New reports on iron related proteins: Molecular characterization of two ferroportin genes in common carp (Cyprinus carpio L.) and its expression pattern.

Iron uptake, transport, and storage require the involvement of several proteins, including ferroportin (fpn), the sole known iron efflux transporter. Due to its critical function fpn has been studied, particularly in humans. Here, we characterized the ferroportin gene in common carp (Cyprinus carpio L.) and performed RNA-seq analysis to evaluate its constitutive transcription levels across different tissues. Our results indicate that C. carpio possesses two functional fpns with distinct expression patterns, highlighting the potential for functional divergence and expression differentiation among fpns in this species.

Re-evaluation of common carp (Cyprinus carpio L.) housekeeping genes for gene expression studies - considering duplicated genes.

Quantitative real-time PCR is one of the most widely used techniques for measuring changes in the expression of target transcripts due to its sensitivity, specificity, and cost-effectiveness. However, the essential step that determines appropriate and correct data interpretation is the selection of proper endogenous control genes. Identifying useful reference genes with stable expression is critical for accurate normalization and precise results. Functional divergence of duplicated genes in tetraploid species, like common carp, can complicate the choice for a proper reference gene. In the present study, we determined the expression stability of duplicated genes of 40s, b2m, ef1α, gapdh, g6pd, and odc1 in different tissues of common carp (Cyprinus carpio L.). Gene expression analysis comprised healthy control fish, fish under bacterial and parasitic infections, and across the early stage of common carp development. Obtained data were compared with the actb gene, which is used widely as a reference in RT-qPCR analysis. The application of the three different algorithms - geNorm, NormFinder, BestKeeper, allowed comparative evaluation of the expression stability of the tested genes. Subsequently, the RefFinder, a web-based tool, was used to rank the examined housekeeping genes comprehensively. We demonstrate variable transcription stability levels in the examined mRNAs as well as differences in expression between paralog gene copies. The 40s, b2m, ef1α and actb genes showed the most stable expression across all physiological conditions and tissues. The gapdh, odc1, and g6pd gene variants demonstrated lower stability. Differences in expression patterns between duplicated genes underline the possibility of functional divergence between them. This aspect should be considered in polyploid species before selecting the reference gene(s). Our study also points on the importance of choice for a reference gene (paralog) when expressing newly identified genes and the spatial expression profile is performed. SUBJECTS: Aquaculture, Molecular Biology, Fish Science.

The many faces of transferrin: Does genotype modulate immune response?

In this study, the expression of pro-inflammatory and iron metabolism genes were analysed under Trypanoplasma borreli (T. borreli) challenge in common carp. Three transferrin (Tf) genotypic groups: two homozygous - DD, GG, and heterozygous DG were intraperitoneally infected with a dose of 2.16 × 105/100 µL parasites. Organ and blood samples were collected at weekly intervals. During the infection period, mortality and parasitaemia were assessed along with measurements of blood iron concentrations and antibody levels. Expression of Tf, Fer, IRP1 and 2, TfR 1a and 1b, Hep, TNF α1 and α2, and IL-1 ß was measured in the peak of parasitaemia and the week preceding the peak. Study revealed, that changes in iron blood level induced by parasite were not correlated with the activities of iron homeostasis genes. Neither iron content nor the specific antibody response correlated with survival. We demonstrate that challenged carp, display three distinct, Tf genotype dependent activity patterns of iron homeostasis genes expression. The expected, "classical" way of up-regulation represented homozygous DD individuals. In contrast, GG individuals demonstrated downward trend, while gene expressions of heterozygous DG carp could be defined as an intermediate. We speculate, whether this phenomenon is related to the transferrin molecule itself or to Tf-genotypes being markers of other factors, that influence the iron homeostasis genes activities. We discussed the role of alarmins in triggering the immune response. Distinct genes activating patterns of homozygous genotypes DD and GG had no consequences in terms of mortality rates caused by T.borreli. The highest mortality was observed in the heterozygous group DG. In conclusion, this study suggest that transferrin variant, but not iron blood concentration, has a significant impact on carp immune response to blood parasite infection. This research sheds a new light on the inflammation process and interaction between a host and invaders.

