From model organism to application: Bacteria-induced growth and development of the green seaweed Ulva and the potential of microbe leveraging in algal aquaculture.

The marine green macroalga Ulva (Chlorophyta, Ulvales), also known as sea lettuce, coexists with a diverse microbiome. Many Ulva species proliferate in nature and form green algal blooms ("green tides"), which can occur when nutrient-rich wastewater from agricultural or densely populated areas is flushed into the sea. Bacteria are necessary for the adhesion of Ulva to its substrate, its growth, and the development of its blade morphology. In the absence of certain bacteria, Ulva mutabilis develops into a callus-like morphotype. However, with the addition of the necessary marine bacteria, the entire morphogenesis can be restored. Surprisingly, just two bacteria isolated from U. mutabilis are sufficient for inducing morphogenesis and establishing the reductionist system of a tripartite community. While one bacterial strain causes algal blade cell division, another causes the differentiation of basal cells into a rhizoid and supports cell wall formation because of a low concentration of the morphogen thallusin (below 10-10 mol/L). This review focuses on the research conducted on this topic since 2015, discusses how U. mutabilis has developed into a model organism in chemical ecology, and explores the questions that have already been addressed and the perspectives that a reductionist model system allows. In particular, the field of systems biology will achieve a comprehensive, quantitative understanding of the dynamic interactions between Ulva and its associated bacteria to better predict the behavior of the system as a whole. The reductionist approach has enabled the study of the bacteria-induced morphogenesis of Ulva. Specific questions regarding the optimization of cultivation conditions as well as the yield of raw materials for the food and animal feed industries can be answered in the laboratory and through applied science. Genome sequencing, the improvement of genetic engineering tools, and the first promising attempts to leverage macroalgae-microbe interactions in aquaculture make this model organism, which has a comparatively short parthenogenetic life cycle, attractive for both fundamental and applied research. The reviewed research paves the way for the synthetic biology of macroalgae-associated microbiomes in sustainable aquacultures.

Diversity, evolution, and emergence of fish viruses.

The production of aquatic animals has more than doubled over the last 50 years and is anticipated to continually increase. While fish are recognized as a valuable and sustainable source of nutrition, particularly in the context of human population growth and climate change, the rapid expansion of aquaculture coincides with the emergence of highly pathogenic viruses that often spread globally through aquacultural practices. Here, we provide an overview of the fish virome and its relevance for disease emergence, with a focus on the insights gained through metagenomic sequencing, noting potential areas for future study. In particular, we describe the diversity and evolution of fish viruses, for which the majority have no known disease associations, and demonstrate how viruses emerge in fish populations, most notably at an expanding domestic-wild interface. We also show how wild fish are a powerful and tractable model system to study virus ecology and evolution more broadly and can be used to identify the major factors that shape vertebrate viromes. Central to this is a process of virus-host co-divergence that proceeds over many millions of years, combined with ongoing cross-species virus transmission.

Keratinocytes drive the epithelial hyperplasia key to sea lice resistance in coho salmon.

BACKGROUND: Salmonid species have followed markedly divergent evolutionary trajectories in their interactions with sea lice. While sea lice parasitism poses significant economic, environmental, and animal welfare challenges for Atlantic salmon (Salmo salar) aquaculture, coho salmon (Oncorhynchus kisutch) exhibit near-complete resistance to sea lice, achieved through a potent epithelial hyperplasia response leading to rapid louse detachment. The molecular mechanisms underlying these divergent responses to sea lice are unknown. RESULTS: We characterized the cellular and molecular responses of Atlantic salmon and coho salmon to sea lice using single-nuclei RNA sequencing. Juvenile fish were exposed to copepodid sea lice (Lepeophtheirus salmonis), and lice-attached pelvic fin and skin samples were collected 12 h, 24 h, 36 h, 48 h, and 60 h after exposure, along with control samples. Comparative analysis of control and treatment samples revealed an immune and wound-healing response that was common to both species, but attenuated in Atlantic salmon, potentially reflecting greater sea louse immunomodulation. Our results revealed unique but complementary roles of three layers of keratinocytes in the epithelial hyperplasia response leading to rapid sea lice rejection in coho salmon. Our results suggest that basal keratinocytes direct the expansion and mobility of intermediate and, especially, superficial keratinocytes, which eventually encapsulate the parasite. CONCLUSIONS: Our results highlight the key role of keratinocytes in coho salmon's sea lice resistance and the diverged biological response of the two salmonid host species when interacting with this parasite. This study has identified key pathways and candidate genes that could be manipulated using various biotechnological solutions to improve Atlantic salmon sea lice resistance.

