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Ornamental aquaculture and the related pet industry are known to be important sources of non-native species worldwide. In the temperate zone, thermal waterbodies are attractive places for irresponsible owners to release unwanted freshwater pets including decapod crustaceans. Several non-native ornamental species have been reported in the thermal locality of Miskolctapolca (a suburb of Miskolc, Hungary). So we surveyed this site in March 2019-November 2021 to update local occurrence records and detect potentially newly released species. A well-established population of Neocaridina denticulata and the occurrence of Caridina cf. babaulti had previously been noted. However, for the first time at this site, we found the shrimps Atyopsis moluccensis, Caridina gracilirostris and C. multidentata, as well as the crayfish Procambarus virginalis, P. clarkii, Cherax quadricarinatus, C. boesemani and C. snowden, and several formally undescribed Cherax species originating from New Guinea. Furthermore, in most species, gravid females carrying eggs were also noticed. Three shrimps, A. moluccensis, C. gracilirostris and C. multidentata, were recorded for the first time in European wild. Further monitoring of this locality and better education of the general public regarding the risks associated with the release of non-native species are strongly recommended.

Aflatoxin B1-contaminated feeds and foods induce various health problems in domesticated animals and humans, including tumor development and hepatotoxicity. Aflatoxin B1 also has embryotoxic effects in different livestock species and humans. However, it is difficult to distinguish between the indirect, maternally-mediated toxic effects and the direct embryotoxicity of aflatoxin B1 in mammals. In the present study, we investigated the aflatoxin B1-induced direct embryotoxic effects in a zebrafish embryo model system combining toxicological, transcriptomic, immunological, and biochemical approaches. Embryonic exposure to aflatoxin B1 induced significant changes at the transcriptome level resulting in elevated expression of inflammatory gene network and repression of lipid metabolism and gastrointestinal tract development-related gene sets. According to the gene expression changes, massive neutrophil granulocyte influx, elevated nitric oxide production, and yolk lipid accumulation were observed in the abdominal region of aflatoxin B1-exposed larvae. In parallel, aflatoxin B1-induced defective gastrointestinal tract development and reduced L-arginine level were found in our model system. Our results revealed the complex direct embryotoxic effects of aflatoxin B1, including inhibited lipid utilization, defective intestinal development, and inflammation.

The present study investigated the effects of chilled storage and cryopreservation on ide sperm motility and fertilizing capacity alongside the longevity of sperm movement. The parameters of motility (progressive motility-pMOT, curvilinear velocity-VCL and straightness-STR) have been recorded during 48 h of chilled storage (4 °C) at 24-h intervals. The longevity of sperm movement was measured following activation for up to 120 s (in a range at 10–120 s) in freshly stripped and thawed sperm. A formerly established cryopreservation method was tested on ide sperm where motility parameters, hatching rate and larval malformation (according to 7 category groups) were investigated. Significant decrement of pMOT has already been observed after 24 h (6 ± 5%) compared to the freshly stripped sperm (49 ± 22%). pMOT and STR showed no significant changes for up to 120 s following activation in fresh sperm, whereas VCL showed significant difference between 10 (51 ± 11 μm/s), 90 (33 ± 3 μm/s) and 120 (31 ± 4 μm/s) seconds as well as between 20 (48 ± 12 μm/s), and 120 s. No negative effect of cryopreservation was recorded on pMOT (fresh: 49 ± 19%, cryopreserved: 22 ± 22%), VCL (fresh: 45 ± 9 μm/s and cryopreserved: 57 ± 5 μm/s), STR (fresh: 81 ± 3% and cryopreserved: 92 ± 1%) hatching rate (fresh: 22 ± 15%, cryopreserved: 33 ± 18%) or larval malformation (fresh: 12 ± 4%, cryopreserved: 12 ± 4%). No significant correlation was found between the three motility parameters and hatching rate. Cryopreservation had no effect on hatching and the prevalence of larval deformity. Furthermore craniofacial and eye deformities were characteristic in the group originating from fertilization with cryopreserved sperm, while edemas (pericardial, yolk) occurred more frequently in the control. The formerly developed cryopreservation protocol (method for cyprinids) was applicable to ide sperm.

