative of a growth-regulatory transition that supported a proactive instead of a reactive behavior in HD fish, which was considered adaptive to preserve an active and synchronized feeding behavior with a minimized risk of oxidative stress and epidermal skin damage

How do novel feed formulations affect growth performance, oxidative stress and immune response of atlantic salmon?

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021.[Introduction]: The aquaculture industry continues to grow faster than any other sector of food production. The need to make aquaculture as sustainable and more environmentally conscious as possible is becoming clearer everyday (FAO, 2020). With this in mind, the replacement of fishmeal and fish oil in aquafeeds has been studied in Atlantic salmon (Salmo salar) (e.g., Bendiksen et al., 2011) with many products emerging as potential alternatives to conventional ones (e.g., Hodar et al., 2020). One of the main objectives of the EU project GAIN is to evaluate new ingredients that are already commercially available using different formulation concepts that consider all the fish nutritional needs. GAIN diets are based on circular economy principles and maximize resource efficiency, while contributing to zero waste in the agri-food value chain, being cost-effective feeds, and having good social acceptability. The present study aims to understand the actual effects of these novel feed formulations on growth performance, nutritional status, immunity and oxidative status.[Methods]: Quadruplicate groups of Atlantic salmon were fed ad libitum with three different diets. Two diets were developed to facilitate the eco-intensification of aquaculture through increased circularity and resource utilization (NOPAP - formula without processed animal protein - and PAP - formula with processed animal protein). The third diet was a commercial-like formulation that was used as a control. After a 96-day feeding trial, plasma samples were collected to evaluate humoral parameters (protease, anti-protease, bactericidal activity, and IgM). Liver and head kidney tissues (collected at day 45 and 96) were used for the simultaneous profiling by PCR array of a panel of 38 or 28 genes, respectively, as markers of growth performance, lipid and energy metabolism, and immune and antioxidant activities. Liver samples were also used to analyse lipid peroxidation. In addition, after 45 and 96 days, the lice count and fish welfare were also assessed by standard methods. The dorsal skin and foregut were collected at days 45 and 96 for mucosal mapping (mucous cell area, density, and barrier status).[Results]: Growth performance was adequate and comparable to commercial standards for the novel diets tested. Other parameters analysed, including those related to key performance indicators, intestinal and skin dorsal mucosal mapping, plasma innate immune defences, and lipid peroxidation in the liver did not significantly differ across diets. Regarding head kidney gene expression, at Day 45, 2 out of 28 genes in the array were differentially expressed (p<0.05). Gene expression of fish fed with novel feed formulations showed a pro-inflammatory profile evidenced by the up regulation of il-8, and a down regulation of il-10.At Day 96, the same genes continued to be differentially expressed, but gene clec1b (membrane protein) was also up-regulated. However, the rest of the analyses do not support this pro-inflammatory profile. A longer trial may bring light to some of the current results. In turn, the liver had a differential gene expression only at the second sampling point (Day96), where 4 out of 38 genes in the array were affected, including growth performance (igf2), lipid metabolism, elongases (elovl4), and energy metabolism (ucp2l and sirt1). These transcriptomic changes may be attributed to an initial response to the experimental diets. Cross-analysis of gene expression by time points and dietary treatment (two-way ANOVA) yielded only 2 out of 38 genes that had significantly different expression across treatments. The differentially expressed genes were related to growth performance (igf2) and lipid metabolism (elovl4).[Conclusions]: The novel feed formulations of the GAIN project for Atlantic Salmon seem to be viable options for the near future. In any case, all results are related to the formulation itself and cannot be attributed to a specific ingredient alteration. More studies are necessary to understand the cost-benefit of these new formulations and their market acceptability to optimize sustainability within the current/predictable European regulatory framework.This project was financed by the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 773330 (GAIN), with additional support from Nord University (Norway) and SPAROS Lda (Portugal)

Evaluation of growth performance, oxidative stress and immune response in gilthead sea bream fed with novel feed formulations

