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- Editorial: microbiome dynamics as biomarkers of welfare status in aquatic speciesPublication . Fonseca, Filomena; Fuentes, JuanThe importance of the microbiome in aquaculture species is widely acknowledged. The fish gut microbiota is essential for host fitness, modulated by diet and environment, and affects animal physiology. The gut microbiome optimizes nutrient uptake and immunomodulation and enhances host resilience against infectious diseases. Understanding microbiome dynamics in aquaculture species is vital in promoting animal health and industry sustainability. In this Research Topic of Frontiers four original papers and one review paper were accepted. This editorial provides an integrated summary of those papers’ main discoveries and clues.
- First report of curvularia trifolii causing curvularia blight in agrostis stolonifera in South of PortugalPublication . Coelho, Luísa; Borrero, C.; Bueno-Pallero, Francisco Angel; Guerrero, Carlos; Fonseca, Filomena; Reis, Mário; Avilés, M.; Dionísio, LídiaAlgarve region (Portugal) has nearly 40 golf courses with a significant economic impact. Summer surveys on 10 golf courses detected an unknown disease on one course in 2009 and on another course in 2012 and 2013 at 29 to 30°C daily average. The second course had symptoms on about 25% of the turf of two putting greens. Diseased bentgrass (Agrostis stolonifera L.) had a green dappled pattern with irregular patches of turfgrass on yellowed leaves. Prior to decaying, affected leaves turned brown and then gray. Crown and leaf sheath infections resulted in dark brown dry rot. No lesions were observed on the roots. Leaves were surface disinfected with 5% commercial bleach (0.225% sodium hypochlorite) and cultured on potato dextrose agar (PDA). Ten fungal colonies grew from the leaf tissue, and brown mycelia, conidiophores, and conidia were observed under a microscope. Conidia were ventricose pyriform, mostly abruptly curved, 20 to 36 µm (30 µm, SD = 4) × 7 to 12 µm (10.5 µm, SD = 1.3) (n = 50), predominantly three-septate, with a prominent hilum and enlarged and darkened central cells. Colonies grown on PDA were black-brown with a black reverse side. Conidia differed in size, 15.4 to 24.6 µm (19.99 µm, SD = 3.00) × 6 to 11 μm (8.68 µm, SD = 1.54) (n = 50) and morphology (cylindrical or slightly curved). These characteristics were consistent with Curvularia trifolii (Kauffm.) Boedijn. (Ellis 1971; Falloon 1976; Khadka 2016). Species identification of the representative isolate A2 1.12 was confirmed by analysis of nucleotide sequences of the ITS1-5.8S-ITS2 region using primers ITS1 and ITS4 (White et al. 1990) and GPDH gene region with primer set gpd (Koike et al. 2013). BLAST searches of GenBank showed a high similarity of the isolate ITS sequence (MG029439) to the reference sequence JN712458 of C. trifolii (99% identity) and GPDH sequence (MK570108) with LT715803.1 (97.88% identity). The maximum likelihood phylogenetic tree showed that our isolate clustered with C. trifolii. The pathogenicity assay of this isolate was conducted in greenhouse on A. stolonifera ‘Penncross’. The isolate was grown on PDA (25°C, 10 days). Five pots (100 ml) were filled with a sand and peat mix (9:1 v/v) with 0.06 g of seeds per pot, covered with a fine sand layer. Turfgrass was cut once a week beginning 2 weeks after seeding and was fertigated with 0.5 g/liter of Peter’s foliar feed (27 + 15 + 12; N + P2O5 + K2O; and micronutrients; Scotts, Heerlen, The Netherlands). To obtain a conidial suspension for inoculation, cultured plates were scraped with a sterilized spreader and water. The suspension was filtered through a sterile gauze. Conidia were counted under a microscope (400×) with a hemocytometer. The suspension was adjusted to 8 × 103 conidia/ml, and 10 ml was sprayed per pot. Pots maintained humidity for 2 days under microtunnels. The first disease symptoms appeared 3 days after inoculation. Bentgrass from the five pots developed Curvularia blight and rotted crown symptoms. Control plants (five pots treated with water) did not display symptoms. This trial was repeated once. On PDA, C. trifolii was reisolated from leaf lesions and morphologically identified, confirming Koch’s postulates. Ellis (1971) referred to the presence of C. trifolii in Portugal, but no region, symptom description, or grass species was detailed. Sivanesan (1987) reported C. trifolii in Portugal only on Lolium multiflorum. Therefore, this is the first report of C. trifolii in Algarve, affecting A. stolonifera. This disease can increase maintenance costs in greens in this area.
