Long-term organogenic callus cultivation of Ranunculus illyricus L.: a blueprint for sustainable ex situ conservation of the species in urban greenery.

The growing trend of introducing wild plant species into urban environments necessitates the identification of novel species adapted to prevailing conditions. A promising reservoir of such species may be xerothermic communities where Ranunculus illyricus occurs. This study aimed to establish a micropropagation protocol for R. illyricus using indirect organogenesis. The protocol includes initiation of culture from various explants, callus proliferation, shoot regeneration, multiplication, and concurrent rooting. Callus appeared on most types of vegetative explants tested, but stolons were considered the best due to their good availability, high disinfection (85%), and robust callus production (maximum increase - 363.1%). The growth rate of the callus fresh matter (CFM) obtained from stolons was calculated. Greater CFM was obtained on the medium with the supplemented picloram 8.0 mg L- 1 with kinetin 5.0 mg L- 1 and in second part of experiment on medium with the addition of 2,4-D (2,4-dichlorophenoxyacetic acid) 2.0 mg L- 1 alone or picloram 6.0 mg L- 1 with kinetin 8.0 mg L- 1. Shoot organogenesis was observed on macronutrients B5 (Gamborg medium), micronutrients MS (Murashige and Skoog) medium with the addition of 2.0 mg L- 1 IBA (indole-3-butyric acid) and 4.0 mg L- 1 BAP (6-benzylaminopurine). To document the process of callus differentiation, microscopic preparations were prepared. Subsequently, the regenerated plants underwent acclimatisation and their growth in an ex situ collection was monitored over three growing seasons. In particular, in vitro-origin plants exhibited developmental patterns similar to those of their seed-origin counterparts. The incorporation of R. illyricus into urban landscapes not only increases aesthetic appeal, but also ensures the preservation of valuable genetic resources for this rare species, potentially contributing to effective ex situ conservation in the future. This marks the first scientific report on in vitro cultures of R. illyricus.

Understanding the host response of farmed fish to blood flukes (Trematoda: Aporocotylidae) for developing new treatment strategies.

Aporocotylids (Trematoda: Digenea), also known as fish blood flukes infect the circulatory system of fish leading to serious health problems and mortality. Aporocotylids are a particular concern for farmed fish as infection intensity can increase within the farming environment and lead to mortalities. In the context of managing these infections, one of the most crucial aspects to consider is the host response of the infected fish against these blood flukes. Understanding the response is essential to improving current treatment strategies that are largely based on the use of anthelmintic praziquantel to manage infections in aquaculture. This review focuses on the current knowledge of farmed fish host responses against the different life stages of aporocotylids. New treatment strategies that are able to provide protection against reinfections should be a long-term goal and is not possible without understanding the fish response to infection and the interactions between host and parasite.

Molecular evidence for stress, inflammation and structural changes in non-specific ulcers in skin of farmed Chinook salmon (Oncorhynchus tshawytscha).

Fish skin is critical to physical defence against pathogens and there is a need to understand the physiological processes impacting ulcers and their healing. Ulcers have been reported in farmed Chinook salmon in New Zealand. This study investigated stress, immune and structural gene expression in farmed Chinook salmon skin with and without ulcers from two sites in New Zealand sampled from February (higher temperature, late summer) to May (lower temperature, late autumn). Skin samples taken adjacent to non-specific ulcers in May and control fish in February demonstrated upregulation of heat shock protein 70 relative to control fish in May. Anterior gradient 2 expression was upregulated in fish with ulcers relative to control fish (both February and May), suggesting increased mucous cell activity. Based on the results of this study, fish with non-specific ulcers showed evidence of stress, inflammation, re-epithelisation, and delayed healing near the ulcer site, elucidating the importance of these processes in the pathogenesis of non-specific ulcers in farmed chinook salmon.

Association between melanin deposits in gill tissue and microbiome across different hatchery reared Atlantic salmon.