Genetic diversity and population structure of farmed and wild Nile tilapia (Oreochromis niloticus) in Uganda: The potential for aquaculture selection and breeding programs.

Nile tilapia is one of the most important aquaculture species globally, providing high-quality animal protein for human nutrition and a source of income to sustain the livelihoods of many people in low- and middle-income countries. This species is native to Africa and nowadays farmed throughout the world. However, the genetic makeup of its native populations remains poorly characterized. Additionally, there has been important introgression and movement of farmed (as well as wild) strains connected to tilapia aquaculture in Africa, yet the relationship between wild and farmed populations is unknown in most of the continent. Genetic characterization of the species in Africa has the potential to support the conservation of the species as well as supporting selective breeding to improve the indigenous strains for sustainable and profitable aquaculture production. In the current study, a total of 382 fish were used to investigate the genetic structure, diversity, and ancestry within and between Ugandan Nile tilapia populations from three major lakes including Lake Albert (L. Albert), Lake Kyoga (L. Kyoga) and Lake Victoria (L. Victoria), and 10 hatchery farms located in the catchment regions of these lakes. Our results showed clear genetic structure of the fish sourced from the lakes, with L. Kyoga and L. Albert populations showing higher genetic similarity. We also observed noticeable genetic structure among farmed populations, with most of them being genetically similar to L. Albert and L. Kyoga fish. Admixture results showed a higher (2.55-52.75%) contribution of L. Albert / L. Kyoga stocks to Uganda's farmed fish than the stock from L. Victoria (2.12-28.02%). We observed relatively high genetic diversity across both wild and farmed populations, but some farms had sizable numbers of highly inbred fish, raising concerns about management practices. In addition, we identified a genomic region on chromosome 5, harbouring the key innate immune gene BPI and the key growth gene GHRH, putatively under selection in the Ugandan Nile tilapia population. This region overlaps with the genomic region previously identified to be associated with growth rate in farmed Nile tilapia.

Hypothalamic transcriptome response to simulated diel earthen pond hypoxia cycles in channel catfish (Ictalurus punctatus).

Commercial culture of channel catfish (Ictalurus punctatus) occurs in earthen ponds that are characterized by diel swings in dissolved oxygen concentration that can fall to severe levels of hypoxia, which can suppress appetite and lead to suboptimal growth. Given the significance of the hypothalamus in regulating these processes in other fishes, an investigation into the hypothalamus transcriptome was conducted to identify specific genes and expression patterns responding to hypoxia. Channel catfish in normoxic water were compared with catfish subjected to 12 h of hypoxia (20% oxygen saturation; 1.8 mg O2/L; 27°C) followed by 12 h of recovery in normoxia to mimic 24 h in a catfish aquaculture pond. Fish were sampled at 0-, 6-, 12-, 18-, and 24-h timepoints, with the 6- and 12-h samplings occurring during hypoxia. A total of 190 genes were differentially expressed during the experiment, with most occurring during hypoxia and returning to baseline values within 6 h of normoxia. Differentially expressed genes were sorted by function into Gene Ontology biological processes and revealed that most were categorized as "response to hypoxia," "sprouting angiogenesis," and "cellular response to xenobiotic stimulus." The patterns of gene expression reported here suggest that transcriptome responses to hypoxia are broad and quickly reversibly with the onset of normoxia. Although no genes commonly reported to modulate appetite were found to be differentially expressed in this experiment, several candidates were identified for future studies investigating the interplay between hypoxia and appetite in channel catfish, including adm, igfbp1a, igfbp7, and stc2b.NEW & NOTEWORTHY Channel catfish are an economically important species that experience diel episodic periods of hypoxia that can reduce appetite. This is the first study to investigate their transcriptome from the hypothalamus in a simulated 24-h span in a commercial catfish pond, with 12 h of hypoxia and 12 h of normoxia. The research revealed functional groups of genes relating to hypoxia, angiogenesis, and glycolysis as well as individual target genes possibly involved in appetite regulation.