- From invasion to fish fodder: inclusion of the brown algae rugulopteryx okamurae in aquafeeds for european sea bass dicentrarchus labrax (L., 1758)Publication . Fonseca, Filomena; Fuentes, Juan; Vizcaíno, Antonio Jesús; Alarcón, Francisco Javier; Mancera, Juan Miguel; Martínez-Rodríguez, Gonzalo; Martos-Sitcha, Juan AntonioIn keeping with the premises of Blue Circular Economy in the European Union, the present study explored the possibility of using the invasive brown alga Rugulopteryx okamurae in aquafeeds for European sea bass (Dicen-trarchus labrax). Assuming the raw algae biomass could negatively impact animal performance, four experi-mental formulations were prepared, by including macroalgae material at 5%, using crude (CR), enzymatically hydrolysed and fermented (EF), enzymatically hydrolysed (E), or fermented (F) R. okamurae biomass, which we tested against a control feed (CT). To evaluate the effects of the experimental diets, besides animal growth performance and biometric parameters, we devised a toolbox focused on the intestine and intestinal function: i) ex-vivo epithelial resistance and permeability in Ussing chambers; ii) microbiota composition through NGS; iii) expression profiles of selected markers for epithelial integrity, transport, metabolism, and immune response, by qPCR. Our results show differentiated allometric growth among diets, coupled with intestinal epithelium al-terations in permeability, integrity, and amino acid transport. Additionally, evidence of microbiota dysbiosis and contrasting immune responses between experimental diets, i.e. pro-inflammatory vs. anti-inflammatory, are also described. In conclusion, we believe that R. okamurae could be a suitable resource for aquafeeds for the European sea bass, although its use requires a pre-treatment before inclusion. Otherwise, while the fish still have a positive growth performance, the gastrointestinal tract pays a toll on the integrity, transport, and inflammatory processes.
- The response of Plantago major ssp pleiosperma to elevated CO2 is modulated by the formation of secondary shootsPublication . Fonseca, Filomena; DenHertog, J; Stulen, IThe effect of elevated CO2 on the relative growth rate (RGR) of Plantago major ssp. pleiosperma was studied during the vegetative stage, in relation to plant development, by growing plants at 350 mu l l(-1) or at 700 mu l l(-1) CO2 in non-limiting nutrient solution with nitrate. To minimize interference by the accumulation of non-structural carbohydrates in the interpretation of results, RGR was expressed on a f. wt basis (RGR(FW)), as were all plant weight ratios. Stimulation of the RGR(FW) Of the whole plant by elevated CO2 was transient, and did not last longer than 8 d. At the same time a transient increase in root weight ratio (RWR) was observed. In order to investigate whether the transient effect of elevated CO2 on RGR(FW) was size-dependent, the data were plotted versus total f. wt (log(e) transformed). The transient period of stimulation of RGR(FW) and of RWR by elevated CO2 was still found, but in both CO2 treatments RGR(FW) decreased after a certain plant size had been reached. This size coincided with the stage at which secondary shoots started to develop, and was reached earlier in plants grown at elevated CO2. The RGR of these secondary shoots (RGR(see)) was Still increased when the period of whole plant stimulation of RGR(FW) had ended, indicating that the development of these new sinks took priority over a continuation of the stimulation of RWR. It is hypothesized that in this Plantago subspecies the response of the RGR(FW) of the whole plants to elevated CO2 is modulated by the formation of secondary shoots. Apparently, partitioning of the extra soluble carbohydrates at elevated CO2 to this tissue takes precedence over partitioning to the roots. resulting in a cessation of stimulation of plant RGR(FW) by elevated CO2.