AIMS: To investigate the relationship between microbial community profiles and gill pathology during a production cycle of Atlantic salmon in two commercial hatcheries. METHODS AND RESULTS: Relationships between gill histology, environmental conditions, and microbiome were determined using high-throughput data, including 16S rDNA amplicon sequencing data, histopathology data, and water quality parameters. Hatchery A used riverine water and operated a mixed system of recirculation aquaculture system (RAS) and flowthrough. Hatchery B was used bore water and operated a RAS. Melanin deposits, hyperplastic, and inflammatory lesions were observed histologically in the gills. A higher prevalence of melanin deposits was detected and correlated to a change in beta diversity of bacterial communities in early time points (fingerling and parr stages). High abundance of Sphaerotilus sp.,Pseudomonas sp.,Nitrospira sp.,Exiguobacterium sp.,Deinococcus sp.,and Comamonas sp. was correlated with a high prevalence of melanin in filaments. Bacterial diversity increased as the fish cohort transitioned from RAS to flowthrough in hatchery A. CONCLUSIONS: Under commercial conditions, the commensal community of gill bacteria was related to melanin prevalence.

Comparative Effects of Two Forms of Chitosan on Selected Phytochemical Properties of Plectranthus amboinicus (Lour.).

In response to stress factors, plants produce a wide range of biologically active substances, from a group of secondary metabolites, which are applied in medicine and health prophylaxis. Chitosan is a well-known elicitor affecting secondary metabolism in plants, but its effect on the phytochemical profile of Plectranthus amboinicus has not been assessed yet. In the present experiment, the effectiveness of the foliar application of two forms of chitosan (chitosan suspension or chitosan lactate) was compared in order to evaluate their potential to induce the accumulation of selected polyphenolic compounds in the aboveground parts of P. amboinicus. It was shown that chitosan lactate had substantially higher elicitation efficiency, as the use of this form exerted a beneficial effect on the analysed quality parameters of the raw material, especially the content of selected polyphenolic compounds (total content of polyphenols, flavonols, anthocyanins, and caffeic acid derivatives) and the free radical-scavenging activity of extracts from elicited plants. Concurrently, it had no phytotoxic effects. Hence, chitosan lactate-based elicitation can be an effective method for optimisation of the production of high-quality P. amboinicus raw material characterised by an increased concentration of health-promoting and antioxidant compounds.

Size-dependent resistance to amoebic gill disease in naïve Atlantic salmon (Salmo salar).

Amoebic gill disease, caused by the protozoan ectoparasite Neoparamoeba perurans, remains a significant threat to commercial Atlantic salmon aquaculture operations worldwide, despite partial control afforded by selective breeding and therapeutic intervention. Anecdotal reports from commercial producers suggest that historically, smaller Atlantic salmon smolts are more susceptible to AGD than larger smolts. Here, large (>350 g) and small (<200 g) commercially sourced, AGD-naïve Atlantic salmon cohorts were experimentally exposed to 50 N. perurans trophozoites L-1 without intervention. Progression and severity of AGD in challenged cohorts was evaluated through gill pathology, using gill score and histological examination, and quantification of gill-associated amoebae burden using qPCR. To determine the potential basis for differences in AGD susceptibility between cohorts, transcriptome analysis was conducted using RNA extracted from whole gill arches. Overall, the large Atlantic salmon cohort had significantly lower gill parasite burdens and reduced AGD-related gross pathology compared to the small cohort. Relative gill load of N. perurans appeared to be proportional to gill score in both size classes, with larger smolts typically observed to have comparatively reduced parasite burdens at a given gill score. Moreover, comparison between gene expression profiles of large and small smolts highlighted upregulation of genes consistent with elevated immune activity in large smolts. Combined, the results presented here provide strong evidence of size-dependent resistance to AGD in AGD-naïve Atlantic salmon.

Transcriptomic characterisation of a common skin lesion in farmed chinook salmon.

Little is known about host responses of farmed Chinook salmon with skin lesions, despite the lesions being associated with increased water temperatures and elevated mortality rates. To address this shortfall, a transcriptomic approach was used to characterise the molecular landscape of spot lesions, the most commonly reported lesion type in New Zealand Chinook salmon, versus healthy appearing skin in fish with and without spot lesions. Many biological (gene ontology) pathways were enriched in lesion adjacent tissue, relative to control skin tissue, including proteolysis, fin regeneration, calcium ion binding, mitochondrial transport, actin cytoskeleton organisation, epithelium development, and tissue development. In terms of specific transcripts of interest, pro-inflammatory cytokines (interleukin 1ß and tumour necrosis factor), annexin A1, mucin 2, and calreticulin were upregulated, while cathepsin H, mucin 5AC, and perforin 1 were downregulated in lesion tissue. In some instances, changes in gene expression were consistent between lesion and healthy appearing skin from the same fish relative to lesion free fish, suggesting that host responses weren't limited to the site of the lesion. Goblet cell density in skin histological sections was not different between skin sample types. Collectively, these results provide insights into the physiological changes associated with common spot lesions in farmed Chinook salmon.

