Photodynamic antimicrobial chemotherapy for pathogenic vibrio control in prawn hatcheries

In the last two decades, the aquaculture industry has grown significantly, providing fish, molluscs and crustaceans to the global market. Microbial pathogens are the principal cause of massive financial losses in many fish and crustacean farms. Disinfection using ozone, chlorine or UV irradiation are expensive and leave toxic byproducts. Biosecurity threats through the introduction of wild brood-stock and live feed is a major concern for the aquaculture industry. Microalgae and/or Artemia nauplii (brine shrimp), have been identified as potential vectors for microbial pathogens that can result in high mortalities, particularly in hatcheries. Bacteria can proliferate rapidly in intensive aquaculture productions due to the high density of target animals, live feed organisms and build-up of biological waste that generate ideal conditions for many potential pathogens. The current inability to bio-secure farmed aquaculture animals through appropriate disinfection strategies of live feed without altering the quality of the final product is an area requiring urgent research, as it is the main barrier to cost-effective product development; the main hurdle to competitive expansions into national and international markets. Recently, photodynamic antimicrobial chemotherapy (PACT) has emerged as a promising water sterilisation technique. PACT uses the activation of photosensitisers by light to generate highly reactive oxygen species (ROS), which indiscriminately oxidises cell wall and cell membrane components (i.e. lipids, proteins, carbohydrates). After entry of the photosensitiser into cells, produced singlet oxygen also targets intracellular components such as organelles, membrane compartments, and nucleic acids (i.e. DNA and RNA). Due to the indiscriminate action of ¹O₂, resistance cannot develop, unlike in the case of antibiotics. The choice of photosensitiser is crucial, and compounds that photobleach with time are preferred for aquaculture purposes to avoid build up in water or the farmed animal. Porphyrins are a group of natural or synthetic compounds that fit the photobleaching requirement, but to date no research has specifically investigated porphyrin-based sterilisation efficiency in prawn hatcheries. In addition, the porphyrin-based antimicrobial efficiency for the ones used in this research has not been tested against luminescent virulent strains of Vibrio harveyi-related species, the main causative agents for luminescent vibriosis, which causes large losses for finfish, bivalves and prawn industries. Each chapter of this thesis addresses specific objectives that, in conjunction with the other chapters, contribute to the principal aim of the research: investigate the potential of using PACT to control Vibrio bacteria in aquaculture. Chapter 2 details objectives relating to method development to select a suitable Vibrio harveyi-related strain to be used in the following chapters. While Chapter 1 reviews PACT history and its application with special interest to aquaculture, the method development chapter (chapter 2), pathogenicity towards prawns after injection and luminescence intensity of two selected Vibrio strains were evaluated. Challenge experiments were performed with Penaeus monodon and Koch's postulates were fulfilled. Precise identification was obtained using molecular techniques including multiplex PCR, housekeeping gene analysis and construction of a phylogenetic tree. The strongly luminescent Vibrio sp ISO7, was demonstrated to be highly virulent towards P. monodon, killing 100% of the injected animals and molecular techniques revealed that this species belongs to the V. campbellii group. In contrast, V. owensii 47666-1, previously described as a luminescent prawn larvae pathogen, caused only 25% mortality in the first challenge experiment. Pathogenicity was regained in bacteria re-isolated from sick prawns, with 100% mortality of P. monodon obtained in a second challenge experiment, however the strain was found to be not luminescent. Hence, V. campbellii ISO7 was selected as the model bacterium based on its high virulence after injection and strong luminescence signal, making it suitable for the development of a fast luminescence-based assay to determine the efficiency of different porphyrin treatment protocols. In chapter 3, the objectives were 1) to compare the photostability of the tetracationic 5,10,15,20-tetrakis (1-methyl-4-pyridinio) porphyrin tetra (p-toluenesulfonate) [TMPyP] and the tetra-anionic 4,4',4",4"-(porphine-5,10,15,20-tetrayl) tetrakis (benzenesulfonic acid) tetrasodium salt hydrate [TPPS₄] porphyrins in seawater to confirm their suitability for aquaculture purposes and 2) to investigate the capacity of TMPyP and TPPS₄ to inactivate the model bacterium Vibrio campbellii ISO7. For the first objective, the photostability of the cationic TMPyP and the anionic TPPS₄ porphyrins were investigated by recording their full light spectra between 350 and 750 nm after irradiating seawater containing different porphyrin concentrations (1, 5, 10 and 20 μM) of porphyrins with high power 150 W LED lights. Results obtained using the maximum peak of absorbance for each porphyrin, showed porphyrin degradation (photobleaching) for both the cationic and the anionic porphyrin within 24 h of irradiation. In addition, the dark control (samples not irradiated) did not photobleach, confirming that porphyrin degradation was due to exposure to light. The results confirmed that both porphyrins were good candidates for aquaculture applications based on their photobleaching properties showing relatively fast degradation(within 24-h at concentration of 1 μM and more than ¼ of absorbance reduction at the concentration of 20 μM). Antimicrobial activity of the porphyrins was investigated using dose-response and time-course experiments. Twenty μM of the cationic porphyrin achieved 100% lethality in the model bacterium (start concentration of V. campbellii ISO7 ~10⁷ CFU·mL⁻¹) after five hours, which was validated using biological activity (luminescence), growth experiments (CFU, absorbance) and 7-day regrowth experiments. Consistent with previous reports, the anionic photosensitizer did not achieve inactivation of the model bacterium and was therefore not investigated further. As demonstrated, water sterilization was achieved after between five and twelve hours depending on the concentration of the cationic porphyrin; however, the effectiveness of sterilization in mixed culture with live feed organisms still needed to be confirmed. In chapters 4 and 5, the potential of using the TMPyP porphyrin to reduce bacteria loads of microalgae cultures (free of the model bacterium) and Artemia cysts was investigated including an evaluation of possible toxic effects caused by singlet oxygen generated during PACT towards the live feed organisms themselves. In chapter 4, the viability of microalgae cells was first evaluated using flow cytometry based on chlorophyll fluorescence and the live-dead stain Propidium Iodide (PI) during a six-hour dose-response treatment with the porphyrin. The treatment time was chosen based on bacterial disinfection results shown in chapter 3. Sensitivity to ¹O₂ was speciesspecific and related to cell wall characteristics. Of the five different microalgae used, only Nannochloropsis oculata was highly resilient to the six-hour treatment with up to 50 μM of the cationic porphyrin. The results were unexpected, as photosynthetic microorganisms possess detoxification systems for ¹O₂ and other reactive oxygen species (ROS). However, ¹O₂ produced externally to the cell may be able to photooxygenate and destabilize cell membrane components, leading to cell death. The thick cell wall of N. oculata most likely protected the cell membrane from fast photooxidation. The highly resilient microalga, N. oculata was therefore used in mixed culture with the model pathogen and treated with 20 μM final concentration of the TMPyP porphyrin. Complete inactivation of the model bacterium was successfully achieved, as verified by absence of luminescent CFUs on agar plates and a species-specific molecular technique that can detect the model organisms with high sensitivity (Multiplex PCR combined with Most Probable Number enrichment). In chapter 5, possible toxicity of the cationic porphyrin and ¹O₂ against two different types of Artemia cysts (magnetic and unmodified) was tested in dose-response experiments using percentage of cyst hatching as the measured variable. Surprisingly, magnetic cysts showed improved hatching under sub-optimal hatching conditions in the presence of porphyrin-generated ROS and the porphyrin TMPyP alone (i.e. independent to the production of ¹O₂) relative to the controls. In contrast, dose-response experiments with unmodified cysts showed a positive hatching response only to TMPyP-generated ¹O₂ but not TMPyP itself. Further investigations showed that when magnetic cysts were mixed with the model pathogen, complete pathogen inactivation was achieved after six hours of incubation with 20 μM of TMPyP in the light. In conclusion, my research demonstrated that PACT is suitable as an additional (or alternative) sterilization method in prawn hatcheries and potentially for aquaculture water treatment in general

