Ádám Staszny

Developmental toxicity and estrogenic potency of zearalenone in zebrafish (Danio rerio)

Zearalenone (ZEA, F2) is one of the most common mycotoxins and the only known mycoestrogen. It enters the food and feed chain from contaminated cereals and infiltrates into sewage or natural waters posing potential threat to exposed livestock, wildlife and humans. Therefore evaluation of its biological effects is of international importance. We performed toxicological tests on zebrafish (Danio rerio) larvae and adults. Developmental toxicity was assessed by an extended (5 days) fish embryo toxicity test (FET). Effects of early ZEA exposure were concentration-dependent with LC50 and LC10 values of 893 and 335 μg/L. In larvae exposed to 500 μg/L and above, ZEA induced similar phenotype to has (heart-and soul) showing defects in heart and eye development and upward curvature of the body axis. From 250 μg/L at 72 hpf the gap in the melanophore streak at the base of the tail fin was missing and the fin fold was abnormal, suggesting disturbance in the development of the adult tail fin primordium. Estrogenic potency was measured on the basis of Vitellogenin (Vtg) protein (adults) levels and relative abundance of vitellogenin-1 mRNA (vtg-1) (larvae and adults). qRT-PCR in larvae proved to be sufficient substitute to adult tests and sensitive enough to detect ZEA in 0.1 μg/L concentrations, that is close to levels observed in wastewaters. Developmental defects reveal that besides direct estrogenic effects, zearalenone might interact with other ontogenic pathways.

Improvement of common carp (Cyprinus carpio) sperm cryopreservation using a programable freezer

The applicability of a programmable freezer for the increased-scale cryopreservation of common carp sperm was investigated. The effect of different equilibration times, cryopreservation methods, extenders, dilution ratios, activating solutions on the post-thaw motility of common carp sperm was investigated. The suitable post-thaw storage time-interval as well as fertilizing capacity of cryopreserved sperm was also examined. The motility, curvilinear velocity (VCL) and straightness (STR) values did not decrease significantly during 60min of equilibration neither in equilibrated nor thawed groups. Motility parameters of thawed sperm were similar using a conventional cryopreservation technique using a polystyrene box [motility (33%), VCL (47μm/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54μm/s) and STR (89%)]. The highest motility and VCL was measured with a sugar based extender (grayling extender) at a ratio 1:9 (motility: 52%, VCL: 76μm/s) and 1:20 (motility: 49%, VCL: 76μm/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2min. A storage time of six hours following thawing did not have a significant effect on the motility parameters of thawed carp sperm. Agglutination was observed during cryopreservation of an elevated volume of sperm whereas motility 47%, VCL 62μm/s and STR 91% were measured after thawing. Fertilization rate with thawed sperm (32%) was significantly lower compared to the control group (73%). According to our results, the developed method using a programmable freezer is suitable for the cryopreservation of elevated number of straws. However, carp sperm agglutination during freezing may have a negative effect on the fertilizing capacity.

Risk assessment of non-native fishes in the catchment of the largest Central-European shallow lake (Lake Balaton, Hungary)

The Fish Invasiveness Screening Kit (FISK) has proved to be a useful tool for assessing and screening the risk posed by potentially invasive fish species in larger risk assessment (RA) areas (i.e. country or multi-country level). In the present study, non-native freshwater fishes were screened for a smaller RA area, the closed and vulnerable but economically important drainage basin of Lake Balaton (Hungary). Receiver operator characteristic analysis of FISK scores for 26 fish species screened by four assessors identified 21 species with scores of ≥11.4 to pose a ‘high risk’ of being invasive, with five species ranked as ‘medium risk’ and none as ‘low risk’. The highest scoring species were gibel carp Carassius gibelio and black bullhead Ameiurus melas, with three Ponto-Caspian Gobiidae identified as amongst the species posing the potentially greatest threat to the catchment. The results of the present study indicate that FISK can be applied to risk assessment areas of smaller geographical scale.

Repeatability, Reproducibility, Separative Power and Subjectivity of Different Fish Morphometric Analysis Methods

We compared the repeatability, reproducibility (intra- and inter-measurer similarity), separative power and subjectivity (measurer effect on results) of four morphometric methods frequently used in ichthyological research, the “traditional” caliper-based (TRA) and truss-network (TRU) distance methods and two geometric methods that compare landmark coordinates on the body (GMB) and scales (GMS). In each case, measurements were performed three times by three measurers on the same specimen of three common cyprinid species (roach Rutilus rutilus (Linnaeus, 1758), bleak Alburnus alburnus (Linnaeus, 1758) and Prussian carp Carassius gibelio (Bloch, 1782)) collected from three closely-situated sites in the Lake Balaton catchment (Hungary) in 2014. TRA measurements were made on conserved specimens using a digital caliper, while TRU, GMB and GMS measurements were undertaken on digital images of the bodies and scales. In most cases, intra-measurer repeatability was similar. While all four methods were able to differentiate the source populations, significant differences were observed in their repeatability, reproducibility and subjectivity. GMB displayed highest overall repeatability and reproducibility and was least burdened by measurer effect. While GMS showed similar repeatability to GMB when fish scales had a characteristic shape, it showed significantly lower reproducability (compared with its repeatability) for each species than the other methods. TRU showed similar repeatability as the GMS. TRA was the least applicable method as measurements were obtained from the fish itself, resulting in poor repeatability and reproducibility. Although all four methods showed some degree of subjectivity, TRA was the only method where population-level detachment was entirely overwritten by measurer effect. Based on these results, we recommend a) avoidance of aggregating different measurer’s datasets when using TRA and GMS methods; and b) use of image-based methods for morphometric surveys. Automation of the morphometric workflow would also reduce any measurer effect and eliminate measurement and data-input errors.

Species-specific markers provide molecular genetic evidence for natural introgression of bullhead catfishes in Hungary

Since three bullhead catfish species were introduced to Europe in the late 19th century, they have spread to most European countries. In Hungary, the brown bullhead (Ameiurus nebulosus) was more widespread in the 1970s–1980s, but the black bullhead (Ameiurus melas) has gradually supplanted since their second introduction in 1980. The introgressive hybridization of the two species has been presumed based on morphological examinations, but it has not previously been supported by genetic evidence. In this study, 11 different Hungarian habitats were screened with a new species-specific nuclear genetic, duplex PCR based, marker system to distinguish the introduced catfish species, Ameiurus nebulosus, Ameiurus melas, and Ameiurus natalis, as well as the hybrids of the first two. More than 460 specimens were analyzed using the above markers and additional mitochondrial sequence analyses were also conducted on >25% of the individuals from each habitat sampled. The results showed that only 7.9% of the specimens from two habitats belonged to Ameiurus nebulosus, and 92.1% were classified as Ameiurus melas of all habitats, whereas the presence of Ameiurus natalis was not detected. Two specimens (>0.4%) showed the presence of both nuclear genomes and they were identified as hybrids of Ameiurus melas and Ameiurus nebulosus. An additional two individuals showed contradicting results from the nuclear and mitochondrial assays as a sign of a possible footprint of introgressive hybridization that might have happened two or more generations before. Surprisingly, the level of hybridization was much smaller than expected based on the analyses of the North American continent’s indigenous stock from the hybrid zones. This phenomenon has been observed in several invasive fish species and it is regarded as an added level of complexity in the management of their rapid adaptation.