Decoding the Arsenal: Protist Effectors and Their Impact on Photosynthetic Hosts.

Interactions between various microbial pathogens including viruses, bacteria, fungi, oomycetes, and their plant hosts have traditionally been the focus of phytopathology. In recent years, a significant and growing interest in the study of eukaryotic microorganisms not classified among fungi or oomycetes has emerged. Many of these protists establish complex interactions with photosynthetic hosts, and understanding these interactions is crucial in understanding the dynamics of these parasites within traditional and emerging types of farming, including marine aquaculture. Many phytopathogenic protists are biotrophs with complex polyphasic life cycles, which makes them difficult or impossible to culture, a fact reflected in a wide gap in the availability of comprehensive genomic data when compared to fungal and oomycete plant pathogens. Furthermore, our ability to use available genomic resources for these protists is limited by the broad taxonomic distance that these organisms span, which makes comparisons with other genomic datasets difficult. The current rapid progress in genomics and computational tools for the prediction of protein functions and interactions is revolutionizing the landscape in plant pathology. This is also opening novel possibilities, specifically for a deeper understanding of protist effectors. Tools like AlphaFold2 enable structure-based function prediction of effector candidates with divergent protein sequences. In turn, this allows us to ask better biological questions and, coupled with innovative experimental strategies, will lead into a new era of effector research, especially for protists, to expand our knowledge on these elusive pathogens and their interactions with photosynthetic hosts. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genomic mining of Vibrio parahaemolyticus highlights prevalence of antimicrobial resistance genes and new genetic markers associated with AHPND and tdh + /trh + genotypes.

BACKGROUND: Acute Hepatopancreatic Necrosis Disease (AHPND) causes significant mortality in shrimp aquaculture. The infection is primarily instigated by Vibrio parahaemolyticus (Vp) strains carrying a plasmid encoding the binary toxin PirAB. Yet, comprehension of supplementary virulence factors associated with this relatively recent disease remains limited. Furthermore, the same holds for gastroenteritis in humans caused by other Vp genotypes. Additionally, given the prevalent use of antibiotics to combat bacterial infections, it becomes imperative to illuminate the presence of antimicrobial resistance genes within these bacteria. RESULTS: A subsampled number of 1,036 Vp genomes was screened for the presence of antimicrobial resistance genes, revealing an average prevalence of 5 ± 2 (SD) genes. Additional phenotypic antimicrobial susceptibility testing of three Vp strains (M0904, TW01, and PV1) sequenced in this study demonstrated resistance to ampicillin by all tested strains. Additionally, Vp M0904 showed multidrug resistance (against ampicillin, tetracycline, and trimethoprim-sulfamethoxazole). With a focus on AHPND, a screening of all Vibrio spp. for the presence of pirA and/or pirB indicates an estimated prevalence of 0.6%, including four V. campbellii, four V. owensii, and a Vibrio sp. next to Vp. Their pirAB-encoding plasmids exhibited a highly conserved backbone, with variations primarily in the region of the Tn3 family transposase. Furthermore, an assessment of the subsampled Vp genomes for the presence of known virulence factors showed a correlation between the presence of the Type 3 Secretion System 2 and tdh, while the presence of the Type 6 Secretion System 1 was clade dependent. Furthermore, a genome-wide association study (GWAS) unveiled (new) genes associated with pirA, pirB, tdh, and trh genotypes. Notable associations with the pirAB genotype included outer membrane proteins, immunoglobulin-like domain containing proteins, and toxin-antitoxin systems. For the tdh + /trh + genotypes (containing tdh, trh, or both genes), associations were found with T3SS2 genes, urease-related genes and nickel-transport system genes, and genes involved in a 'minimal' type I-F CRISPR mechanism. CONCLUSIONS: This study highlights the prevalence of antimicrobial resistance and virulence genes in Vp, identifying novel genetic markers associated with AHPND and tdh + /trh + genotypes. These findings contribute valuable insights into the genomic basis of these genotypes, with implications for shrimp aquaculture and food safety.