- First molecular characterization of grapevine virus B (GVB) in portuguese grapevine cultivars and improvement of the RT-PCR detection assayPublication . Fonseca, Filomena; Fernandes Duarte, Vilma Vanessa; Santos, Margarida Teixeira; Brazão, João; Eiras-Dias, EduardoThis work describes the first molecular characterization of grapevine virus B (GVB) in Portuguese grapevine cultivars. During a routine screening of 44 accessions in the National Collection of Grapevine Varieties (CAN PRT051), 17 were found infected with GVB in DAS-ELISA assays with commercial antibodies. However, only six of the corresponding isolates were successfully amplified using primer pairs described in the literature. The sequence variants (ORF4-3'UTR, 1147 nt) retrieved from these isolates segregated into two phylogenetic groups, which included sequences from complete genomes available in GenBank. The highly discrepant results obtained using serological and RT-PCR-based diagnostic tools led to the design of a primer pair for detection of GVB, which allowed the amplification of a 606-bp GVB-specific fragment from all DAS-ELISA-positive isolates and also revealed the existence of false negatives in the serological testing.
- Impact of ocean acidification on the intestinal microbiota of the marine sea bream (Sparus aurata L.)Publication . Fonseca, Filomena; Cerqueira, Ricardo; Fuentes, JuanWithin a scenario of increasing atmospheric CO2 and ocean acidification (OA), it is highly relevant to investigate its impacts not only on fish performance but also on fish intestinal microbiome and how that reflects on host performance and health. The main objective of this study was to establish if the intestinal microbiota of the sea bream (Sparus aurata) was affected by high level of CO2 in line with the predictions for this century. The bacterial communities of the intestinal fluid were characterized in animals kept at the present-day level of CO2 (400 μatm) and in animals switched to high CO2 (1200 μatm) for 1 month. Bacterial taxa identification was based on molecular methods, using the DNA coding for the 16S ribosomal RNA and primers targeting the regions V1-V3. Amplicons obtained from DNA samples of animals in the same tank were combined, cloned to obtain a bacterial DNA library, and the clones were sequenced. No significant differences were found between the two treatments for alpha diversity. However, beta diversity analysis revealed distinct dysbiosis in response to hypercapnia, with phylum Firmicutes absent from the bacterial communities of fish exposed to 1200 μatm CO2, whereas Proteobacteria relative abundance was increased at elevated CO2, due to the presence of Gammaproteobacteria (Vibrionaceae and Alteromonadaceae), a class not present in the control samples. This study provides a first glimpse at the impact of OA in fish intestinal microbiota and highlights potential downstream effects to the general condition of fishes under hypercapnia.
- Genetic variants of Grapevine leafroll-associated virus 2 infecting Portuguese grapevine cultivarsPublication . Fonseca, Filomena; Esteves, Filipa; Teixeira Santos, Margarida; Brazão, João; Eiras-Dias, José EduardoGenetic variability of 19 isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) from Portuguese grapevine cultivars was characterized by sequencing the entire capsid protein (CP) gene of the virus. Global phylogenetic analysis of the CP gene, which included nucleotide sequences obtained in this study and complete homologous sequences from GenBank, showed segregation of GLRaV-2 variants from Portuguese isolates into three major phylogroups (PN, 93/955 and H4). The novelty of these phylogenetic results is the evidence of well-supported subdivision within H4 as well as within PN, with subgroup PN3 composed exclusively of variants from a Portuguese isolate. These findings and the genetic analysis of global phylogroups indicate demographic expansion, mainly within PN and 93/955. Because the existence of a mixture of variants from different phylogroups was detected in some of the isolates, a typification assay based on reverse transcription reaction followed by polymerase chain reaction and restriction fragment length polymorphism analysis, was developed to complement molecular detection assay of the virus. This protocol discriminates variants from the phylogroups identified in this study, and is appropriate for routine testing for GLRaV-2.