In our study, a traditionally used (Grayling, already used in cyprinid species) and a newly tested (Pike) extender was tested to avoid sperm agglutination phenomenon following thawing during carp sperm cryopreservation. A large-scale (elevated volume of sperm) freezing method in a controlled-rate freezer using 5 ml straw and 10 ml cryotube was also systematically established. In all experiments, the sperm cryopreserved in using Grayling extender (except only one sample) showed an agglutination phenomenon (damaged and intact cells adhered to each other) after thawing where Pike extender resulted the regular cell suspension. No significant difference was observed between the two cryopreserved groups (Pike and Grayling extender) in all motility parameters using the 0.5 ml straw and the polystyrene box. Similarly, motility parameters did not show a significant difference in the two frozen groups with the 5 ml straw, also in the polystyrene box. A significantly higher progressive motility (pMOT, Grayling: 54% ± 8%, Pike: 37% ± 5%), straight line velocity (VSL, Grayling: 50 ± 5 µm/s, Pike: 39 ± 4 µm/s) and beat cross frequency (BCF, Grayling: 20 ± 1 Hz, Pike: 17 ± 1 Hz) was observed in the case of the grayling extender by the 5 ml straw cryopreserved in a controlled-rate freezer (CRF) compare to the pike extender. A significantly higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryotube than with the pike extender. Despite the randomly occurring differences in a few parameters, our new controlled freezing method using the newly tested Pike extender, the 5 ml straw or the 10 ml cryotube can be a good solution for the preservation of elevated volume of carp sperm.

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.

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 60 min 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 (47 mu m/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54 mu m/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: 76 mu m/s) and 1:20 (motility: 49%, VCL: 76 mu m/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2 min. 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 62 mu m/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.

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.

The quality and fertilizing capacity of perch (Perca fluviatilis) sperm collected outside of the spawning season (off-season) and cryopreserved at a commercial scale, were tested. Basic parameters (equilibration time, dilution ratio, sperm concentration, post-thaw motility duration) which can have a significant effect on cryopreservation success were systematically investigated for effects on sperm quality using computer assisted sperm analysis (CASA). No significant decrease in progressive motility (pMOT) and straightness (STR) of fresh-diluted sperm was recorded among groups equilibrated for 0, 30 or 60min in an extender with cryoprotectants. Curvilinear velocity (VCL) was reduced significantly after 30min (30min: 146±15μm/s, 60min: 124±18μm/s) of equilibration compared to the control (174±9μm/s). After thawing, no decrease in pMOT or VCL was observed at different equilibration times in any of the analyzed groups. No correlation was observed among progressive motility, dilution ratios (p=0.7) and cell concentrations (p=0.1). The use of different activating solutions resulted in similar pMOT and VCL in the first 120s post-thaw. Nevertheless, post-thaw sperm motility was reduced after 30s using all activators. Motility parameters with low variation were recorded after thawing of 57 straws (pMOT: 37±7%, VCL: 92±10μm/s, STR: 89±3%). Ten randomly selected straws from commercial-scale cryopreservation resulted in a high fertilization rate (cryopreserved sperm: 72±14%, fresh control: 94±2%). An optimized commercial-scale cryopreservation protocol was successfully developed for Eurasian perch. The applicability of the off-season collected perch sperm for cryopreservation and fertilization was demonstrated.

Landmark-based geometric morphometric analysis revealed differences in scale shape between European sardine Sardina pilchardus and round sardinella Sardinella aurita as well as among the local populations of each species. Fish scale measurements from four different areas in the central and eastern Mediterranean Sea showed that the mean scale shape of the two species using landmark data could be differentiated with high certainty. Populations of S. aurita from the central and eastern Mediterranean Sea could be separated reliably (P0·001) with an average discrimination rate of 91%, whereas the average discrimination of the S. pilchardus populations was lower (80%), albeit still high.

Intraspecific morphological variability may reflect either genetic divergence among groups of individuals or response of individuals to environmental circumstances within the frame of phenotypic plasticity. Several studies were able to discriminate wild fish populations based on their scale shape. Here we examine whether the variations in the scale shape in fish populations could be related to genetic or environmental factors, or to both of them. In the first experiment, two inbred lines of zebrafish, Danio rerio (Hamilton 1822) reared under identical environmental conditions were compared. Secondly, to find out what effect environmental factors might have, offsprings were divided into two groups and reared on different diets for 12 weeks. Potential recovery of scales from an environmental effect was also assessed. Experimental groups could successfully be distinguished according to the shape of scales in both experiments, and the results showed that both genetic and environmental factors may notably influence scale shape. It was concluded that scale shape analysis might be used as an explanatory tool to detect potential variability of environmental influences impacting genetically homogeneous groups of fish. However, due to its sensitivity to environmental heterogeneity, the applicability of this technique in identifying intraspecific stock membership of fish could be limited.