Instituto de Acuicultura (IA)

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Draft Genome Sequence of Edwardsiella piscicida Strain ACC35.1 Isolated from 1 Diseased Turbot (Scophthalmus maximus) in Europe
(American Society for Microbiology, 2017) Buján Gómez, Noemí; Estévez Toranzo, María Alicia Carolina; Magariños Ferro, Beatriz; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA)
Edwardsiella piscicida is a bacterial fish pathogen with a high degree of virulence. The strain ACC35.1 was isolated from diseased turbot in Europe. The draft genome sequence comprises 3.84 Mb with a G+C content of 59.8% and >3,450 protein-coding genes.
Insights into the virulence-related genes of Edwardsiella tarda isolated from turbot in Europe: genetic homogeneity and evidence for vibrioferrin production
(Wiley, 2016) Castro, Nuria; Rodríguez Osorio, Carlos; Buján Gómez, Noemí; Fuentes, Juan Carlos; Rodríguez, Jaime; Romero Bernárdez, Manuel; Jiménez, Carlos; Estévez Toranzo, María Alicia Carolina; Magariños Ferro, Beatriz; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA)
Edwardsiella tarda has long been known as a pathogen that causes severe economic losses in aquaculture industry. Insights gained on E. tarda pathogenesis may prove useful in the development of new methods for the treatment of infections as well as preventive measures against future outbreaks. In this report, we have established the correlation between the presence of virulence genes, related with three aspects typically involved in bacterial pathogenesis (chondroitinase activity, quorum sensing and siderophore-mediated ferric uptake systems), in the genome of E. tarda strains isolated from turbot in Europe and their phenotypic traits. A total of 8 genes were tested by PCR for their presence in 73 E. tarda isolates. High homogeneity was observed in the presence/absence pattern of all the strains. Positive results in the amplification of virulence-related genes were correlated with the detection of chondroitinase activity in agar plates, in vivo AHL production during fish infection and determination of type of siderophore produced by E. tarda. To the best of our knowledge, this is the first study carried out with European strains on potential virulence factors. Furthermore, we demonstrated for the first time that E. tarda produces the siderophore vibrioferrin
Experimental susceptibility of European sea bass and Senegalese sole to different betanodavirus isolates
(Elsevier, 2015-03-04) Souto Pereira, Sandra; López Jimena, Benjamín; Alonso, Maria Carmen; García-Rosado, Esther; Bandín Matos, Isabel; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA)
The susceptibility of juvenile European sea bass and Senegalese sole to three VNNV isolates (a reassortant RGNNV/SJNNV, as well as the parental RGNNV and SJNNV genotypes) has been evaluated by challenges using two inoculation ways (bath and intramuscular injection). The results demonstrate that these two fish species are susceptible to all the VNNV isolates tested. In European sea bass, RGNNV caused the highest cumulative mortality, reaching maximum values of viral RNA and titres. Although the SJNNV isolate did not provoke mortality or clinical signs of disease in this fish species, viral production in survivor fish was determined; on the other hand the reassortant isolate did cause mortality and clinical signs of disease, although less evident than those recorded after RGNNV infection. These results suggest that the changes suffered by the SJNNV RNA2 segment of the reassortant isolate, compared to the parental SJNNV, may have involved host-specificity and/or virulence determinants for European sea bass. Regarding Senegalese sole, although the three isolates caused 100% mortality, the reassortant strain provoked the most acute symptoms, and more quickly, especially in the bath challenge. This was also the isolate showing less difference between the number of RNA copies and viral titre, reaching the highest titres of infective viral particles in nervous tissue of infected animals. The RGNNV isolate produced the lowest values of infective viral particles. All these results suggest that the RGNNV and the reassortant isolates are the most suited for infecting European sea bass and Senegalese sole, respectively.
Cold-blooded vertebrates evolved organized germinal center–like structures
(American Association for the Advancement of Science, 2023-11-01) Shibasaki, Yasuhiri; Afanasyev, Sergei; Fernández Montero, Álvaro; Ding, Yang; Watanabe, Shota; Takizawa, Fumio; Lamas Fernández, Jesús; Fontenla Iglesias, Francisco; Leiro Vidal, José Manuel; Krasnov, Aleksei; Boudinot, Pierre; Sunyer, J. Oriol; Universidade de Santiago de Compostela. Departamento de Bioloxía Funcional; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA)
Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)–specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non–M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA–associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igμ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.