Molecular Approaches Detect Early Signals of Programmed Cell Death in Hippolyte inermis Leach.

The protandric shrimp Hippolyte inermis is the only known marine invertebrate whose sex determination is strongly influenced by the composition of its food. In H. inermis, a sex reversal is triggered by the ingestion of diatoms of the genus Cocconeis associated with leaves of the seagrass Posidonia oceanica. These diatoms contain compounds that promote programmed cell death (PCD) in H. inermis and also in human cancer cells. Transcriptomic analyses suggested that ferroptosis is the primary trigger of the shrimp's sex reversal, leading to the rapid destruction of the androgen gland (AG) followed by a chain of apoptotic events transforming the testes into ovaries. Here, we propose a molecular approach to detect the effects of compounds stimulating the PCD. An RNA extraction method, suitable for young shrimp post-larvae (five days after metamorphosis; PL5 stage), was established. In addition, six genes involved in apoptosis, four involved in ferroptosis, and seven involved in the AG switch were mined from the transcriptome, and their expression levels were followed using real-time qPCR in PL5 fed on Cocconeis spp., compared to PL5 fed on a basic control feed. Our molecular approach, which detected early signals of sex reversal, represents a powerful instrument for investigating physiological progression and patterns of PCD in marine invertebrates. It exemplifies the physiological changes that may start a few days after the settlement of post-larvae and determine the life destiny of an individual.

Redefining piscine lactococcosis.

Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae. However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)-L. garvieae, L. petauri, and L. formosensis-which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis. Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention. IMPORTANCE: Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

Antagonistic activity of Phaeobacter piscinae against the emerging fish pathogen Vibrio crassostreae in aquaculture feed algae.

Aquaculture provides a rich resource of high-quality protein; however, the production is challenged by emerging pathogens such as Vibrio crassostreae. While probiotic bacteria have been proposed as a sustainable solution to reduce pathogen load in aquaculture, their application requires a comprehensive assessment across the aquaculture food chain. The purpose of this study was to determine the antagonistic effect of the potential probiotic bacterium Phaeobacter piscinae against the emerging fish pathogen V. crassostreae in aquaculture feed algae that can be an entry point for pathogens in fish and shellfish aquaculture. P. piscinae strain S26 produces the antibacterial compound tropodithietic acid (TDA). In a plate-based assay, P. piscinae S26 was equally to more effective than the well-studied Phaeobacter inhibens DSM17395 in its inhibition of the fish pathogens Vibrio anguillarum 90-11-286 and V. crassostreae DMC-1. When co-cultured with the microalgae Tetraselmis suecica and Isochrysis galbana, P. piscinae S26 reduced the maximum cell density of V. crassostreae DMC-1 by 2 log and 3-4 log fold, respectively. A TDA-deficient mutant of P. piscinae S26 inhibited V. crassostreae DMC-1 to a lesser extent than the wild type, suggesting that the antagonistic effect involves TDA and other factors. TDA is the prime antagonistic agent of the inhibition of V. anguillarum 90-11-286. Comparative genomics of V. anguillarum 90-11-286 and V. crassostreae DMC-1 revealed that V. crassostreae DMC-1 carries a greater arsenal of antibiotic resistance genes potentially contributing to the reduced effect of TDA. In conclusion, P. piscinae S26 is a promising new candidate for inhibition of emerging pathogens such as V. crassostreae DMC-1 in algal feed systems and could contribute to a more sustainable aquaculture industry.IMPORTANCEThe globally important production of fish and shellfish in aquaculture is challenged by disease outbreaks caused by pathogens such as Vibrio crassostreae. These outbreaks not only lead to substantial economic loss and environmental damage, but treatment with antibiotics can also lead to antibiotic resistance affecting human health. Here, we evaluated the potential of probiotic bacteria, specifically the newly identified strain Phaeobacter piscinae S26, to counteract these threats in a sustainable manner. Through a systematic assessment of the antagonistic effect of P. piscinae S26 against V. crassostreae DMC-1, particularly within the context of algal feed systems, the study demonstrates the effectiveness of P. piscinae S26 as probiotic and thereby provides a strategic pathway for addressing disease outbreaks in aquaculture. This finding has the potential of significantly contributing to the long-term stability of the industry, highlighting the potential of probiotics as an efficient and environmentally conscious approach to safeguarding aquaculture productivity against the adverse impact of pathogens.

