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Symbiotic relationships are ubiquitous throughout the world’s oceans, yet for many marine organisms, including those in the high latitudes, little is understood about symbiotic associations and functional relationships. From a recently determined genome sequence of a filter-feeding basket star from Argentina, Gorgonocephalus chilensis , we discovered a novel Mycoplasma species with a 796Kb genome (CheckM completeness of 97.9%, G+C content = 30.1%). Similar to other Mycoplasma spp. within Mycoplasmatota, genomic analysis of the novel organism revealed reduced metabolic pathways including incomplete biosynthetic pathways, suggesting an obligate association with their basket star host. Results of 16S rRNA and multi-locus phylogenetic analyses revealed that this organism belonged to a recently characterized non-free-living lineage of Mycoplasma spp. specifically associated with marine invertebrate animals. Thus, the name “ Candidatus Mycoplasma mahonii” is proposed for this novel species. Based on 16S rRNA PCR-screening, we found that Ca . M. mahonii also occurs in Gorgonocephalus eucnemis from the Northwest Pacific and other Gorgonocephalus chilensis from Argentinian waters. The level of sequence conservation within Ca . M. mahonii is considerable between widely disparate high-latitude Gorgonocephalus species, suggesting that oceanic dispersal of this microbe may be greater than excepted. 

Abstract Flavobacterium covaeand virulentAeromonas hydrophilaare prevalent bacterial pathogens within the US catfish industry that can cause high mortality in production ponds. An assessment of in vivo bacterial coinfection with virulentA. hydrophila(ML09‐119) andF. covae(ALG‐00‐530) was conducted in juvenile channel catfish (Ictalurus punctatus). Catfish were divided into seven treatments: (1) mock control; (2) and (3) high and low doses of virulentA. hydrophila; (4) and (5) high and low doses ofF. covae; (6) and (7) simultaneous challenge with high and low doses of virulentA. hydrophilaandF. covae. In addition to the mortality assessment, anterior kidney and spleen were collected to evaluate immune gene expression, as well as quantify bacterial load by qPCR. At 96 h post‐challenge (hpc), the high dose of virulentA. hydrophilainfection (immersed in 2.3 × 10⁷ CFU mL⁻¹) resulted in cumulative percent mortality (CPM) of 28.3 ± 9.5%, while the high dose ofF. covae(immersed in 5.2 × 10⁶ CFU mL⁻¹) yielded CPM of 23.3 ± 12.9%. When these pathogens were delivered in combination, CPM significantly increased for both the high‐ (98.3 ± 1.36%) and low‐dose combinations (76.7 ± 17.05%) (p < .001). Lysozyme activity was found to be different at 24 and 48 hpc, with the high‐dose vAh group demonstrating greater levels than unexposed control fish at each time point. Three proinflammatory cytokines (tnfα,il8,il1b) demonstrated increased expression levels at 48 hpc. These results demonstrate the additive effects on mortality when these two pathogens are combined. The synthesis of these mortality and health metrics advances our understanding of coinfections of these two important catfish pathogens and will aid fish health diagnosticians and channel catfish producers in developing therapeutants and prevention methods to control bacterial coinfections. 

Contrary to the understanding that divalent cations only result in under-estimation of gene quantification via DNA hybridization-based assays, we have discovered that Mg 2+ could cause either under or over-estimation at different concentrations. Its switchable inhibitory behavior is likely due to its rigid first solvation (hydrated) shell and hence it is inclined to form non-direct binding with DNA. At low concentrations, it caused under-estimation by occupying the hybridization sites. At high concentrations, it caused probe, signaling and target DNA to aggregate non-specifically via Coulomb forces. By quantifying target DNAs at a range of Mg 2+ concentrations using a gene quantification assay (NanoGene assay), a Mg 2+ inflection concentration of ∼10 −3 M was observed for both target ssDNA and dsDNA. Field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FT-IR) were employed to observe Mg 2+ -induced non-specific binding in the complexes that mimicked the presence of target DNA. Together with two other divalent cations Ca 2+ and Cu 2+ , they were further examined via zeta potential measurements as well as NanoGene assay. This study revealed the importance of Mg 2+ in achieving accurate gene quantification. Through a better mechanistic understanding of this phenomenon, it will be possible to develop strategies to mitigate the impact of Mg 2+ on DNA hybridization-based gene quantification. 