A virulence gene typing scheme for Photobacterium damselae subsp. piscicida, the causative agent of fish photobacteriosis, reveals a high prevalence of plasmid-encoded virulence factors and of type III secretion system genes
(Elsevier, 2020-05-15) Abushattal, Saqr; Vences Lorenzo, Ana; Rodríguez Osorio, Carlos; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
Fish photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) constitutes one of the major diseases affecting marine aquaculture worldwide. Although several virulence factors have been described in this pathogen, its pathobiology is still poorly understood. Two virulence factors, the exotoxin AIP56 and the siderophore piscibactin, are encoded by plasmids. Notably, presence of genes of a type III secretion system (T3SS) has been recently reported for the first time in this subspecies. However, the prevalence of these virulence genes in the populations of Pdp remains unstudied. Here, we have designed a PCR-based typing scheme for the detection of plasmid (pPHDP10)-borne aip56 gene encoding AIP56 exotoxin, plasmid (pPHDP70)-borne irp2 and frpA genes encoding piscibactin biosynthesis and transport proteins respectively, and for detection of the T3SS genes vscD and vcrD. The analysis of 103 isolates between 1963 and 2015, from different host species and geographical locations, has revealed that aip56 is highly prevalent, with only 6 isolates negative for aip56. The virulence plasmid pPHDP70 was present in 89 European and 2 Japanese isolates, refuting previous observations of a linkage of this plasmid exclusively to European strains. Most notably, 74 isolates tested positive for the T3SS genes, and their presence in photobacteriosis outbreaks could be traced back to 1980. Remarkably, we noticed that spontaneous rifampicin-resistant Pdp colonies underwent the loss of the T3SS genes vscD and vcrD, concomitantly with the loss of additional T3SS gene markers and of the putative plasmid-related genes parAB and traC, suggesting that the Pdp T3SS system is encoded within an unstable plasmid. This study unveils an unsuspected high prevalence of a putative plasmid-borne T3SS in this pathogen, and provides evidence that photobacteriosis outbreaks can be caused by distinct Pdp genotypes.
Highly Transferable pAQU-Related Plasmids Encoding Multidrug Resistance Are Widespread in the Human and Fish Pathogen Photobacterium damselae subsp. damselae in Aquaculture Areas in the Black Sea
(Springer Nature, 2020-05-08) Vences Lorenzo, Ana; Abushattal, Saqr; Matanza Fente, Xosé Manuel; Dubert Pérez, Javier; Uzun, Ecren; Ogut, Hamdi; Rodríguez Osorio, Carlos; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
The marine bacterium Photobacterium damselae subsp. damselae is a pathogen that causes disease in diverse marine animals, and is also a serious opportunistic human pathogen that can cause fatal infections. Strains of this pathogen isolated from diseased European sea bass in aquaculture facilities in the Turkish coast of the Black Sea were found to exhibit reduced sensitivity to multiple antimicrobials. Selected representative strains were subjected to complete genome sequencing and plasmid characterization. It was found that multidrug resistant (MDR) isolates harboured large conjugative plasmids sharing part of their sequence backbone with pAQU-group plasmids, hitherto reported exclusively in China and Japan. Four new pAQU-group versions of plasmids were identified in the present study, containing distinct combinations of the resistance determinants tetB, floR, sul2, qnrVC, dfrA and strAB. Conjugative transfer of pPHDD2-OG2, a representative plasmid of 170,998 bp, occurred at high frequencies (2.2 × 10-2 transconjugants per donor cell), to E. coli and to pathogenic P. damselae subsp. damselae and subsp. piscicida strains. Upon transfer, pPHDD2-OG2 conferred reduced susceptibility to a number of antimicrobials to the recipient strains. Comparative genomics analysis of host strains suggested that these MDR plasmids of the pAQU-group were acquired by different genetic lineages of Pdd. This study provides evidence that P. damselae subsp. damselae isolated from diseased fish constitute a reservoir for conjugative MDR pAQU-group plasmids in the Mediterranean basin, and have the potential to spread to diverse bacterial species.