Acyclovir inhibits Cyprinid herpesvirus 3 multiplication in vitro.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is an aetiological agent of a virulent and lethal disease in common and koi carp. In this study, we examined in vitro the anti-CyHV-3 activity of acyclovir (ACV), nucleoside analogue commonly used against human herpesviruses, as well as acyclovir monophospate (ACV-MP). The cytotoxicity of the ACV and the ACV-MP for two common carp cell lines, CCB (Common carp brain) and KF1 (Koi carp fin 1), was determined by means of MTT and crystal violet assays. In subsequent studies, the concentration of 66.67 µM was applied. The ACV and the ACV-MP (66.67 µM) inhibited a cytopathic effect (CPE) induced by the CyHV-3 virus in the CCB (ACV by 66%, ACV-MP by 58%) and the KF1 (ACV by 25%, ACV-MP by 37%). The viral load measured by the means of TaqMan qPCR was reduced in a range of 67%-93% depending on the analogue, the cell line and the time of incubation. The expression of viral genes (ORF149, ORF3, ORF134 and ORF78) in CCB cells infected with the CyHV-3 was strongly downregulated within the range of 78%-91%. In summary, both the ACV and the ACV-MP can inhibit CyHV-3 replication in vitro.

Hematological Alterations as a Response to Exposure to Selected Fungicides in Common Carp (Cyprinus carpio L.).

The aim of the present study was to assess the hematological response of common carp to fungicides and to determine recovery patterns in fungicide-free water. Fish were exposed to mancozeb, prochloraz or tebuconazole (at concentrations of 1.0, 1.0 and 2.5 mg 1⁻¹, respectively) for 14 days followed by a 30-day recovery period. The following hematological parameters were examined after 1, 3 and 14 days of exposure as well as after recovery time: red blood cells (RBC), hematocrit (Het), total hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), total number of leukocytes (WBC) and leukograms. All analyzed parameters revealed alterations in relation to control samples. The pattern of these changes was irregular, showing either an increase or decrease at different time points of the experiment and not all observed differences were statistically significant. The most noticeable fungicide-specific changes were,observed on the 1st and 14th days of chemical exposure. The majority of the parameters under investigation returned to the control levels after a detoxication period. However, some of the exerted effects were irreversible (Hb, MCH, MCHC and WBC for fish subjected to mancozeb; Hb, MCH, MCHC and monocyte count for fish subjected to prochloraz; Hct and monocyte number for fish subjected to tebuconazole). All of the observed hematoloaical changes were not toxin-soecific.

The localisation of HSP70 and oxidative stress indices in heads of Spodoptera exigua larvae in a cadmium-exposed population.

The effects of cadmium toxicity may vary between animals with different history of metal exposure. The aim of our study was to examine HSP70, protein carbonyl levels, catalase activity and total antioxidant capacity in the heads of Spodoptera exigua (Hübner) larvae originated from undergoing 1- and 44-generational cadmium treatment and in control (those that were not exposed to cadmium). We also measured the cadmium concentration and DNA damage level in the larvae. We observed higher level of heat shock proteins (HSPs) in the heads of larvae derived from multi-generational metal treatment than in the heads of those from one-generational treatment (derived from the control rearing). Analysis of HSP localisation in the larval brain suggests that these changes could be important for protecting the neural function of larval mushroom bodies for animals selected during multigenerational metal exposure. Animals from one-generational treatment had, in turn, higher total antioxidant capacity than animals from multigenerational treatment. Anyway, animals from one- and 44-generational metal treatments did not differ in metal accumulation in the heads and the whole larval bodies, catalase activity or DNA damage level. All these measurements were higher than for control larvae and cadmium accumulation in the heads was much lower than in the whole bodies.

Roundup in the Reproduction of Crucian Carp (Carassius carassius): An In Vitro Effect on the Pituitary Gland and Ovary.