Edwardsiella ictaluri and Flavobacterium covae are pervasive bacterial pathogens associated with significant losses in catfish aquaculture. Bacterial coinfections have the potential to increase outbreak severity and can worsen on-farm mortality. A preliminary assessment of in vivo bacterial coinfection with E. ictaluri (S97-773) and F. covae (ALG-00-530) was conducted using juvenile channel catfish (Ictalurus punctatus). Catfish were divided into five treatment groups: (1) mock control; (2) E. ictaluri full dose (immersion; 5.4 × 105 CFU mL−1); (3) F. covae full dose (immersion; 3.6 × 106 CFU mL−1); (4) E. ictaluri half dose (immersion; 2.7 × 105 CFU mL−1) followed by half dose F. covae (immersion; 1.8 × 106 CFU mL−1); and (5) F. covae half dose followed by half dose E. ictaluri. In the coinfection challenges, the second inoculum was delivered 48 h after the initial exposure. At 21 days post-challenge (DPC), the single dose E. ictaluri infection yielded a cumulative percent mortality (CPM) of 90.0 ± 4.1%, compared with 13.3 ± 5.9% in the F. covae group. Mortality patterns in coinfection challenges mimicked the single dose E. ictaluri challenge, with CPM of 93.3 ± 5.4% for fish initially challenged with E. ictaluri followed by F. covae, and 93.3 ± 2.7% for fish exposed to F. covae and subsequently challenged with E. ictaluri. Despite similarities in the final CPM within the coinfection groups, the onset of peak mortality was delayed in fish exposed to F. covae first but was congruent with mortality trends in the E. ictaluri challenge. Catfish exposed to E. ictaluri in both the single and coinfected treatments displayed increased serum lysozyme activity at 4-DPC (p < 0.001). Three pro-inflammatory cytokines (il8, tnfα, il1β) were evaluated for gene expression, revealing an increase in expression at 7-DPC in all E. ictaluri exposed treatments (p < 0.05). These data enhance our understanding of the dynamics of E. ictaluri and F. covae coinfections in US farm-raised catfish. 

The channel catfish (Ictalurus punctatus) farming industry is the largest and one of the oldest aquaculture industries in the United States. Despite being an established industry, production issues stemming from disease outbreaks remain problematic for producers. Supplementing fish diets with probiotics to enhance the immune system and growth potential is one approach to mitigating disease. Although considerable laboratory data demonstrate efficacy, these results do not always translate to natural modes of disease transmission. Hence, the present work was conducted in the laboratory but incorporated flow-through water from large catfish pond production systems, allowing for natural exposure to pathogens. Two feeding trials were conducted in an 18-tank aquaria system housing two different sizes, 34.8 ± 12.5 g and 0.36 ± 0.03 g, of channel catfish. Channel catfish in the first trial were fed three experimental diets over six weeks. Commercial diets were top-coated with two selected spore-forming Bacillus spp. probiotics, Bacillus velezensis AP193 (1 × 106 CFU g−1) and BiOWiSH (3.6 × 104 CFU g−1), or a basal diet that contained no dietary additive. In the second eight-week trial, diets were top-coated with BiOWiSH at three concentrations (1.8, 3.6, and 7.3 × 104 CFU g−1), along with one basal diet (no probiotic). At the completion of these studies, growth performance, survival, hematocrit, blood chemistry, and immune expression of interleukin 1β (il1β), tumor necrosis factor-alpha (tnf-α), interleukin-8 (il8), transforming-growth factor β1 (tgf-β1), and toll-like receptor 9 (tlr9) were evaluated using qPCR. Trial results revealed no differences (p > 0.05) among treatments concerning growth, survival, or hematological parameters. For immune gene expression, interesting trends were discerned, with substantial downregulation observed in B. velezensis AP193-fed fish for il1β, tnf-α, and tlr9 expression within splenic tissue, compared to that of the basal and BiOWiSH diets (p < 0.05). However, the results were not statistically significant for anterior kidney tissue in the first trial. In the second trial, varied levels of probiotic inclusion revealed no significant impact of BiOWiSH’s products on the expression of il1β, tnf-α, il8, and tgf-β1 in both spleen and kidney tissue at any rate of probiotic inclusion (p > 0.05). Based on these findings, more research on utilizing probiotics in flow-through systems with natural infection conditions is crucial to ensure consistency from a controlled laboratory scale to real-world practices. 

ABSTRACT Flavobacterium covae is one of four Flavobacterium spp. that cause columnaris disease in teleost fish. Here, we report the draft genomes of two isolates, LSU-066-04 and LV-359-01, and their predicted virulence factors. 

ABSTRACT Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo -inositol as the sole carbon source. A total of 113 myo- inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish ( Pangasius pangasius ) and striped catfish ( Pangasianodon hypophthalmus ) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo- inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo -inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.