Wastewater Based Epidemiology for the Surveillance of Illicit Drug and Substance of Abuse Use in Prison Settings: A Critical Review
(Wiley, 2025-04-07) Egaña, Iker; Nogales-García, Maite; Akhrimenko, Vladimir; González Gómez, Xiana; Quintana Álvarez, José Benito; Villanueva-Blasco, Víctor José; Orive, Gorka; Lertxundi, Unax; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Investigación e Análises Alimentarias; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA)
People held in prison suffer from drug use disorders, with important health and safety implications. In order to design strategies to reduce drug consumption and for the provision of services, reliable data is necessary. In this study, a review of published works that have applied wastewater-based epidemiology (WBE) in prison settings was performed in order to evaluate its utility. The study also explored future options for WBE in correctional settings. A search of MEDLINE was conducted using the following PubMed query terms: (“Wastewater” OR “Sewage”) AND (“Prisons” OR “Health Care Correctionals”) AND (“Abuse, substance” OR “illicit drugs”) since 2005 (first report of WBE applied to illicit drug consumption) to February 26, 2025. Then, it was complemented by a Google Scholar citation analysis. We found 8 studies, half of which have been conducted in Australia. Cocaine, cannabis, methamphetamine, and methadone have been the most widely monitored substances (5 studies). Different studies have also monitored various new psychoactive substances (NPS). So far, estimated consumption in prison settings has generally been lower than in the community, with the exception of cannabis and cocaine in French prisons. WBE has proven to be a valuable complementary tool to inform drug consumption in prison settings, as it can offer objective data. However, the number of publications is still limited. Studies focusing on gabapentinoids, synthetic cannabinoids, or enantiomeric profiling should be considered in future research. Additionally, the use of WBE to monitor the effectiveness of interventions offers a great potential as well.
Role of the Vibriolysin VemA Secreted by the Emergent Pathogen Vibrio europaeus in the Colonization of Manila Clam Mucus
(MDPI, 2022-12-01) Martínez, Clara; Rodriguez, Sergio; Vences, Ana; Barja Pérez, Juan Luis; Estévez Toranzo, María Alicia Carolina; Dubert Pérez, Javier; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
Vibrio europaeus is an emergent pathogen affecting clams, oysters and scallops produced in the most important countries for bivalve aquaculture. Studies concerning virulence factors involved in the virulence of V. europaeus are very scarce despite its global significance for aquaculture. Zinc-metalloproteases have been described as a major virulence factor in some Vibrio spp., although their contribution and role in the virulence of V. europaeus is not clear. To address this, we have studied an extracellular zinc-metalloprotease (VemA) encoded by V. europaeus, which was identified as a vibriolysin, highly conserved in this species and homologous in other pathogenic and non-pathogenic species. Virulence challenge experiments demonstrated that infection processes were faster when Manila clam larvae and juveniles were infected with the wildtype rather than with a mutant defective in the vemA gene (ΔvemA). V. europaeus was able to resist the bactericidal action of mucus and displayed a chemotaxis ability favoured by VemA to colonize the body mucus of clams and form a biofilm. The overall results suggest that VemA, although it is not a major virulence factor, plays a role in the colonization of the Manila clam mucus, and thus boosts the infection process as we observed in virulence challenge experiments.
Following the infection process of vibriosis in Manila clam (Ruditapes philippinarum) larvae through GFP-tagged pathogenic Vibrio species
(Elsevier, 2016-01-01) Dubert Pérez, Javier; Nelson, David. R.; Spinard, Edward J.; Kessner, Linda; Gomez-Chiarri, Marta; da Costa, Fiz; Prado Plana, Susana; Barja Pérez, Juan Luis; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA)
Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2 h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6–8 h), colonizing the larvae completely at the peak of infection (Stage III) (14–24 h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was demonstrated in fish and homeothermic cell lines for the first time. That activity was lost after heat treatment.
Beneficial effects of carpet shell clam (Ruditapes decussatus) depuration during short periods of conditioning in shellfish hatchery: Role of the temperature and phytoplankton on reduction and diversity of vibrios
(Elsevier, 2016-06-01) Dubert Pérez, Javier; da Costa, Fiz; Aranda-Burgos, Jose A.; Martínez-Patiño, Dorotea; Prado Plana, Susana; Barja Pérez, Juan Luis; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA)
Broodstock conditioning in hatcheries is the step previous to spawning and its optimization may be a key to the success of larval cultures. Cleaning and brushing of the broodstock and the utilization of antibiotics to reduce their vibrios load are used regularly as routine prophylactic measures prior to spawning induction. The development of protocols to reduce these bacteria using cheap and harmless techniques is of utmost importance for commercial bivalve production in hatcheries. With this aim, we have evaluated initially different conditionings (A–D) during short periods (a total of four weeks): first two weeks under gradient temperature (increasing + 0.3 °C day− 1 from 14.5 °C to 20 °C), without (A) or with phytoplankton (B), and constant temperature (20 °C) without (C) or with phytoplankton (D). Afterwards, all conditionings were kept at 20 °C and fed for two more weeks. Furthermore, broodstock optimal feeding time was re-evaluated during a second trial series. In all conditionings, bacterial loads were determined in terms of marine heterotrophic bacteria (MHB) and presumptive vibrios (PV). Broodstock under the optimal short period of conditioning (conditioning C) obtained the best gonadal development and a significant reduction in PV load at a lower expense. A total of 61 PV were isolated from all conditionings and identified by sequencing the 16S rRNA gene. Splendidus clade was dominant in the samples coming from natural beds. Diversity of vibrios changed throughout the conditionings in the hatchery favoured by exogenous factors whose effect was mainly observed at the end of the trials: Splendidus clade was also dominant in the broodstock conditioned at gradient temperature and Mediterranei and Harveyi clades were prevalent at constant temperature. Moreover, the percentage of transformation to D-larvae was estimated and the vertical transmission of vibrios from broodstock to eggs and D-larvae was suggested. Implementation of conditioning C reduces considerably the Vibrio load of clams without using antibiotics, and thus it represents a novel, cheap, environmental friendly and harmless methodology that can be easily transferred to commercial hatchery.
Persistence of antibiotic resistant Vibrio spp. in shellfish hatchery environment
(Springer, 2016-11-01) Dubert Pérez, Javier; Rodríguez Osorio, Carlos; Prado Plana, Susana; Barja Pérez, Juan Luis; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA); Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
The characterization of antibiotic-resistant vibrios isolated from shellfish aquaculture is necessary to elucidate the potential transfer of resistance and to establish effective strategies against vibriosis. With this aim, we analyzed a collection of bacterial isolates obtained from 15 failed hatchery larval cultures that, for the most part, had been treated experimentally with chloramphenicol to prevent vibriosis. Isolates were obtained during a 2-year study from experimental cultures of five different clam species. Among a total of 121 Vibrio isolates studied, 28 were found to be chloramphenicol resistant, suggesting that the shellfish hatchery had been using a sublethal concentration of the antibiotic. Interestingly, chloramphenicol-resistant vibrios showed also resistance to tetracycline and amoxicillin (group A; n = 19) or to streptomycin (group B; n = 9). Chloramphenicol-resistant vibrios were subjected to a PCR amplification and DNA sequencing of the chloramphenicol acetyltransferase genes (cat), and the same approach was followed to study the tetracycline resistance markers (tet). 16S ribosomal RNA (rRNA) gene sequencing revealed that chloramphenicol-resistant vibrios pertained mostly to the Splendidus clade. Conjugation assays demonstrated that various R-plasmids which harbored the cat II/tet(D) genes and cat III gene in groups A and B respectively, were transferred to E. coli and bivalve pathogenic vibrios. Most interestingly, transconjugants exhibited the antibiotic resistance patterns of the donors, despite having been selected only on the basis of chloramphenicol resistance. This is the first report carried out in a bivalve hatchery elucidating the persistence of resistant v
Integration of a 3D-printed electrochemical reactor with a tubular membrane photoreactor to promote sulfate-based advanced oxidation processes
(Elsevier, 2024-10-24) Olivera, Agustina R. de; Montes Goyanes, Rosa; Quintana Álvarez, José Benito; Rodil Rodríguez, María del Rosario; Vilar, Vítor J. P.; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Investigación e Análises Alimentarias; Universidade de Santiago de Compostela. Instituto de Acuicultura (IA)
This study investigates the integration of an in-house 3D printed electrochemical cell − SERPIC-UCLM® cell – for the in situ generation of peroxymonosulfuric acid (PMSA) with a lab-scale tubular membrane photoreactor (TMPr) to evaluate the effectiveness of sulfate-radical advanced oxidation processes (SR-AOPs) in eliminating contaminants of emerging concern (CECs) from reverse osmosis and nanofiltration concentrates (ROC and NFC, respectively). First, the SERPIC-UCLM® cell was evaluated in terms of mass transport features employing the limiting current technique, demonstrating favorable volumetric mass transport rates (kmA ∼ 10–3 s–1) and Sherwood values (Sh > 300) under the laminar flow regime (110 < Reynolds (Re) < 790). Afterward, the effect of the electrolyte (sulfuric acid, H2SO4) initial pH in the electrochemical generation of PMSA was studied, with an initial pH of 1 selected as optimal. PMSA is a highly reactive peroxyacid that undergoes self-decomposition at neutral pH media (e.g., ROC and NFC with a pH of 7.6 and 7.9, respectively), primarily existing in the form of peroxomonosulfate (PMS). Additionally, the phototreatment of the ROC and NFC was assessed using the electrogenerated PMS and commercial peroxydisulfate (PDS) under the same conditions. The results indicated comparable degradation patterns for CECs in both ROC and NFC. Furthermore, the application of 2.4 mM PMS resulted in removals higher than 60 % for 7 of the 11 CECs identified in the NFC, and ensured compliance with wastewater discharge regulations for pH, chemical oxygen demand (COD), and total suspended solids (TSS) levels. These findings emphasize the importance of this technology, showing its advantages in terms of versatility and logistics.
Bioaccessibility of plastic-related compounds from polymeric particles in marine settings: Are microplastics the principal vector of phthalate ester congeners and bisphenol A towards marine vertebrates?
(Elsevier, 2024-09-25) López Vázquez, Javier; Miró, Manuel; Quintana Álvarez, José Benito; Cela Torrijos, Rafael; Ferriol, Pere; Rodil Rodríguez, María del Rosario; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura; Universidade de Santiago de Compostela. Instituto de Investigación e Análises Alimentarias
Marine vertebrates are known to ingest significant amounts of microplastics (MPs). Once ingested, MPs might cause gastrointestinal injuries and serve as a path of harmful plastic components, such as phthalate esters (PAEs) and bisphenol A (BPA) in the food chain. However, there is a lack of standardized in-vitro methods capable of simulating fish uptake of chemicals from MPs in the environment as potential vectors of such contaminants. In this work, leaching and in-vitro oral bioaccessibility testing of PAEs and BPA from MPs were conducted batchwise using artificial seawater and gut fluids mimicking gastric, intestinal, and gastrointestinal compartments of marine vertebrates at physiological temperature. The environmental and physiologically relevant extraction tests were applied to medium-density polyethylene (PE) and polyvinyl chloride (PVC) certified reference materials containing eight PAEs of varying hydrophobicity, namely, dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate, benzylbutyl phthalate, diethylhexyl phthalate, di-n-octyl phthalate, diisononyl phthalate and diisodecyl phthalate, and BPA (only in PE) as MP surrogates with realistic analyte concentrations of additives for primary MPs. The analysis of the leachates/gut fluid extracts was performed via dilute-and-shoot by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Only the most hydrophilic compounds, i.e. DMP, DEP and BPA, were found to get released significantly in saline waters, and exhibited the highest oral bioaccessibility rates (34–83 %). Based on our results, a dual-compartment physiologically relevant gastrointestinal test is recommended for appropriate estimation of fish bioaccessibility. The fish daily intakes of DMP, DEP and BPA from MPs, and seawater ingestion as well were estimated using several contamination scenarios (10th percentile as the low level, 50th percentile as the medium level and 90th percentile as the high level) based on probabilistic distributions and cumulative probability curves of measured environmental concentrations of (i) MPs in seawater throughout the world, (ii) DMP, DEP and BPA in beached MPs and those sampled in the open ocean (including both incurred and adsorbed contaminants), and (iii) DMP, DEP and BPA in seawater as reported in recent literature. Under a medium-level concentration scenario (50th percentile) in marine settings, and taking the gastrointestinal bioaccessibility factor into account, the daily intake of DMP, DEP and BPA from MPs accounted for a mere 0.02 % of the waterborne contribution. Hence, the ingestion of MPs should not be considered the primary route of fish exposure to BPA and the most polar PAEs in marine environments. However, more studies on the local and the global scales for mass concentrations of MPs and additives in marine settings are needed for further confirmation of our findings.