Trabajo presentado en Aquaculture Europe 2020, celebrado en modalidad virtual del 12 al 15 de abril de 2021.[Introduction]: As the aquaculture sector continues to expand while being more environmentally conscious, the development of sustainable aquafeeds is becoming increasingly important (FAO, 2020). Tolerance to the replacement of fishmeal and fish oil in feeds has been largely studied in gilthead seabream (Sparus aurata) (Gasco et al., 2018; Karapanagiotidis, Psofakis, Mente, Malandrakis, & Golomazou, 2019), and many products emerge now as potential alternatives to ingredients used in conventional formulations. A main goal of GAIN EU project is to evaluate emerging ingredients, already commercially available, using different formulation concepts that consider all fish nutritional requirements. GAIN diets are based on circularity principles, maximizing resource efficiency, while contributing towards zero waste in the agro-food value chain, feed cost-effectiveness, and having good social acceptance. The present study aims to understand the real impacts of these novel feed formulations on growth performance, nutritional condition, immunity, and oxidative status using biomarkers.[Methods]: Quadruplicate groups of gilthead seabream (Sparus aurata) were fed ad libitum with four different diets. Three of them have been designed to facilitate aquaculture eco-intensification through increased circularity and resource utilization: NOPAP - formula without terrestrial animal by-products processed animal protein; PAP - formula with terrestrial animal by-products processed animal protein; and MIX - a mixture of NOPAP and PAP. The fourth feed followed a standard commercial formulation and was used as a control diet. After a 77-day feeding trial, plasma samples were collected to evaluate humoral parameters (protease, anti-protease, bactericidal activity and IgM). Liver and head kidney tissues were collected for the simultaneous profiling of a panel of 42 (liver) or 29 (head kidney) genes, as markers of growth performance, lipid and energy metabolism, and immune and antioxidant activities by qPCR. Liver samples were also used to analyse oxidative biomarker (Lipid peroxidation and catalase).[Results]: Tested feed formulations did not affect growth performance or feed intake. However, fish fed PAP and MIX diets had a higher feed conversion ratio (FCR) and protein efficiency ratio than control and NOPAP groups. This impairment was accompanied by a decreased hepatic expression of igf-i and ghr1. NOPAP diet slightly increased innate immunity parameters, showing better results on bactericidal, IgM, and anti-protease activity, as well as a significant up-regulation of il-8 in head kidney. Fish fed with PAP diet displayed an up-regulation of pro-inflammatory genes, namely il-8 and other cytokines (il-1β, tnf-α), chemokines (ck8), and chemokine receptors (ccr3). The same pattern was found for the T-cell markers cd3x, cd4, and cd8a. The activity of the antioxidant enzyme catalase was significantly lower in fish fed with PAP and MIX diet, being a possible indication of decreased antioxidant defences. This is supported by the observed regulation of antioxidant genes (mn-sod/sod2, gpr-170, gpr-94, and gpr-75), although not statistically significant.[Discussion]: The similar performance of novel formulations and the control diet indicates that they can be considered as viable options for seabream feeds. Differences in FCR suggest that NOPAP can promote a better bioavailability and/or increased absorption of key nutrients than PAP and MIX diets. Indeed, this impairment was also evidenced by their hepatic expression pattern of markers of growth performance. In general, PAP exhibited an opposite response to the NOPAP group. NOPAP was closer to the control diet, and MIX showed intermediate values between PAP and NOPAP in almost all parameters. The markedly pro-inflammatory head kidney expression profile in PAP fish may be also indicative of an impaired response at the mucosal level. In any case, the low proportion of differentially expressed genes between the experimental diets and control (18 out of 71) constitutes an additional and indirect confirmation of their suitability.[Conclusions]: Novel feed formulations for gilthead seabream seem to be viable options for a near future. In any case, all results are related to the formulation itself and cannot be attributed to a specific ingredient alteration. More studies are necessary to understand the cost-benefit of these new formulations and their market acceptability to optimize sustainability within the current/predictable European regulatory framework.“This project was financed from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 773330 (GAIN), with additional support from Nord university (Norway) and Sparos SA (Portugal)”