Roundup, the most popular herbicide in global agriculture, is regarded as an endocrine disruptor causing alterations of important hormones at the hypothalamic-pituitary-gonadal axis as well as impairment of gametogenesis. The whole pituitary glands of crucian carp (Carassius carassius) were incubated for 3 h in the medium containing Roundup (0-control, 1 and 10 ng/mL). The level of luteinizing hormone (LH), and mRNA transcript abundance of kisspeptin (kiss-1) and its receptor (gpr54), were determined. The isolated ovarian fragments were incubated for 24 h in the presence of Roundup and the following effects on reproductive parameters were determined: the final oocyte maturation and ovulation, structural changes in follicles, secretion of 17,20ß-progesterone (17,20ß-P) as well as mRNA transcript abundance of the luteinizing hormone receptor (lhr), estrogen receptors (erα, erß1, erß2), and zona radiata (chorion) proteins (zp2 and zp3). Roundup inhibited final oocyte maturation and decreased the percentage of ovulated eggs, and furthermore, caused structural changes in the ovarian follicular components. There were no significant changes in the measured hormone levels and analyzed genes mRNA transcript abundance. Summing up, obtained results indicate that Roundup may adversely affect oocyte maturation and the quality of eggs, suggesting that exposure to this herbicide can lead to reproductive disorders in fish.

Anti-CyHV-3 Effect of Fluorescent, Tricyclic Derivative of Acyclovir 6-(4-MeOPh)-TACV in vitro.

INTRODUCTION: Cyprinid herpesvirus 3 (CyHV-3) is a virus infecting carp with disease symptoms of gill necrosis, fish discoloration, sunken eyes, and mortality reaching 90%. Several research groups have examined how to potentially abate the consequences of viral activity. Recently we showed that acyclovir inhibits CyHV-3 replication in vitro and in the present study we examined the anti-CyHV-3 activity of the tricyclic derivative of acyclovir 6-(4-MeOPh)-TACV (T-ACV), a fluorescent molecule known for higher lipophilicity than acyclovir, and therefore potentially better candidate for application in vivo. MATERIAL AND METHODS: CCB and KF1 cell lines were incubated with T-ACV at concentrations of 0, 66.67, and 133.33 µM for three days and toxicity examined with MTT and CV assays. To investigate the antiviral activity of T-ACV, the lines were infected with CyHV-3 or mock infected and incubated for three days with the drug at concentrations of 0 or 66.67 µM. The activity of T-ACV was evaluated by plaque assay and TaqMan qPCR. RESULTS: T-ACV at a concentration of 66.67 µM displayed low toxicity and inhibited CyHV-3 activity by 13-29%, varying by cell line and method. CONCLUSION: The low anti-CyHV-3 activity of T-ACV indicates that it would be reasonable to screen several tricyclic derivatives of acyclovir for such activity.

Influence of the Genetic Makeup of Common Carp on the Expression of Iron-related Genes During Trypanoplasma Borreli Infection.

INTRODUCTION: Genes related to iron metabolism play an important role in inflammatory response. The objective of this study was to investigate the role of ferritin, transferrin receptors 1a and 1b, and transferrin genes in the response to blood parasite infection in common carp (Cyprinuscarpio L.). MATERIAL AND METHODS: Two genetically distinct carp groups were used: R3 carp, which are established as being sensitive to parasitic infection, and SA carp (Cyprinus carpio haematopterus) of wild origin. An established challenge model with Trypanoplasma borreli was applied. Challenged carp were sampled to determine their expression levels of transferrin receptors 1a and 1b, ferritin, and transferrin mRNA. Mortality and serum iron concentration were also measured. RESULTS: The study revealed contrasting differences in the expression profiles of all key iron regulatory genes except the transferrin gene. In the case of other parameters, significant differences were also observed. CONCLUSION: Our results demonstrate that the level of parasitic infection depends on the blood iron status. This parameter was related to the origin of the fish.