Metabolic pathway engineering of high-salinity-induced overproduction of L-proline improves high-salinity stress tolerance of an ectoine-deficient Halomonas elongata.

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018's Ect biosynthetic operon with the artificial self-cloned proBm1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GKD118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proBm1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GKD118N/D119N and γ-GKD154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GKD107N and γ-GKE143A, the γ-GKD118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.

Low salinity stress increases the risk of Vibrio parahaemolyticus infection and gut microbiota dysbiosis in Pacific white shrimp.

BACKGROUND: Extreme precipitation events often cause sudden drops in salinity, leading to disease outbreaks in shrimp aquaculture. Evidence suggests that environmental stress increases animal host susceptibility to pathogens. However, the mechanisms of how low salinity stress induces disease susceptibility remain poorly understood. METHODS: We investigated the acute response of shrimp gut microbiota exposed to pathogens under low salinity stress. For comparison, shrimp were exposed to Vibrio infection under two salinity conditions: optimal salinity (Control group) and low salinity stress (Stress group). High throughput 16S rRNA sequencing and real-time PCR were employed to characterize the shrimp gut microbiota and quantify the severity level of Vibrio infection. RESULTS: The results showed that low salinity stress increased Vibrio infection levels, reduced gut microbiota species richness, and perturbed microbial functions in the shrimp gut, leading to significant changes in lipopolysaccharide biosynthesis that promoted the growth of pathogens. Gut microbiota of the bacterial genera Candidatus Bacilliplasma, Cellvibrio, and Photobacterium were identified as biomarkers of the Stress group. The functions of the gut microbiota in the Stress group were primarily associated with cellular processes and the metabolism of lipid-related compounds. CONCLUSIONS: Our findings reveal how environmental stress, particularly low salinity, increases shrimp susceptibility to Vibrio infection by affecting the gut microbiota. This highlights the importance of avoiding low salinity stress and promoting gut microbiota resilience to maintain the health of shrimp.

Progress and future directions for seaweed holobiont research.

In the marine environment, seaweeds (i.e. marine macroalgae) provide a wide range of ecological services and economic benefits. Like land plants, seaweeds do not provide these services in isolation, rather they rely on their associated microbial communities, which together with the host form the seaweed holobiont. However, there is a poor understanding of the mechanisms shaping these complex seaweed-microbe interactions, and of the evolutionary processes underlying these interactions. Here, we identify the current research challenges and opportunities in the field of seaweed holobiont biology. We argue that identifying the key microbial partners, knowing how they are recruited, and understanding their specific function and their relevance across all seaweed life history stages are among the knowledge gaps that are particularly important to address, especially in the context of the environmental challenges threatening seaweeds. We further discuss future approaches to study seaweed holobionts, and how we can apply the holobiont concept to natural or engineered seaweed ecosystems.

Effect of diets containing probiotic yeast Cystobasidium benthicum and fruit Cyrtocarpa edulis on growth and immune parameters of Nile tilapia (Oreochromisniloticus).