Element concentrations, histology and serum biochemistry of arctic char (Salvelinus alpinus) and shorthorn sculpins (Myoxocephalus scorpius) in northwest Greenland.

The increasing exploratory efforts in the Greenland mineral industry, and in particular, the proposed rare earth element (REE) mining projects, requires an urgent need to generate data on baseline REE concentrations and their potential environmental impacts. Herein, we have investigated REE concentrations in anadromous Arctic char (Salvelinus alpinus) and shorthorn sculpins (Myoxocephalus scorpius) from uncontaminated sites in Northwest Greenland, along with the relationships between the element concentrations in gills and liver, and gill histology and serum biochemical parameters. Concentrations of arsenic, silver, cadmium, cerium, chromium, copper, dysprosium, mercury, lanthanum, neodymium, lead, selenium, yttrium, and zinc in gills, liver and muscle are presented. No significant statistical correlations were observed between element concentrations in different organs and gill histology or serum biochemical parameters. However, we observed positive relationships between age and histopathology, emphasizing the importance of including age as a co-variable in histological studies of fish. Despite no element-induced effects were observed, this study is considered an important baseline study, which can be used as a reference for the assessment of impacts of potential future REE mine sites in Greenland.

Urinary calculi in larvae of striped trumpeter, Latris lineata.

Urinary calculi are observed in some cultured marine fish larvae and may negatively impact larval health and survival. This study assessed urinary calculi in striped trumpeter Latris lineata larvae from hatching to 28 days post-hatching (dph). The prevalence of urinary calculi was variable over time and ranged from 15% to 50%, whereas the average size of calculi increased with larval age. Urinary calculi were semi-translucent, light cream to white colour with irregular morphology and a uniform internal structure. The calculi resulted in pressure atrophy causing a distended epithelium of the urinary bladder of fish with calculi. The calculi were predominantly formed of calcium hydrogen phosphate (CaHPO4 ). Routine assessment of the prevalence of urinary calculi in marine fish larvae is recommended, along with other parameters, to monitor larval quality and inform hatchery management actions.

Prevalence of epitheliocystis in freshwater Atlantic salmon reared in flow-through and recirculation aquaculture systems.

Epitheliocystis, an intracellular bacterial infection in the gills and skin epithelium, has been frequently reported in Atlantic salmon (Salmo salar) during freshwater production in a number of countries. This study describes the prevalence and intensity of a natural epitheliocystis infection present in the gills of two strains of Atlantic salmon reared in either a flow-through (FT) or a recirculation aquaculture system (RAS) in Ireland. Repeated sampling of gills prior to and throughout seawater transfer, histology and quantitative real-time PCR were used to determine infection prevalence and intensity. Despite no clinical gill disease, and minor histopathological changes, epitheliocystis lesions were identified in histology at all time points. Specific PCR confirmed the presence of Candidatus Clavichlamydia salmonicola in both strains and its number of copies was correlated with intensity of epitheliocystis lesions. A significant interaction between hatchery system and fish strain on the prevalence and intensity of gill epitheliocystis was found both using histological and molecular methods. Specifically, fish from FT had higher prevalence and intensity than RAS reared fish and within FT, the Irish cohort were more affected than Icelandic.

Effects of exposure to environmentally relevant concentrations of lead (Pb) on expression of stress and immune-related genes, and microRNAs in shorthorn sculpins (Myoxocephalus scorpius).

Old lead-zinc (Pb-Zn) mining sites in Greenland have increased the environmental concentration of Pb in local marine organisms, including the shorthorn sculpin. Organ metal concentrations and histopathology have been used in environmental monitoring programs to evaluate metal exposure and subsequent effects in shorthorn sculpins. So far, no study has reported the impact of heavy metals on gene expression involved in metal-related stress and immune responses in sculpins. The aim of this study was to investigate the effect of exposure to environmentally relevant waterborne Pb (0.73 ± 0.35 µg/L) on hepatic gene expression of metallothionein (mt), immunoglobulin M (igm), and microRNAs (miRNAs; mir132 and mir155) associated with immune responses in the shorthorn sculpin compared to a control group. The mt and igm expression were upregulated in the Pb-exposed group compared to the control group. The transcripts of mir132 and mir155 were not different in sculpins between the Pb-exposed and control group; however, miRNA levels were significantly correlated with Pb liver concentrations. Furthermore, there was a positive correlation between liver Pb concentrations and igm, and a positive relationship between igm and mir155. The results indicate that exposure to Pb similar to those concentrations reported in in marine waters around Greenland Pb-Zn mine sites influences the mt and immune responses in shorthorn sculpins. This is the first study to identify candidate molecular markers in the shorthorn sculpins exposed to waterborne environmentally relevant Pb suggesting mt and igm as potential molecular markers of exposure to be applied in future assessments of the marine environment near Arctic mining sites.

KSK-74: Dual Histamine H3 and Sigma-2 Receptor Ligand with Anti-Obesity Potential.

Many studies involving compounds that enhance histamine release, such as histamine H3 receptor (H3R) antagonists, have shown efficacy in inhibiting weight gain, but none have passed clinical trials. As part of the search for H3 receptor ligands that have additional properties, the aim of this study is to evaluate the activity in the reduction in weight gain in a rat model of excessive eating, as well as the impact on selected metabolic parameters, and the number and size of adipocytes of two new H3R antagonists, KSK-60 and KSK-74, which also exert a significant affinity at the sigma-2 receptor. Compounds KSK-60 and KSK-74 are homologues and the elongation of the distal part of the molecule resulted in an approximate two-fold reduction in affinity at H3R, but simultaneously an almost two-fold increase in affinity at the sigma-2 receptor. Animals fed palatable feed and receiving KSK-60 or KSK-74 both at 10 mg/kg b.w. gained significantly less weight than animals in the control obese group. Moreover, KSK-74 significantly compensated for metabolic disturbances that accompany obesity, such as an increase in plasma triglyceride, resistin, and leptin levels; improved glucose tolerance; and protected experimental animals against adipocyte hypertrophy. Furthermore, KSK-74 inhibited the development of inflammation in obesity-exposed adipose tissue. The in vivo pharmacological activity of the tested ligands appears to correlate with the affinity at the sigma-2 receptors; however, the explanation of this phenomenon requires further and extended research.

Protective, Biostimulating, and Eliciting Effects of Chitosan and Its Derivatives on Crop Plants.

Chitosan is a biodegradable and biocompatible polysaccharide obtained by partial deacetylation of chitin. This polymer has been gaining increasing popularity due to its natural origin, favorable physicochemical properties, and multidirectional bioactivity. In agriculture, the greatest hopes are raised by the possibility of using chitosan as a biostimulant, a plant protection product, an elicitor, or an agent to increase the storage stability of plant raw materials. The most important properties of chitosan include induction of plant defense mechanisms and regulation of metabolic processes. Additionally, it has antifungal, antibacterial, antiviral, and antioxidant activity. The effectiveness of chitosan interactions is determined by its origin, deacetylation degree and acetylation pattern, molecular weight, type of chemical modifications, pH, concentration, and solubility. There is a need to conduct research on alternative sources of chitosan, extraction methods, optimization of physicochemical properties, and commercial implementation of scientific progress outcomes in this field. Moreover, studies are necessary to assess the bioactivity and toxicity of chitosan nanoparticles and chitosan conjugates with other substances and to evaluate the consequences of the large-scale use thereof. This review presents the unique properties of chitosan and its derivatives that have the greatest importance for plant production and yield quality as well as the benefits and limitations of their application.

Experimental exposure to low concentrations of Neoparamoeba perurans induces amoebic gill disease in Atlantic salmon.

Amoebic gill disease (AGD) is a significant issue in Atlantic salmon mariculture. Research on the development of treatments or vaccines uses experimental challenges where salmon is exposed to amoebae concentrations ranging from 500 to 5,000/L. However, the water concentrations of N. perurans on affected salmon farms are much lower. The lowest concentration of N. perurans previously reported to cause AGD was 10/L. Here, we report that concentrations as low as 0.1/L of N. perurans can cause AGD. We propose that concentrations of N. perurans that reflect those measured on salmon farms should be used for future experimental challenges.

Case definitions for skin lesion syndromes in chinook salmon farmed in Marlborough Sounds, New Zealand.

Skin lesions are commonly reported in farmed salmonids. Chinook salmon (Oncorhynchus tshawytscha), introduced from California USA, is the only salmonid species commercially farmed in New Zealand, although trout are cultured for release by Fish and Game New Zealand. There are several farming areas in New Zealand, including Marlborough Sounds, Canterbury and Stewart Island. While the industry has not been affected by any of the major production diseases seen internationally, skin lesions have been recorded with an increase in prevalence from 2012 onwards in the Marlborough Sounds and were associated with elevated summer mortalities on farms in 2015. Here we are proposing case definitions for different types of skin lesions for future monitoring and research. Based on our current understanding of the above skin conditions, we developed case definitions for spots, spreading spots, ulcerated spreading spots and Regular Outline Flank Ulcers defining a positive case for three study units, including individual fish, pen/unit and farm.

NaCl-Induced Elicitation Alters Physiology and Increases Accumulation of Phenolic Compounds in Melissa officinalis L.

In nature, plants usually produce secondary metabolites as a defense mechanism against environmental stresses. Different stresses determine the chemical diversity of plant-specialized metabolism products. In this study, we applied an abiotic elicitor, i.e., NaCl, to enhance the biosynthesis and accumulation of phenolic secondary metabolites in Melissa officinalis L. Plants were subjected to salt stress treatment by application of NaCl solutions (0, 50, or 100 mM) to the pots. Generally, the NaCl treatments were found to inhibit the growth of plants, simultaneously enhancing the accumulation of phenolic compounds (total phenolics, soluble flavonols, anthocyanins, phenolic acids), especially at 100 mM NaCl. However, the salt stress did not disturb the accumulation of photosynthetic pigments and proper functioning of the PS II photosystem. Therefore, the proposed method of elicitation represents a convenient alternative to cell suspension or hydroponic techniques as it is easier and cheaper with simple application in lemon balm pot cultivation. The improvement of lemon balm quality by NaCl elicitation can potentially increase the level of health-promoting phytochemicals and the bioactivity of low-processed herbal products.

High Temperature Alters Secondary Metabolites and Photosynthetic Efficiency in Heracleum sosnowskyi.

Due to global warming, invasive species have spread across the world. We therefore studied the impact of short-term (1 day or 2 days) and longer (7 days) heat stress on photosynthesis and secondary metabolites in Heracleum sosnowskyi, one of the important invasive species in the European Union. H. sosnowskyi leaves exposed to short-term heat stress (35 °C/1 d) showed a decrease in chlorophyll and maximum potential quantum efficiency of photosystem II (Fv/Fm) compared to control, 35 °C/2 d, or 30 °C/7 d treatments. In turn, the high level of lipid peroxidation and increased H2O2 accumulation indicated that the 30 °C/7 d stress induced oxidative damage. The contents of xanthotoxin and bergapten were elevated in the 2 d and 7 d treatments, while isopimpinellin was detected only in the heat-stressed plants. Additionally, the levels of free proline and anthocyanins significantly increased in response to high temperature, with a substantially higher increase in the 7 d (30 °C) treatment. The results indicate that the accumulation of proline, anthocyanins, and furanocoumarins, but not of phenolic acids or flavonols, contributes to protection of H. sosnowskyi plants against heat stress. Further studies could focus on the suppression of these metabolites to suppress the spread of this invasive species.

Microbial Ecology of Atlantic Salmon (Salmo salar) Hatcheries: Impacts of the Built Environment on Fish Mucosal Microbiota.

Successful rearing of fish in hatcheries is critical for conservation, recreational fishing, commercial fishing through wild stock enhancements, and aquaculture production. Flowthrough (FT) hatcheries require more water than recirculating aquaculture systems (RAS), which enable up to 99% of their water to be recycled, thus significantly reducing environmental impacts. Here, we evaluated the biological and physical microbiome interactions of three Atlantic salmon hatcheries (RAS n = 2, FT n = 1). Gill, skin, and digesta from six juvenile fish along with tank biofilms and water were sampled from tanks in each of the hatcheries (60 fish across 10 tanks) to assess the built environment and mucosal microbiota using 16S rRNA gene sequencing. The water and tank biofilm had more microbial richness than fish mucus, while skin and digesta from RAS fish had 2 times the richness of FT fish. Body sites each had unique microbiomes (P < 0.001) and were influenced by hatchery system type (P < 0.001), with RAS being more similar. A strong association between the tank and fish microbiome was observed. Water and tank biofilm richness was positively correlated with skin and digesta richness. Strikingly, the gill, skin, and digesta communities were more similar to that in the origin tank biofilm than those in all other experimental tanks, suggesting that the tank biofilm has a direct influence on fish-associated microbial communities. Lastly, microbial diversity and mucous cell density were positively associated with fish growth and length. The results from this study provide evidence for a link between the tank microbiome and the fish microbiome, with the skin microbiome as an important intermediate.IMPORTANCE Atlantic salmon, Salmo salar, is the most farmed marine fish worldwide, with an annual production of 2,248 million metric tons in 2016. Salmon hatcheries are increasingly changing from flowthrough toward recirculating aquaculture system (RAS) design to accommodate more control over production along with improved environmental sustainability due to lower impacts on water consumption. To date, microbiome studies of hatcheries have focused either on the fish mucosal microbiota or on the built environment microbiota but have not combined the two to understand their interactions. Our study evaluates how the water and tank biofilm microbiota influences the fish microbiota across three mucosal environments (gill, skin, and digesta). Results from this study highlight how the built environment is a unique source of microbes to colonize fish mucus and, furthermore, how this can influence fish health. Further studies can use this knowledge to engineer built environments to modulate fish microbiota for beneficial phenotypes.

Correction to: Comparative Assessment of the New PDE7 Inhibitor - GRMS-55 and Lisofylline in Animal Models of Immune-Related Disorders: A PK/PD Modeling Approach.

There was a mistake in the unit of clearance (Cl) in Table II. In addition, the descriptions of V1(ROL) and V1(GRMS-55) were imprecise and the reference number in the footnote below this table should be (9). The corrected Table appears below.

Comparative Assessment of the New PDE7 Inhibitor - GRMS-55 and Lisofylline in Animal Models of Immune-Related Disorders: A PK/PD Modeling Approach.

PURPOSE: This study aimed to assess the activity of two phosphodiesterase (PDE) inhibitors, namely GRMS-55 and racemic lisofylline ((±)-LSF)) in vitro and in animal models of immune-mediated disorders. METHODS: Inhibition of human recombinant (hr)PDEs and TNF-alpha release from LPS-stimulated whole rat blood by the studied compounds were assessed in vitro. LPS-induced endotoxemia, concanavalin A (ConA)-induced hepatitis, and collagen-induced arthritis (CIA) animal models were used for in vivo evaluation. The potency of the investigated compounds was evaluated using PK/PD and PK/PD/disease progression modeling. RESULTS: GRMS-55 is a potent hrPDE7A and hrPDE1B inhibitor, while (±)-LSF most strongly inhibits hrPDE3A and hrPDE4B. GRMS-55 decreased TNF-alpha levels in vivo and CIA progression with IC50 of 1.06 and 0.26 mg/L, while (±)-LSF with IC50 of 5.80 and 1.06 mg/L, respectively. Moreover, GRMS-55 significantly ameliorated symptoms of ConA-induced hepatitis. CONCLUSIONS: PDE4B but not PDE4D inhibition appears to be mainly engaged in anti-inflammatory activity of the studied compounds. GRMS-55 and (±)-LSF seem to be promising candidates for future studies on the treatment of immune-related diseases. The developed PK/PD models may be used to assess the anti-inflammatory and anti-arthritic potency of new compounds for the treatment of rheumatoid arthritis and other inflammatory disorders.