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The Pacific whiteleg shrimp Penaeus (Litopenaeus) vannamei is a highly relevant species for the world's aquaculture development, for which an incomplete genome is available in public databases. In this work, PacBio long-reads from 14 publicly available genomic libraries (131.2 Gb) were mined to improve the reference genome assembly. The libraries were assembled, polished using Illumina short-reads, and scaffolded with P. vannamei, Feneropenaeus chinensis, and Penaeus monodon genomes. The reference-guided assembly, organized into 44 pseudo-chromosomes and 15,682 scaffolds, showed an improvement from previous reference genomes with a genome size of 2.055 Gb, N50 of 40.14 Mb, L50 of 21, and the longest scaffold of 65.79 Mb. Most orthologous genes (92.6%) of the Arthropoda_odb10 database were detected as "complete," and BRAKER predicted 21,816 gene models; from these, we detected 1,814 single-copy orthologues conserved across the genomic references for Marsupenaeus japonicus, F. chinensis, and P. monodon. Transcriptomic-assembly data aligned in more than 99% to the new reference-guided assembly. The collinearity analysis of the assembled pseudo-chromosomes against the P. vannamei and P. monodon reference genomes showed high conservation in different sets of pseudo-chromosomes. In addition, more than 21,000 publicly available genetic marker sequences were mapped to single-site positions. This new assembly represents a step forward to previously reported P. vannamei assemblies. It will be helpful as a reference genome for future studies on the evolutionary history of the species, the genetic architecture of physiological and sex-determination traits, and the analysis of the changes in genetic diversity and composition of cultivated stocks.

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Piscirickettsiosis is the main cause of mortality in salmonids of commercial importance in Chile, which is caused by Piscirickettsia salmonis, a Gram-negative, γ-proteobacteria that can produce biofilm as one of its virulence factors. The Chilean salmon industry uses large amounts of antibiotics to control piscirickettsiosis outbreaks, which has raised concern about its environmental impact and the potential to induce antibiotic resistance. Thus, the use of phytogenic feed additives (PFA) with antibacterial activity emerges as an interesting alternative to antimicrobials. Our study describes the antimicrobial action of an Andrographis paniculate-extracted PFA on P. salmonis planktonic growth and biofilm formation. We observed complete inhibition of planktonic and biofilm growth with 500 and 400 µg/mL of PFA for P. salmonis LF-89 and EM-90-like strains, respectively. Furthermore, 500 µg/mL of PFA was bactericidal for both evaluated bacterial strains. Sub-inhibitory doses of PFA increase the transcript levels of stress (groEL), biofilm (pslD), and efflux pump (acrB) genes for both P. salmonis strains in planktonic and sessile conditions. In conclusion, our results demonstrate the antibacterial effect of PFA against P. salmonis in vitro, highlighting the potential of PFA as an alternative to control Piscirickettsiosis.

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Introducción: El pargo mancha es un pez marino de alto consumo e interés comercial en Costa Rica que está sometido a una fuerte presión pesquera, la cual puede afectar la diversidad genética y generar problemas por depresión endogámica. Objetivo: Evaluar el estado genético de la población de Lutjanus guttatus mediante el uso microsatélites. Métodos: Se recolectaron muestras entre el 2018 y 2019 y se estudiaron 44 individuos de cada una de las localidades del Golfo de Nicoya y Golfo Dulce. Se realizó la extracción de ADN y la amplificación de diez loci con microsatélites mediante PCR, para la determinación del genotipo, análisis de diversidad genética y estructura poblacional. Resultados: Los parámetros de diversidad indican un elevado polimorfismo asociado con un alto número de alelos obtenidos por locus, pero con bajos niveles de heterocigosidad observada en comparación con la esperada (Ho= 0.774 y 0.800 y He= 0.948 y 0.954 para Golfo de Nicoya y Golfo Dulce, respectivamente). No hay evidencia suficiente para decir que las dos poblaciones son distintas (FST= 0.00264, P > 0.05). La desviación del Equilibrio de Hardy-Weinberg indica la posible mezcla de organismos de origen distinto a los del medio silvestre. Conclusiones: L. guttatus tiene niveles altos de diversidad genética, no hay evidencia de diferenciación en subpoblaciones genéticas, lo que en manejo pesquerías se considera una sola población panmíctica. La posible mezcla de individuos de origen distinto al silvestre sugiere la presencia de organismos de un programa de repoblación o de cultivos comerciales en la región. El uso de marcadores genéticos se recomienda para el monitoreo, además, en programas de repoblación y evaluar su efecto.