This study investigates Cystobasidium benthicum (Cb) probiotic yeast and Cyrtocarpa edulis (Ce) fruit dietary effects, single (0.5 %) or combined (Cb:Ce, 0.25:0.25 %), on growth performance, humoral immunity in serum and skin mucus, and intestinal morphology of Nile tilapia (Oreochromis niloticus) after 14 and 28 days. The Cb group presented the highest (P < 0.05) specific growth rate, weight gain, and absolute growth rate with respect to the control group. Immunological assays indicated that Cb, Ce and Cb:Ce groups increased serum nitric oxide concentration compared to the control group (P < 0.05). Cb and Cb:Ce groups showed the highest serum myeloperoxidase enzyme activity at day 14 and 28, respectively (P < 0.05); whereas, Cb:Ce group had the highest (P < 0.05) myeloperoxidase activity in skin mucus. The superoxide dismutase enzyme activity was unaffected. On day 28, Cb, Ce, and Cb:Ce groups showed higher and lower (P < 0.05) catalase enzyme activity in serum and skin mucus, respectively, compared with the control group. Only the Cb group had higher (P < 0.05) total protein concentration in serum (day 14) and skin mucus (day 14 and 28) with respect to the control group. The lysozyme activity in serum (day 28) and skin mucus (day 14) was higher (P < 0.05) in the Cb group compared to the control group. Only the skin mucus of Ce group showed bactericidal activity against Aeromonas dhakensis (P < 0.05). Histological studies indicated that Cb and Cb:Ce groups increased microvilli height, and Cb, Ce and Cb:Ce augmented goblet cell area at day 14 compared to the control group (P < 0.05). At day 28, microvilli height was higher in all groups and the number of intraepithelial leukocytes increased in Cb and Ce groups with respect to the control group (P < 0.05). The ex vivo assay revealed that A. dhakensis in leukocytes decreased cell viability similar to the control group (P < 0.05). A principal component analysis (PCA) confirmed the results. In conclusion, C. benthicum in the diet was the best supplement to improve the growth and immunity of Nile tilapia.

Molecular characterization, antibiotic resistant pattern and biofilm forming potentiality of bacterial community associated with Ompok pabda fish farming in southwestern Bangladesh.

Ompok pabda is gaining popularity in the aquaculture industry due to its increasing demand; however research on microbial diversity and antibiotic susceptibility remains limited. The present study was designed to identify the bacterial pathogens commonly found in the pabda farming system with their biofilm forming potential and antibiotic susceptibility. Different bacterial strains were isolated from water, sediments and gut, gill of pabda fish and the isolates were identified based on their morphological traits, biochemical and molecular analysis. Antibiotic susceptibilities, antibiotic resistance gene determination and biofilm formation capabilities were evaluated by disc diffusion method, PCR amplification and Microtiter plate (MTP) assay, respectively. The respective isolates of gill and gut of pabda aquaculture and their environments were: Exiguobacterium spp. (25 %), Enterococcus spp. (20 %), Bacillus spp. (10 %), Acinetobacter spp. (10 %), Enterobacter spp. (10 %), Aeromonas spp. (10 %), Lactococcus spp. (5 %), Klebsiella spp. (5 %) and Kurthia spp. (5 %). Antibiotic resistance frequencies were found to be relatively high, especially for trimethoprim (95 %), sulfafurazole (75 %), ampicillin (60 %), amoxicillin-clavulanic acid (55 %), and cephradine (50 %). 30 % isolates were categorized as DR bacteria followed by 30 % isolates were MDR bacteria and 40 % were classified as XDR bacteria. Moreover, 4 antibiotic resistant genes were detected with sul1 (30 %), dfrA1 (10 %), tetC (40 %), and qnrA (5 %) of isolates. Based on the microtiter plate method, 20 %, 25 %, and 30 % of isolates were found to produce strong, moderate, and weak biofilms, respectively. The findings suggest that biofilm forming bacterial strains found in O. pabda fish farm may be a potential source of numerous antibiotic-resistant bacteria. The study sheds new light on antibiotic resistance genes, which are typically inherited by bacteria and play an important role in developing effective treatments or control strategies.