- High plant protein diet impairs growth performance and intestinal integrity in greater amberjack (Seriola dumerili): Molecular and physiological insightsPublication . Fuentes, Juan; Fonseca, Filomena; Gregorio, Silvia; Kussaba Miguel, Larissa; Perera, Erick; Alarcón-López, Francisco J.; Martos-Sitcha, Juan A.The Mediterranean aquaculture industry that produces mainly seabream and seabass is exploring alternative plant-based protein sources for fish feeds together with the diversification of fish species. Effective plant-based formulations require diverse sources and additives to maintain fish welfare and growth, which is especially important for carnivorous species. In this scenario, the objective of the present study was to evaluate the impact of a high percentage of fishmeal replacement with protein of plant origin in Seriola dumerili, a fast-growing species with a high protein requirement. Two diets were developed: MAP (marine animal protein) with a protein content of 92 % of animal origin and PPB (plant protein blend) with a 50 % replacement of protein of animal origin with plant protein. We combined electrophysiology measurements with expression analysis of claudins and members of the SLC superfamily of solute carriers to unravel and characterise putative markers of intestinal integrity and absorption. The replacement resulted in shorter and lighter fish with a reduction in growth rate (SGR) from 2.6 to 2. We simultaneously observed lower transepithelial tissue resistance (TER), lower permeability, and decreased cld12 expression in the anterior intestine. In addition, we demonstrated a strong regiondependent electrogenic transport of essential amino acids, with the mid-intestine having the highest transport capacity. The comparative study performed in the mid-intestine with fish fed the MAP or the PPB diets exposed a negative effect of dietary replacement with plant protein. No significant dietary impact on di- and tri-peptide transporters (SLC15) was found at the molecular level. However, a clear region-dependent expression pattern of slc15a1, slc15a2, and slc15a4 was observed, which warrants further investigation. The expression pattern of slc7a5 showed the effect of the diet, but in contrast, the diet and intestinal region affected the expression of its functionally associated slc3a1 and slc3a2 for amino acid antiport, with the strongest effects in the posterior intestine. The integrity and absorption impairments detected in response to protein source replacement will likely underlie the significant growth differences observed between the two dietary regimes.
- Digestive and metabolic consequences of on-growing greater amberjack (Seriola dumerili) juveniles at different temperatures. In-vivo and ex-vivo assessmentPublication . Navarro-Guillén, C.; Perera, E.; Pérez-Hilario, D.; Martos-Sitcha, J.A.; Molina-Roque, L.; Gregorio, Silvia; Fonseca, Filomena; Fuentes, J.; Yúfera, M.Aiming to elucidate the effects of temperature on different aspects of the fish digestive physiology that may affect the growth, we examined, firstly, variations in growth performance, feed intake, digestive enzyme activities, expression of transport related genes, metabolites in plasma and liver, and oxidative stress response in juveniles of greater amberjack (Seriola dumerili) growing during two months at 18, 22 and 26 degrees C of water temperature. Secondly, we analysed the epithelial function of the intestinal mucosa by electrophysiological ex-vivo experiments. As expected, body growth increased with increasing temperature in the tested range. Highest relative feed intake was observed at 26 degrees C, but the food conversion ratio was the same at 22 and 26 degrees C and less favourable at 18 degrees C. Digestive proteases activities were similar at 22 and 26 degrees C, while an evident effect of temperature was observed on lipids digestive capacity, being lipase activity undetectable at 18 degrees C. Electrophysiological assays revealed a relationship between temperature and intestinal mucosa plasticity. Temperature increase promoted epithelial functionality through higher tissue resistance and short-circuit current in mid-intestine at 26 degrees C, as well as better electrogenic amino acids transport. On the other hand, mRNA expression of peptide transporters tended to be higher in fish that grew at 18 degrees C, probably to reinforce the transport capacity. Plasma circulating levels of metabolites demonstrated higher energy and protein mobilization with the increasing temperature, where a hypometabolic state was denoted by lower cortisol levels at 18 degrees C together with an apparent switch from lipids to carbohydrate usage as energy source and increased oxidative stress in the liver at the lowest temperature. Altogether indicates that the tested temperatures are within the tolerance range for the species, although 22 and 26 degrees C appear as optimal temperatures for on-growing greater amberjack juveniles. Changes in nutrient digestion and absorption induced by temperature are related with both hydrolytic activity and remodelling of the intestinal mucosa. Impairing growth capacity and initial evidences of welfare compromise were observed at 18 degrees C.