Introduction: The spotted snapper is a high-consumption and commercially important marine fish in Costa Rica, subjected to heavy fishing pressures, which can affect genetic diversity and generate problems due to inbreeding depression. Objective: To evaluate the genetic status of the population of Lutjanus guttatus using microsatellites. Methods: Samples were collected between 2018 and 2019, and 44 individuals from each of the localities of the Gulf of Nicoya and the Gulf of Dulce were studied. DNA extraction and amplification of ten loci with microsatellites using PCR were performed, followed by genotyping, analysis of genetic diversity, and population structure. Results: Diversity parameters indicate a high polymorphism associated with a high number of alleles obtained per locus, but with low levels of observed heterozygosity compared to expected (Ho= 0.774 and 0.800, and He= 0.948 and 0.954 for the Gulf of Nicoya and Gulf of Dulce, respectively). There is not enough evidence to say that the two populations are distinct (FST= 0.00264, P > 0.05). Deviation from Hardy-Weinberg equilibrium was recorded, indicating possible mixing of organisms of different origin from the wild environment. Conclusions: L. guttatus presents high levels of genetic diversity, without evidence of differentiation in genetic subpopulations. For fisheries management purposes, they would be considered a single panmictic population. The possible mixing with wild individuals suggests the presence of organisms derived from a restocking or commercial cultivation program carried out in the region. The use of genetic markers is recommended to maintain monitoring, follow up on restocking programs and evaluate their effect.

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Nutritional immunity regulates the homeostasis of micronutrients such as iron, manganese, and zinc at the systemic and cellular levels, preventing the invading microorganisms from gaining access and thereby limiting their growth. Therefore, the objective of this study was to evaluate the activation of nutritional immunity in specimens of Atlantic salmon (Salmo salar) that are intraperitoneally stimulated with both live and inactivated Piscirickettsia salmonis. The study used liver tissue and blood/plasma samples on days 3, 7, and 14 post-injections (dpi) for the analysis. Genetic material (DNA) of P. salmonis was detected in the liver tissue of fish stimulated with both live and inactivated P. salmonis at 14 dpi. Additionally, the hematocrit percentage decreased at 3 and 7 dpi in fish stimulated with live P. salmonis, unchanged in fish challenged with inactivated P. salmonis. On the other hand, plasma iron content decreased during the experimental course in fish stimulated with both live and inactivated P. salmonis, although this decrease was statistically significant only at 3 dpi. Regarding the immune-nutritional markers such as tfr1, dmt1, and ireg1 were modulated in the two experimental conditions, compared to zip8, ft-h, and hamp, which were down-regulated in fish stimulated with live and inactivated P. salmonis during the course experimental. Finally, the intracellular iron content in the liver increased at 7 and 14 dpi in fish stimulated with live and inactivated P. salmonis, while the zinc content decreased at 14 dpi under both experimental conditions. However, stimulation with live and inactivated P. salmonis did not alter the manganese content in the fish. The results suggest that nutritional immunity does not distinguish between live and inactivated P. salmonis and elicits a similar immune response. Probably, this immune mechanism would be self-activated with the detection of PAMPs, instead of a sequestration and/or competition of micronutrients by the living microorganism.

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Renibacterium salmoninarum is one of the oldest known fish bacterial pathogens. This Gram-positive bacterium is the causative agent of Bacterial Kidney Disease (BKD), a chronic infection that primarily infects salmonids at low temperatures. Externally, infected fish may show exophthalmos, skin blisters, ulcerations, and hemorrhages at the base of the fins and along the lateral line. Internally, the kidney, heart, spleen, and liver may show signs of inflammation. The best characterized virulence factor of R. salmoninarum is p57, a 57 kDa protein located on the bacterial cell surface and secreted into surrounding fish tissue. The p57 protein in fish is the main mediator in suppressing the immune system, reducing antibody production, and intervening in cytokine activity. In this review, we will discuss aspects such as single nucleotide polymorphisms (SNPs) that modify the DNA sequence, variants in the number of copies of MSA genes, physical-chemical properties of the signal peptides, and the limited iron conditions that can modify p57 expression and increase the virulence of R. salmoninarum.