Quercetin disrupts biofilm formation and attenuates virulence of Aeromonas hydrophila.

Aeromonas hydrophila poses significant health and economic challenges in aquaculture owing to its pathogenicity and prevalence. Overuse of antibiotics has led to multidrug resistance and environmental pollution, necessitating alternative strategies. This study investigated the antibacterial and antibiofilm potentials of quercetin against A. hydrophila. Efficacy was assessed using various assays, including antibacterial activity, biofilm inhibition, specific growth time, hemolysis inhibition, autoaggregation, and microscopic evaluation. Additionally, docking analysis was performed to explore potential interactions between quercetin and virulence proteins of A. hydrophila, including proaerolysin, chaperone needle-subunit complex of the type III secretion system, and alpha-pore forming toxin (PDB ID: 1PRE, 2Q1K, 6GRK). Quercetin exhibited potent antibacterial activity with 21.1 ± 1.1 mm zone of inhibition at 1.5 mg mL-1. It also demonstrated significant antibiofilm activity, reducing biofilm formation by 46.3 ± 1.3% at the MIC and attenuating autoaggregation by 55.9 ± 1.5%. Hemolysis was inhibited by 41 ± 1.8%. Microscopic analysis revealed the disintegration of the A. hydrophila biofilm matrix. Docking studies indicated active hydrogen bond interactions between quercetin and the targeted virulence proteins with the binding energy -3.2, -5.6, and -5.1 kcal mol⁻1, respectively. These results suggest that quercetin is an excellent alternative to antibiotics for combating A. hydrophila infection in aquaculture. The multifaceted efficacy of quercetin in inhibiting bacterial growth, biofilm formation, virulence factors, and autoaggregation highlights the potential for aquaculture health and sustainability. Future research should delve into the precise mechanisms of action and explore synergistic combinations with other compounds for enhanced efficacy and targeted interventions.

Cardiovascular physiology of decapod crustaceans: from scientific inquiry to practical applications.

Until recently, the decapod crustacean heart was regarded as a simple, single ventricle, contraction of which forces haemolymph out into seven arteries. Differential tissue perfusion is achieved by contraction and relaxation of valves at the base of each artery. In this Review, we discuss recent work that has shown that the heart is bifurcated by muscular sheets that may effectively divide the single ventricle into 'chambers'. Preliminary research shows that these chambers may contract differentially; whether this enables selective tissue perfusion remains to be seen. Crustaceans are unusual in that they can stop their heart for extended periods. These periods of cardiac arrest can become remarkably rhythmic, accounting for a significant portion of the cardiac repertoire. As we discuss in this Review, in crustaceans, changes in heart rate have been used extensively as a measurement of stress and metabolism. We suggest that the periods of cardiac pausing should also be quantified in this context. In the past three decades, an exponential increase in crustacean aquaculture has occurred and heart rate (and changes thereof) is being used to understand the stress responses of farmed crustaceans, as well as providing an indicator of disease progression. Furthermore, as summarized in this Review, heart rate is now being used as an effective indicator of humane methods to anaesthetize, stun or euthanize crustaceans destined for the table or for use in scientific research. We believe that incorporation of new biomedical technology and new animal welfare policies will guide future research directions in this field.

Flavobacterium acetivorans sp. nov. and Flavobacterium galactosidilyticum sp. nov., psychrotolerant flavobacteria isolated from cultivated fish.