Determination of the urinary concentrations of six bisphenols in public servants by online solid-phase extraction-liquid chromatography tandem mass spectrometry
(Springer, 2024-06-18) Estévez-Danta, Andrea; Rodil Rodríguez, María del Rosario; Quintana Álvarez, José Benito; Montes Goyanes, Rosa; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura; Universidade de Santiago de Compostela. Instituto de Investigación e Análises Alimentarias
Bisphenols are widely used as monomers and additives in plastic production. Thus, bisphenol A (BPA) and its most prominent substitutes have been detected in many environmental and human samples. This study proposes an online solid-phase extraction analytical methodology coupled to liquid chromatography with tandem mass spectrometry for the determination of six bisphenols (BPA and bisphenols F (BPF), S (BPS), AF (BPAF), B (BPB), and E (BPE)) in urine samples as an efficient and automated methodology. The method was developed and validated for all bisphenols with good recoveries (92–112%) and repeatability (RSD ≤ 10%) despite the variable matrix effects, except BPAF (which would require a dedicated internal standard), achieving method quantification limits in the 0.05–2.2 ng mL−1 range. The methodology was subsequently applied to 435 urine samples from a non-occupational exposure population (civil servants for the regional government) from Santiago de Compostela (Galicia, Spain). Only BPA, BPF, and BPS were positively detected; the last two presented higher detection frequencies than BPA. When the urinary concentrations are extrapolated to human intake and compared to the European Food Safety Agency (EFSA) tolerable daily intake (TDI) of 2 × 10−4 µg kg−1 day−1 (TDI), all BPA positively identified samples would surpass this threshold. Although no TDI exists currently for the other two identified bisphenols, it is evident that human exposure to bisphenols should be limited. Finally, the results stratification by gender revealed higher levels of exposure to BPF in the women group.
Detection of different Betanodavirus genotypes in wild fish from Spanish Atlantic coastal waters (Galicia, northwestern Spain)
(American Fisheries Society, 2023) Vázquez Salgado, Lucía; Olveira Hermida, José Gabriel; Pereira Dopazo, Carlos; Bandín Matos, Isabel; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura
Objective The nervous necrosis virus (NNV; genus Betanodavirus) is an aquatic pathogen that is responsible for a neurological disease affecting marine fish. Despite its almost worldwide distribution, global warming could favor the spread of NNV to new areas, highlighting the importance of conducting epidemiological surveys on both wild and farmed marine fish species. In this study, we assessed NNV prevalence in wild fish caught along the Galician Atlantic coast. Methods In total, 1277 fish were analyzed by reverse transcription real-time polymerase chain reaction. Result Twenty two (1.72%) of those fish tested positive for NNV, including two species in which the pathogen had not yet been reported. Conclusion The reassortant RGNNV/SJNNV (red-spotted grouper NNV/striped jack NNV) was detected in 55% of NNV-positive individuals, while the remaining 45% harbored the SJNNV-type genome. Moreover, from European Pilchard Sardina pilchardus and Atlantic Mackerel Scomber scombrus, we isolated four reassortant strains that carried amino acid mutations at key sites related to NNV–host interaction.
Screening of organic chemicals associated to virgin low-density polyethylene microplastic pellets exposed to the Mediterranean Sea environment by combining gas chromatography and liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry
(Elsevier, 2024-03-01) López Vázquez, Javier; Rodil Rodríguez, María del Rosario; Álvarez, Elvira; Alomar, Carme; Cela Torrijos, Rafael; Miró, Manuel; Quintana Álvarez, José Benito; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura
In this work, organic chemicals associated with microplastics (MPs) exposed to a coastal anthropogenized environment for up to eight weeks have been screened for, in order to discern the (de)sorption dynamics of chemicals in the marine ecosystem. Low-density polyethylene (LDPE) pellets were studied since they represent primary MPs used by the plastic industry and a relevant input of MPs into the oceans. To maximize the coverage of chemicals that could be detected, both liquid and gas chromatography coupled to quadrupole-time-of-flight (GC-QTOF and LC-QTOF, respectively) were used. In the case of LC-QTOF, an electrospray ionization source was employed, and the compounds were investigated by combining suspect and non-target screening workflows. The GC-QTOF was equipped with an electron ionization source and compounds were screened in raw and derivatized (silylated) extracts by deconvolution and contrast to high- and low-resolution libraries. A total of 50 compounds of multifarious classes were tentatively identified. Among them, melamine and 2-ethylhexyl salicylate (EHS) were detected in the original MPs but were rapidly desorbed. Melamine was completely released into the marine environment, while EHS was partly released but a portion remained bound to the MPs. On the other hand, many other chemicals of both anthropogenic (e.g. phenanthrene or benzophenone) and natural origin (e.g. betaine and several fatty acids) accumulated onto MPs over time. Quantification of 12 unequivocally identified chemicals resulted into a total concentration of 810 μg/kg after MPs exposure for 8 weeks.
Insights into the application of the anodic oxidation process for the removal of per- and polyfluoroalkyl substances (PFAS) in water matrices
(Elsevier, 2024-01-23) López Vázquez, Javier; Santos, Carla S.; Montes Goyanes, Rosa; Rodil Rodríguez, María del Rosario; Quintana Álvarez, José Benito; Gäbler, J.; Schäfer, L.; Moreira, Francisca C.; Vilar, Vítor J. P.; Universidade de Santiago de Compostela. Departamento de Química Analítica, Nutrición e Bromatoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura
The current study aimed to investigate major knowledge gaps regarding the application of the anodic oxidation (AO) process with boron-doped diamond (BDD) anodes for the remediation of water matrices contaminated with per- and polyfluoroalkyl substances (PFAS). This included: (i) the degradation of ultrashort-chain (C1-C3) and long-chain (C9-C13) PFAS in addition to short- and medium-chain (C4-C8) PFAS, (ii) the application of multi-solute systems with different PFAS content (0.2 µg L−1 versus 2.0 µg L−1) and diversity (24 C1-C13 versus 8 C1-C8) in addition to single-solute systems, (iii) the use of real water matrices in addition to pure water, and (iv) the application of current densities (j) up to 250 mA cm−2 in addition to usual j (≤20 mA cm−2). C1-C4 PFAS with a sulfonated headgroup were the most recalcitrant compounds. By contrast, PFAS ≥ C9 with a sulfonated headgroup and PFAS ≥ C12 with a carboxylic headgroup were potentially instantaneously degraded. The content and diversity of PFAS mainly affected the degradation kinetics of PFEtS (C2), PFPrA (C3), and PFBA (C4). Four real water matrices were under focus: drinking water (DW), urban wastewater after secondary treatment (UWW), and nanofiltration concentrate (NF) and reverse osmosis concentrate (RO) from urban wastewater polishing step. PFAS degradation typically benefited from using real matrices primarily due to the presence of chloride ions and consequent electrogeneration of active chlorine species. However, for waters with a high organic content, namely a chemical oxygen demand (COD) of 319 mg O2 L−1, PFAS degradation was hindered. Furthermore, the removal of most PFAS benefited from the application of j > 20 mA cm−2, and some specific PFAS required the use of j ≥ 250 mA cm−2 to have maximized removal rates.