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The spotted rose snapper, Lutjanus guttatus, is an important fishery species with high potential for aquaculture. Genetic characterization of its natural populations is necessary to avoid stock collapse and loss of genetic diversity. Previous studies carried out in the Tropical Eastern Pacific (TEP), however, have shown contrasting results in the genetic structure of fish populations, particularly in species of Lutjanidae. Therefore, to understand the genetic structure of spotted rose snapper in the TEP, twelve microsatellite loci were used to assess the genetic diversity and explore the hypothesis of population genetic structure in samples of the species collected throughout the TEP. Fin clips from 186 sampled individuals (27 to 49 per site) were analyzed from five sites in the three regional biogeographic provinces, delimited by shoreline reef habitat breaks: La Paz (Cortez province), Colima and Oaxaca (Mexican province), Chiriqui and Port of Panama (Panamic province). Results of global Analysis of Molecular Variance (AMOVA), population pairwise FST, hierarchical AMOVA, and a discriminant analysis of principal components (DAPC) reflected a panmictic population involving the entire set of sampled sites. The role of larval dispersal, post-recruitment migration, and marine current dynamics as drivers of genetic connectivity in this species is discussed.(AU)

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The innate immune system can limit the growth of invading pathogens by depleting micronutrients at a cellular and tissue level. However, it is not known whether nutrient depletion mechanisms discriminate between living pathogens (which require nutrients) and pathogen-associated molecular patterns (PAMPs) (which do not). We stimulated SHK-1 cells with different PAMPs (outer membrane vesicles of Piscirickettsia salmonis "OMVs", protein extract of P. salmonis "TP" and lipopolysaccharides of P. salmonis "LPS") isolated from P. salmonis and evaluated transcriptional changes in nutritional immunity associated genes. Our experimental treatments were: Control (SHK-1 stimulated with bacterial culture medium), OMVs (SHK-1 stimulated with 1µg of outer membrane vesicles), TP (SHK-1 stimulated with 1µg of total protein extract) and LPS (SHK-1 stimulated with 1µg of lipopolysaccharides). Cells were sampled at 15-, 30-, 60- and 120-minutes post-stimulation. We detected increased transcription of zip8, zip14, irp1, irp2 and tfr1 in all three experimental conditions and increased transcription of dmt1 in cells stimulated with OMVs and TP, but not LPS. Additionally, we observed generally increased transcription of ireg-1, il-6, hamp, irp1, ft-h and ft-m in all three experimental conditions, but we also detected decreased transcription of these markers in cells stimulated with TP and LPS at specific time points. Our results demonstrate that SHK-1 cells stimulated with P. salmonis PAMPs increase transcription of markers involved in the transport, uptake, storage and regulation of micronutrients such as iron, manganese and zinc.

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Extracellular traps (ETs) are webs of DNA, citrullinated histones, anti-microbial peptides, and proteins that were not previously reported in Atlantic salmon (Salmo salar). ETs are mainly released from polymorphonuclear neutrophils (PMN) and are considered a novel PMN-derived effector mechanism against different invasive pathogens. Here, we showed that Atlantic salmon-derived PMN released ETs-like structures in vitro in response to highly pathogenic facultative intracellular rickettsial bacteria Piscirickettsia salmonis. PMN were isolated from pre-smolt Atlantic salmon and stimulated in vitro with oleic acid and P. salmonis. Extracellular DNA was measured using the PicoGreen™ dye, while immunofluorescence image analysis was used to confirm the classical components of salmonid-extruded ETs. Future studies are required to better understand the role of Atlantic salmon-derived ETs orchestrating innate/adaptive immunity and the knowledge on regulation pathways involved in this cell death process. Thus, comprehension of salmonid-derived ETs against P. salmonis might represent novel alternative strategies to improve host innate defense mechanisms of farmed salmon against closely related rickettsial bacteria, as a complement to disease prevention and control strategies.

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The tropical eastern Pacific (TEP) is a highly dynamic region and a model system to study how habitat discontinuities affect the distribution of shorefishes, particularly for species that display ontogenetic habitat shifts, including snappers (Lutjanidae). To evaluate the genetic structure of the Pacific red snapper (Lutjanus peru) and the yellow snapper (Lutjanus argentiventris) throughout their distribution range along the TEP, 13 and 11 microsatellite loci were analysed, respectively. The genetic diversity of L. peru (N = 446) and L. argentiventris (N = 170) was evaluated in 10 and 5 localities, respectively, showing slightly higher but non-significant values in the Gulf of California for both species. The genetic structure analysis identified the presence of significant genetic structure in both species, but the locations of the identified barriers for the gene flow differed between species. The principal driver for the genetic structure at large scales >2500 km was isolation by distance. At smaller scales (<250 km), the habitat discontinuity for juveniles and adults and the environmental differences throughout the distribution range represented potential barriers to gene flow between populations for both species.

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Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and sessile LF-89 generated the highest virulence levels, associated with differential expression of il-1ß, il-8, nf-κb, and iκb-α genes in SHK-1 cells. These results suggest that there is no single virulence pattern or gene expression profile induced by the planktonic or sessile condition of P. salmonis, which are dependent on each strain and bacterial condition used.