Relaciones entre talla del otolito y talla del pez en algunos peces mesopelágicos y batipelágicos del Mediterráneo (estrecho de Messina, Italia)

The length-weight relationships and the regressions between otolith size (length and width) and fish length of some mesopelagic and bathypelagic fishes living in the central Mediterranean Sea were provided. Images and morphological description of otoliths (sagittae) from 16 species belonging to the families of Gonostomatidae (1), Microstomatidae (2), Myctophidae (8), Phosichthyidae (2), Sternoptychidae (2) and Stomiidae (1) were given. The length-weight relationship showed an isometric growth in 13 species. No differences between right and left otolith sizes were detected by t-test, so a single linear regression was plotted against standard length (SL) for otolith length (OL) and otolith width (OW). Data fitted well to the regression model for both OL and OW to SL, for each species (R2 > 0.8). These relationships offer a helpful tool in feeding studies and also provide support to palaeontologists in their research on fish fossils.En este trabajo se presentan las relaciones talla-peso y las regresiones entre la talla del otolito (longitud y anchura) y la talla del pez (SL) para algunos peces meso y batipelágicos que viven en el Mediterráneo central. Se incluyen imágenes y descripciones morfológicas de los otolitos (sagittae) de 16 especies de las familias Gonostomatidae (1), Microstomatidae (2), Myctophidae (8), Phosichthyidae (2), Sternoptychidae (2) y Stomiidae (1). La relación talla-peso muestra un crecimiento isométrico en 13 especies. No se encontraron diferencias relevantes (t-test) entre la talla del otolito derecho e izquierdo, por lo que se representó una sola regresión lineal entre la longitud estándar y los siguientes parámetros: longitud de otolite (OL) y la anchura (OW). Los datos se ajustaron bien al modelo de regresión lineal para OL y OW, para todas las especies (R2 > 0.8). Las relaciones obtenidas puede ser utilizadas como instrumento en estudios de alimentación y para sustentar estudios de paleontología sobre peces fósiles

Edad y crecimiento del pámpano, Trachinotus ovatus, del Estrecho de Messina (Mediterráneo central)

This is the first paper to provide detailed information on the age and growth of Trachinotus ovatus. The size of the 244 individuals collected in the Strait of Messina ranged from 2.7 to 30.4 cm in fork length (LF) and 0.31 to 508.6 g in body mass (M). The relationship between these parameters (M - LF) was investigated and showed a good fit. Age estimation based on vertebrae and otoliths yielded similar results, suggesting a maximum age of five years. However, the precision and accuracy tests, such as percentage of agreement (PA), mean coefficient of variation (ACV) and average percent error (APE) indicated that the otolith readings (97.83% PA, 0.54% ACV and 0.38% APE) were more reliable for age estimation than vertebrae readings (82.17% PA, 5.33% ACV and 3.77% APE). The multi-model inference approach allowed us to compare different non-linear growth models. The von Bertalanffy model (L∞=29.139, k=0.496 and t0=−0.347) fitted the length-at-age data best. This species has a relatively rapid growth and an estimated longevity of five to seven years. This information could be used for management and first stock assessment studies on T. ovatus in the Mediterranean Sea.Este es el primer artículo que proporciona información detallada sobre la edad y el crecimiento de Trachinotus ovatus. El tamaño de los 244 individuos recolectados en el Estrecho de Messina osciló entre 2.7 y 30.4 cm de longitud a la horquilla (LF) y 0.31 a 508.6 g de masa corporal (M). Se investigó la relación entre estos parámetros (M - LF) y mostró un buen ajuste. La estimación de la edad basada en vértebras y otolitos arrojó resultados similares, lo que sugiere una edad máxima de cinco años. Sin embargo, las pruebas de precisión y exactitud, como el porcentaje de concordancia (PA), el coeficiente medio de variación (ACV) y el porcentaje de error medio (APE), indicaron que las lecturas del otolito (97.83% PA, 0.54% ACV y 0.38 % APE) fueron más confiables para la estimación de la edad que las lecturas de vértebras (82.17% PA, 5.33% ACV y 3.77% APE). El enfoque de inferencia de modelos múltiples nos permitió comparar diferentes modelos de crecimiento no lineal. El modelo de von Bertalanffy (L∞=29.139, k=0.496 and t0=−0.347) se ajusta mejor a los datos de talla por edad. Esta especie tiene un crecimiento relativamente rápido y una longevidad estimada de cinco a siete años. Esta información podría utilizarse para la ordenación y los primeros estudios de evaluación de poblaciones de T. ovatus en el mar Mediterráneo