Genetic diferentiation of a critically endangered population of the limpet Patella candei candei d’Orbigny, 1840, in the Canary Islands
(Springer, 2022) Quinteiro Vázquez, Javier; González-Lorenzo, Gustavo; Hernández Reyes, Dailos; Quinteiro, Lara; Herrera Pérez, Rogelio; Martínez Barrio, Juan; González Ramos, Antonio Juan; Rey Méndez, Manuel; González Henríquez, Nieves; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Instituto de Acuicultura
The adoption of measures to protect the viability of threatened populations should be supported by empirical data identifying appropriate conservation units and management strategies. The global population of the majorera limpet, P. candei candei d’Orbigny, 1840, is restricted to the Macaronesian islands in the NE Atlantic, including near-to-extinct and healthy populations in Fuerteventura and Selvagens, respectively. The taxonomic position, genetic diversity and intra- and interspecific relationships of these populations are unclear, which is hindering the implementation of a recovery plan for the overexploited majorera limpet on Fuerteventura. In this study, ddRAD-based genome scanning was used to overcome the limitations of mitochondrial DNA-based analysis. As a result, P. candei candei was genetically differentiated from the closely related P. candei crenata for the first time. Moreover, genetic differentiation was detected between P. candei candei samples from Selvagens and Fuerteventura, indicating that translocations from the healthy Selvagens source population are inadvisable. In conclusion, the majorera limpet requires population-specific management focused on the preservation of exceptional genetic diversity with which to face future environmental challenges
Vibrio neptunius produces piscibactin and amphibactin and both siderophores contribute significantly to virulence for clams
(Frontiers, 2021) Galvis Serrano, Nestor Fabián; Ageitos, Lucía; Rodríguez González, Jaime; Jiménez González, Carlos; Barja Pérez, Juan Luis; Balado Dacosta, Miguel; Lemos Ramos, Manuel Luis; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura
Vibrio neptunius is an inhabitant of mollusc microbiota and an opportunistic pathogen causing disease outbreaks in marine bivalve mollusc species including oysters and clams. Virulence of mollusc pathogenic vibrios is mainly associated with the production of extracellular products. However, siderophore production is a common feature in pathogenic marine bacteria but its role in fitness and virulence of mollusc pathogens remains unknown. We previously found that V. neptunius produces amphibactin, one of the most abundant siderophores in marine microbes. In this work, synthesis of the siderophore piscibactin was identified as the second siderophore produced by V. neptunius. Single and double mutants in biosynthetic genes of each siderophore system, piscibactin and amphibactin, were constructed in V. neptunius and their role in growth ability and virulence was characterized. Although the High Pathogenicity Island encoding piscibactin is a major virulence factor in vibrios pathogenic for fish, the V. neptunius wild type did not cause mortality in turbot. The results showed that amphibactin contributes more than piscibactin to bacterial fitness in vitro. However, infection challenges showed that each siderophore system contributes equally to virulence for molluscs. The V. neptunius strain unable to produce any siderophore was severely impaired to cause vibriosis in clams. Although the inactivation of one of the two siderophore systems (either amphibactin or piscibactin) significantly reduced virulence compared to the wild type strain, the ability to produce both siderophores simultaneously maximised the degree of virulence. Evaluation of the gene expression pattern of each siderophore system showed that they are simultaneously expressed when V. neptunius is cultivated under low iron availability in vitro and ex vivo. Finally, the analysis of the distribution of siderophore systems in genomes of Vibrio spp. pathogenic for molluscs showed that the gene clusters encoding amphibactin and piscibactin are widespread in the Coralliilyticus clade. Thus, siderophore production would constitute a key virulence factor for bivalve molluscs pathogenic vibrios
The Vibriolysin-Like Protease VnpA and the Collagenase ColA Are Required for Full Virulence of the Bivalve Mollusks Pathogen Vibrio neptunius
(MDPI, 2021) Galvis Serrano, Nestor Fabián; Barja Pérez, Juan Luis; Balado Dacosta, Miguel; Lemos Ramos, Manuel Luis; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Instituto de Acuicultura
Vibrio neptunius is an important pathogen of bivalve mollusks worldwide. Several metalloproteases have been described as virulence factors in species of Vibrio that are pathogenic to bivalves, but little is known about the contribution of these potential virulence factors to Vibrio neptunius pathogenesis. In silico analysis of the genome of V. neptunius strain PP-145.98 led to the identification of two hitherto uncharacterized chromosomal loci encoding a probable vibriolysin-like metalloprotease and a putative collagenase, which were designated VnpA and ColA, respectively. Single defective mutants of each gene were obtained in V. neptunius PP-145.98, and the phospholipase, esterase and collagenase activities were studied and compared with those of the wild-type strain. The results showed that the single inactivation of vnpA resulted in a 3-fold reduction in phospholipase/esterase activity. Inactivation of colA reduced the collagenase activity by 50%. Finally, infection challenges performed in oyster larvae showed that ΔvnpA and ΔcolA—single mutant strains of V. neptunius—are between 2–3-fold less virulent than the wild-type strain. Thus, the present work demonstrates that the production of both VnpA and ColA is required for the full virulence of the bivalve pathogen V. neptunius

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