Two yellow-coloured strains, F-29T and F-340T, were isolated from fish farms in Antalya and Mugla in 2015 and 2017 during surveillance studies. The 16S rRNA gene sequence analysis showed that both strains belong to the genus Flavobacterium. A polyphasic approach involving a comprehensive genome analysis was employed to ascertain the taxonomic provenance of the strains. The overall genome-relatedness indices of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) between the strains and the other members of the genus Flavobacterium were found to be well below the established thresholds of 70 and 95 %, respectively. The whole-genome-based phylogenetic analysis revealed that strain F-29T is closely related to Flavobacterium granuli (dDDH 39.3 % and ANI 89.4 %), while strain F-340T has a close relationship with the type strain of Flavobacterium pygoscelis (dDDH 25.6 % and ANI 81.5 %). Both strains were psychrotolerant with an optimum growth temperature of 25 °C. The chemotaxonomic characteristics of the strains were typical of the genus Flavobacterium. Both strains had phosphatidylethanolamine, aminolipids and unidentified lipids in their polar lipid profile and MK-6 as the isoprenoid quinone. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The genome size of the strains was 3.5 Mb, while G+C contents were 35.3 mol% for strain F-29T and 33.4 mol% for strain F-340T. Overall, the characterizations confirmed that both strains are representatives of two novel species within the genus Flavobacterium, for which the names Flavobacterium acetivorans sp. nov. and Flavobacterium galactosidilyticum sp. nov. are proposed, with F-29T (JCM 34193T=KCTC 82253T) and F-340T (JCM 34203T=KCTC 82263T) as the type strains, respectively.

Fish Iridoviridae: infection, vaccination and immune response.

Each year, due to climate change, an increasing number of new pathogens are being discovered and studied, leading to an increase in the number of known diseases affecting various fish species in different regions of the world. Viruses from the family Iridoviridae, which consist of the genera Megalocytivirus, Lymphocystivirus, and Ranavirus, cause epizootic outbreaks in farmed and wild, marine, and freshwater fish species (including ornamental fish). Diseases caused by fish viruses of the family Iridoviridae have a significant economic impact, especially in the aquaculture sector. Consequently, vaccines have been developed in recent decades, and their administration methods have improved. To date, various types of vaccines are available to control and prevent Iridoviridae infections in fish populations. Notably, two vaccines, specifically targeting Red Sea bream iridoviral disease and iridoviruses (formalin-killed vaccine and AQUAVAC® IridoV, respectively), are commercially available. In addition to exploring these themes, this review examines the immune responses in fish following viral infections or vaccination procedures. In general, the evasion mechanisms observed in iridovirus infections are characterised by a systemic absence of inflammatory responses and a reduction in the expression of genes associated with the adaptive immune response. Finally, this review also explores prophylactic procedure trends in fish vaccination strategies, focusing on future advances in the field.

Salmonid Rickettsial Septicemia (SRS) disease dynamics and Atlantic salmon immune response to Piscirickettsia salmonis LF-89 and EM-90 co-infection.

In Chile, Piscirickettsia salmonis contains two genetically isolated genogroups, LF-89 and EM-90. However, the impact of a potential co-infection with these two variants on Salmonid Rickettsial Septicemia (SRS) in Atlantic salmon (Salmo salar) remains largely unexplored. In our study, we evaluated the effect of P. salmonis LF-89-like and EM-90-like co-infection on post-smolt Atlantic salmon after an intraperitoneal challenge to compare changes in disease dynamics and host immune response. Co-infected fish had a significantly lower survival rate (24.1%) at 21 days post-challenge (dpc), compared with EM-90-like single-infected fish (40.3%). In contrast, all the LF-89-like single-infected fish survived. In addition, co-infected fish presented a higher presence of clinical lesions than any of the single-infected fish. The gene expression of salmon immune-related biomarkers evaluated in the head kidney, spleen, and liver showed that the EM-90-like isolate and the co-infection induced the up-regulation of cytokines (e.g., il-1ß, ifnγ, il8, il10), antimicrobial peptides (hepdicin) and pattern recognition receptors (PRRs), such as TLR5s. Furthermore, in serum samples from EM-90-like and co-infected fish, an increase in the total IgM level was observed. Interestingly, specific IgM against P. salmonis showed greater detection of EM-90-like antigens in LF-89-like infected fish serum (cross-reaction). These data provide evidence that P. salmonis LF-89-like and EM-90-like interactions can modulate SRS disease dynamics in Atlantic salmon, causing a synergistic effect that increases the severity of the disease and the mortality rate of the fish. Overall, this study contributes to achieving a better understanding of P. salmonis population dynamics.