When opportunistic predators interact with swordfish harpoon fishing activities: shark depredation over catches in the Strait of Messina (central Mediterranean Sea)

We describe the interaction between harpoon fishing activity and sharks, which opportunistically depredated harpoon catches in the Strait of Messina. Shark bite marks were observed on harpooned swordfish during the period 2014–2020, with different damages to the catches. Most of the depredation events have focused on large swordfish, generally weighing more than 60 kg. Data on direct observations were implemented by interviews and questionnaires to fishermen aimed to recover the information on their local fishing and ecological knowledge. Fishermen provided additional data on shark-harpoon fishing interactions also supplying information on by-catch species (i.e., bluefin tuna). Therefore, these results suggest that sharks migrating through the Strait of Messina are occasionally attracted by injured prey, due to their ability to detect chemical cues, fish distress stimuli and body fluids (i.e. blood) in the water. In addition, our investigations showed an increase in shark attacks on harpooned fish over time, likely due to an increase in harpoon swordfish catches. This may be related to the effects of the driftnets' ban enforced by European Regulations in the last decades

Evidence of a predation event on a tagged Mediterranean spearfish (Tetrapturus belone; Pisces, Istiophoridae), inferred from pop-up satellite tagging data

The Strait of Messina is located at the centre of the Mediterranean Sea and is considered a biodiversity hotspot and an obligatory seasonal passage for different pelagic species such as sharks, marine mammals, and billfishes. For the first time, in the Strait of Messina, our research group tagged a Mediterranean spearfish (Tetrapturus belone) using a pop-up satellite archival tag (PSAT). The observation of abiotic parameters (depth, light, and temperature) recorded by the PSAT confirmed that the tagged specimen was predated after about nine hours. The tag was then regurgitated 14 days after the tag deployment date. The analysis of collected data seems to indicate that the predator may be an ectothermic shark, most likely the bluntnose sixgill shark (Hexanchus griseus)

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Combined searches for the production of supersymmetric top quark partners in proton-proton collisions at root s=13 TeV

A combination of searches for top squark pair production using proton-proton collision data at a center-of-mass energy of 13 TeV at the CERN LHC, corresponding to an integrated luminosity of 137 fb(-1) collected by the CMS experiment, is presented. Signatures with at least 2 jets and large missing transverse momentum are categorized into events with 0, 1, or 2 leptons. New results for regions of parameter space where the kinematical properties of top squark pair production and top quark pair production are very similar are presented. Depending on themodel, the combined result excludes a top squarkmass up to 1325 GeV for amassless neutralino, and a neutralinomass up to 700 GeV for a top squarkmass of 1150 GeV. Top squarks with masses from 145 to 295 GeV, for neutralino masses from 0 to 100 GeV, with a mass difference between the top squark and the neutralino in a window of 30 GeV around the mass of the top quark, are excluded for the first time with CMS data. The results of theses searches are also interpreted in an alternative signal model of dark matter production via a spin-0 mediator in association with a top quark pair. Upper limits are set on the cross section for mediator particle masses of up to 420 GeV

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe