Genes and elements involved in the regulation of the nervous system and growth affect the development of spinal deformity in Cyprinus carpio.

Spinal deformity is a serious economic and animal welfare problem in intensive fish farming systems, which will be a significant unsolved problem for the fish sector. The aim of this study was to determine the relative expression of genes (Akt1 substrate 1, Calreticulin, Collagen type I alpha 2 chain, Corticotropin-releasing hormone, Chromodomain-Helicase DNA-binding, Growth hormone, Insulin like growth factor 1, Myostatin, Sine oculis-related homeobox 3, Toll-like receptor 2) in different tissues associated with spinal deformity and to determine the macroelement (calcium, magnesium, phosphorus, potassium, sodium, sulfur) and microelement (barium, copper, iron, manganese, strontium, zinc) content of spine in healthy and deformed common carps (Cyprinus carpio) in Hungary. The mRNA levels of the genes were measured in 7 different tissues (abdominal fat, blood, brain, dorsal muscle, genitals, heart, liver) by qRT-PCR. Correlations between gene expression and element content were analyzed by using linear regression and Spearman rank correlation. In a total of 15 cases, we found a statistically significant connection between gene expression in a tissue and the macro- or microelement content of the spine. In these contexts, the genes Akt1 substrate 1 (3), Collagen type I alpha 2 chain (2), Corticotropin-releasing hormone (4), Insulin-like growth factor 1 (4), and Myostatin (2), the tissue's blood (3), brain (6), heart (5), and liver (1), the macroelements sodium (4), magnesium (4), phosphorus (1) and sulfur (2) as well as the microelement iron (4) were involved. We also found statistically significant mRNA level differences between healthy and deformed common carps in tissues that were not directly affected by the deformation. Based on our results, genes regulating the nervous system and growth, elements, and tissues are the most associated components in the phenomenon of spinal deformity. With our study, we wish to give direction to and momentum for the exploration of these complex processes.

Association study between relative expression levels of eight genes and growth rate in Hungarian common carp (Cyprinus carpio).

One of the most important issues in improving the competitiveness of the fish production sector is to improve the growth rate of fish. The genetic background to this trait is at present poorly understood. In this study, we compared the relative gene expression levels of the Akt1s1, FGF, GH, IGF1, MSTN, TLR2, TLR4 and TLR5 genes in blood in groups of common carps (Cyprinus carpio), which belonged to different growth types and phenotypes. Fish were divided into groups based on growth rate (normal group: n = 6; slow group: n = 6) and phenotype (scaled group: n = 6; mirror group: n = 6). In the first 18 weeks, we measured significant differences (p < 0.05) between groups in terms of body weight and body length. Over the next 18 weeks, the fish in the slow group showed more intense development. In the same period, the slow group was characterized by lower expression levels for most genes, whereas GH and IGF1 mRNA levels were higher compared to the normal group. We found that phenotype was not a determining factor in differences of relative expression levels of the genes studied.

First report of Sclerotinia sclerotiorum on watercress (Nasturtium officinale) in aquaponic system in Hungary.

Watercress (Nasturtium officinale) is an aquatic dicotyledonous vegetable belonging to Brassicaceae (Aiton 1812). Watercress was grown in an aquaponic system on fired clay ball medium at the Aquaponic Research Station of the University of Debrecen, in the city of Debrecen (Hungary). During January 2020, 3-month-old plants showed symptoms in aquaponic cultivation. A visual survey showed 30% of plants with symptoms. Leaves and stems withered and showed white cotton-like mycelium. Mycelia from infected plants were placed on potato dextrose agar (PDA) and incubated at 25°C for seven days. Single hyphal tips were transferred to produce a pure culture. All ten fungal isolates showed similar morphological characteristics on PDA. Colonies consisted of white mycelia after three days and globoid to irregular and black 2.5 to 7 (average, 3) mm (n = 100 from ten plates) sclerotia formed ten days later, which are the typical morphological features of Sclerotinia sclerotiorum (Mordue et al. 1976). Molecular identification was performed with one of the ten isolates (Scl_B). Mycelia were grown in 250 ml of potato dextrose broth in a rotary shaker at 175 rpm at 24°C for six days. DNA was extracted from mycelium using a Nucleospin plant II (Macherey-Nagel, Germany) according to the manufacturer's protocol. PCR amplification (Kim et al. 2014) was performed with primers ITS1/ITS4 for the internal transcribed spacer region (White et al. 1990) on a Primus 96 thermal cycler (MWG Biotech, Germany). Specific polymerase chain reaction was performed with primers SSasprF/SSasprR (Abd-Elmagid et al. 2013). PCR products were sequenced by Microsynth Austria GmbH. NCBI BLAST analysis of the 440-bp ITS sequence (Genbank MW012403.1) showed 100% identity with the sequence of S. sclerotiorum (MT177267.1, etc.). The 170-bp specific gene sequence (Genbank MW959042.1) had a 100% similarity to hypothetical proteins (Genbank MK028159.1), with a 99.4% similarity to a portion of the S. sclerotiorum aspartyl protease gene (AF271387.1). Pathogenicity tests were carried out by inoculating surface-disinfested, 30-day-old watercress plants in plastic pots (15x15x12 cm). In three repeated experiments 90 watercress plants were measured. 15 plants (one plant per pot) were planted into the five-times autoclaved substrate (Biorgmix: pH 6.1±0.5%, N:1.5%, P2O5:0.7%, K2O:0.5%, organic matter content:50%) and inoculated by ten wheat kernels that were colonized by S. sclerotiorum (Scl_B) (Garibaldi et al. 2019). 15 plants were planted into the substrate with ten non-inoculated kernels as a control. Plants were kept in an MLR-352 climatic test chamber (PHCbi, Japan) at 21 ± 1°C for 12 hr light:dark cycle. On the first day of the experiment complex nutrient solution (Tek-Land: N:5%, P2O5:5%, K2O:5%, B:0.01%, Cu:0,01%, Mn:0.02%, Mo:0.002%, Zn:0.016%) was used, then autoclaved water daily. Eight days later white mycelium appeared on every inoculated plant and five days later dark sclerotia formed on the stems. Based on the morphological characteristics the re-isolated pathogen was S. sclerotiorum. Similar results were detected in three repeated experiments with white mold fungus being reisolated from all 45 infected watercress plants. The 45 non-inoculated plants did not show any symptoms and any diseases. This pathogen has already been reported on watercress in the field (Farr et al. 1989; Boland and Hall 1994; Garibaldi et al. 2019). This is the first reported case of white mold on watercress in aquaponic system in Hungary.

Effects of phytonutrient-supplemented diets on the intestinal microbiota of Cyprinus carpio.

In the aquaculture sector, a strategy for the more efficient use of resources and proper disease control is needed to overcome the challenges of meat production worldwide. Modulation of the gastrointestinal tract microbiota is a promising approach for promoting animal health and preventing infection. This feeding experiment was conducted to discover the phytonutrient-induced changes in the gastrointestinal tract microbiota of common carp (Cyprinus carpio). Acclimatized animals aged 7 months (30 weeks) were divided randomly into five experimental groups to investigate the effects of the applied feed additives. The dietary supplements were manufactured from anthocyanin-containing processing wastes from the food industry, specifically the production of Hungarian sour cherry extract, synbiotics from fermented corn, and fermentable oligosaccharides from Hungarian sweet red pepper seeds and carotenoids from Hungarian sweet red pepper pulps, applied at a dose of 1%. The gut contents of the animals were collected at four time points throughout the 6-week study period. To track the compositional and diversity changes in the microbiota of the carp intestinal tract, V3-V4 16S rRNA gene-based metagenomic sequencing was performed. The growth performance of common carp juveniles was not significantly affected by supplementation of the basal diet with plant extracts. Phytonutrients improve the community diversity, increase the Clostridium and Lactobacillus abundances and decrease the abundances of potentially pathogenic and spoilage bacteria, such as Shewanella, Pseudomonas, Acinetobacter and Aeromonas. The phyla Proteobacteria, Tenericutes and Chlamydiae were positively correlated with the body weight, whereas Spirochaetes and Firmicutes exhibited negatively correlations with the body weight. We hypothesize that the application of phytonutrients in aquaculture settings might be a reasonable green approach for easing the usage of antibiotics.

Iron and Manganese Retention of Juvenile Zebrafish (Danio rerio) Exposed to Contaminated Dietary Zooplankton (Daphnia pulex)-a Model Experiment.

In present study the effect of iron (Fe) and manganese (Mn) contamination was assessed by modeling a freshwater food web of water, zooplankton (Daphnia pulex), and zebrafish (Danio rerio) under laboratory conditions. Metals were added to the rearing media of D. pulex, and enriched zooplankton was fed to zebrafish in a feeding trial. The elemental analysis of rearing water, zooplankton, and fish revealed significant difference in the treatments compared to the control. In D. pulex the Mn level increased almost in parallel with the dose of supplementation, as well as the Fe level differed statistically. A negative influence of the supplementation on the fish growth was observed: specific growth rate (SGR%) and weight gain (WG) decreased in Fe and Mn containing treatments. The redundancy analysis (RDA) of concentration data showed strong correlation between the rearing water and D. pulex, as well as the prey organism of Fe- and Mn-enriched D. pulex and the predator organism of D. rerio. The bioconcentration factors (BCF) calculated for water to zooplankton further proved the relationship between the Fe and Mn dosage applied in the treatments and measured in D. pulex. Trophic transfer factor (TTF) results also indicate that significant retention of the metals occurred in D. rerio individuals, however, in a much lower extent than in the water to zooplankton stage. Our study suggests that Fe and Mn significantly accumulate in the lower part of the trophic chain and retention is effective through the digestive track of zebrafish, yet no biomagnification occurs. Graphical abstract.

Genetic Diversity and Structure of Common Carp (Cyprinus carpio L.) in the Centre of Carpathian Basin: Implications for Conservation.

Hungary is one of the largest common carp-production countries in Europe and now, there is a large number of local breeds and strains in the country. For proper maintenance of the animal genetic resources, information on their genetic diversity and structure is essential. At present, few data are available on the genetic purity and variability of the Hungarian common carp. In this study, we genetically analyzed 13 strains in Hungary and, in addition, the Amur wild carp, using 12 microsatellite markers. A total of 117 unique alleles were detected in 630 individuals. Low levels of genetic differentiation (Fst and Cavalli-Sforza and Edwards distance) were estimated among strains. The AMOVA showed the low but significant level of genetic differentiation among strains (3.79%). Bayesian clustering analysis using STRUCTURE classified the strains into 14 different clusters. The assignment test showed that 93.64% of the individuals could be assigned correctly into their original strain. Overall, our findings can be contributed to complementing scientific knowledge for conservation and management of threatened strains of common carp.

Accumulation of Metals in Juvenile Carp (Cyprinus carpio) Exposed to Sublethal Levels of Iron and Manganese: Survival, Body Weight and Tissue.

Many oxbows are contaminated by Fe and Mn as a consequence of the elemental concentration of sediment and water originating from the Upper Tisza Region of Hungary. The phenomenon is partly caused by anthropogenic activities and mainly due to the geochemical characteristics of the region. The effects of Fe and Mn on the aquatic ecosystem of these wetlands were investigated in a model experiments in this study. Survival, individual body weight and the elemental concentrations of organs were determined in common carp (Cyprinus carpio) juveniles reared in Fe and Mn contaminated media (treatment 1: Fe 0.57 mg L-1, Mn 0.29 mg L-1, treatment 2: Fe 0.57 mg L-1, Mn 0.625 mg L-1, treatment 3: Fe 1.50 mg L-1, Mn 0.29 mg L-1, treatment 4: Fe 1.50 mg L-1, Mn 0.625 mg L-1 and control: Fe 0.005 mg L-1, Mn 0.003 mg L-1), for rearing time of 49 days. The treatment with Fe and Mn did not have any effect on the survival data and individual body weight in the levels tested. The highest concentration of Fe and Mn was found in the liver and brain of carp juveniles, while the lowest concentration of these elements occurred in the muscular tissue and gills. The treatment where Fe and Mn were applied in the highest concentrations resulted in a statistically higher level of these elements in the brain, grills and muscle tissues. The treatment where only Mn was present in the highest concentration caused increased level of Mn only in the liver. We found metal accumulation in almost every organ; however, the applied concentrations and exposure time did not affect the survival and average body weight of carp juveniles.