Removal of dead fish eggs by Asellus aquaticus as a potential biological control in aquaculture.

The objective of the present experimental study was to gain a better understanding of the foraging activity of Asellus aquaticus during fish egg incubation. A. aquaticus were introduced into experimental setups of dead eggs, viable eggs and hatched larvae of zebrafish (Danio rerio), a commonly used model organism. The amount of A. aquaticus and the duration of their exposure to the eggs significantly affected the proportion of consumed dead eggs in each experimental cycle. A. aquaticus belongs to the group of aquatic detritivores, and no predatory behavior was observed during the experiments. These crustaceans could distinguish between the dead eggs and those containing living embryos. Furthermore, zebrafish larvae remained unharmed by A. aquaticus, even in the absence of alternative food source. These findings underscore the potential sanitary role of these crustaceans in natural waters and offer new perspectives on their possible use as biological control organisms in aquaculture hatcheries. Additionally, our results suggest a potential application of A. aquaticus in combating pathogens by reducing the growth substrates for bacteria and fungi.

Growth of two invasive cichlids (Perciformes: Cichlidae) in a natural thermal water habitat of temperate Central Europe (Lake Hévíz, Hungary).

The outflow of the natural thermal Lake of Hévíz is habitat of several fish species, with conservation relevance. In the past few years, numerous thermophile (tropically originated) fishes were reported in this waterbody, from which two species Parachromis managuensis (Günther, 1867), Vieja melanurus (Günther, 1862) characterized with strong, self-sustaining population. The aim of our research was to provide basic population data and to study their individual growth. The standard length of jaguar cichlid ranged from 37 to 283 mm (mean SL = 110.21 ± 65.4 mm), the redhead cichlid standard length varied between 30 and 203 mm (mean SL = 93.91 ± 40.0 mm). Slightly positive allometry (b > 3) was found in the case of both species. The von Bertalanffy Growth Function can be described as the following Lt = 343.6[1 - e-0.196(t+0.973)] in jaguar cichlid and Lt = 298.9[1 - e-0.113(t+0.997)] in the case of redhead cichlid. The Bertalanffy growth equations show slow growth for both species. Fulton's condition factor (K) values varied between 1.376 and 2.11 (mean K = 1.701 ± 0.17) in the case of jaguar cichlid, and between 1.391 and 3.033 (mean K = 2.237 ± 0.24) for redhead cichlid. These baseline population biology data from the first known self-sustaining, temperate-zone populations of two tropical cichlids provide information e.g., for future ecological risk assessments or comparative growth analyzes.

The pet trade as a source of non-native decapods: the case of crayfish and shrimps in a thermal waterbody in Hungary.

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.

Pharmaceuticals in water and sediment of small streams under the pressure of urbanization: Concentrations, interactions, and risks.

Small streams are crucial but vulnerable elements of ecological networks. To better understand the occurrence of pharmaceutically active compounds (PhACs) in streams, this study focused on the occurrence, distribution, and environmental risk of 111 PhACs and 7 trace elements based on a total of 141 water and sediment samples from small streams located in the urbanizing region of Budapest, Hungary. Eighty-one PhACs were detected in the aqueous phase, whereas sixty-two compounds were detected in the sediment. Carbamazepine (CBZ) was the most frequently identified PhAC in water, and was found in 91.5% of all samples. However, the highest concentrations were measured for lamotrigine (344.8 µg·L-1) and caffeine (221.4 µg·L-1). Lidocaine was the most frequently occurring PhAC in sediment (73.8%), but the maximum concentrations were detected for CBZ (395.9 ng·g-1) and tiapride (187.7 ng·g-1). In both water and sediment, more PhACs were found downstream of the wastewater treatment plants (WWTPs) than in the samples not affected by treated wastewater, even though no relationship was observed between the total amount of treated wastewater and the number of detected PhACs. The PhAC concentrations were also independent of the distance from the WWTP effluents. PhAC-polluted samples were detected upstream of the WWTPs, thereby suggesting the relevance of diffuse emissions in addition to WWTP outlets. The most frequently detected PhACs in the sediment were usually also present in the water samples collected at the same place and time. The varying concentrations of PhACs and the fluctuating water-sediment properties resulted in a lack of correlation between the general chemical properties and the concentrations of PhACs, which makes it difficult to predict PhAC contamination and risks in urbanized small streams. The environmental risk assessment indicated that diclofenac had the highest risk in the sampling area.

The mixed phylogenetic origin of northern pike (Esox lucius Linnaeus 1758) populations in the Middle Danubian drainage.

BACKGROUND: Pikes, members of genus Esox, are widespread freshwater predators of the northern hemisphere, and important sport fish also. From the Carpathian basin only one species, the northern pike (E. lucius) is noted. At the same time the pike stocks living in this area show high level of phenotypic variance (e.g. various body pattern) and its growth varies highly both among and within populations. These features usually explained by the environmental diversity of the area. Whereas we think that genetic reasons -e.g. the appearance of other/new pike species in the area- may also be responsible for these observed features. Since as no detailed information have been published from the pike populations of this area, so far; we conducted phylogenetic and morphological assay on 88 pike specimens, collected from 49 Middle Danubian sampling sites. RESULTS: Our phylogenetic surveys showed that the northern pike appear in the study area solely, but all the three of its major lineages (Northern, Circumpolar, Southern) were indicated. Only six specimens represent the Northern lineage, collected from the western part of the study area. The Circumpolar and Southern lineages were common in the Carpathian basin, but the Southern lineage showed higher levels of haplotype diversity than the Circumpolar clade. Which indicates that only the Southern lineage is native in the area, while the other two groups could have appeared in the Middle Danubian system either spontaneously or by human introduction. Moreover, the different clades appeared in the same populations, suggesting the opportunity of inter-lineage hybridisation. From the studied morphologicalal features, the number of scales on the lateral line and the head length showed significant differences among the lineages. At the same time the body pattern of the studied individuals seems to be rather influenced by the ontogenic changes than phylogeny. CONCLUSIONS: The high phenotypic variability of Middle Danubian northern pike populations may be due that all of its three major clades appeared and came in secondary contact in the area. In the within watershed spread of the non-native lineages the human stocking/transfer may play a considerable role.

Mitochondrial sequence diversity reveals the hybrid origin of invasive gibel carp (Carassius gibelio) populations in Hungary.

BACKGROUND: Invasive gibel carp, Carassius gibelio (Bloch, 1782) has become well-established in the Hungarian waters and now are spreading in the European waters. On major concern now is the potential hybridization between gibel carp and the other invasive species in the Carassius auratus complex (CAC), which may further accelerate the spread of the whole invasive species complex. The identification of gibel carp and their hybrids is difficult because of its morphological similarity to the other species in CAC. Here we carry out a genomic assessment to understand the history of gibel carp invasion and its phylogenetic relationship with the other species in CAC. Three loci of the mitochondrial genome (D-loop, CoI, Cytb) were used to determine the phylogenetic origin of individuals and relarionship among six gibel carp populations and the other species in the CAC. METHODOLGY: A total of 132 gibel carp samples from six locations in Southern Transdanubia (Hungary) were collected after phenotypic identification to measure the genetic diversity within and among gibel carp populations of Southern Transdanubia (Hungary). The genetic background was examined by the sequences of the mitochondrial genome: D-loop, Cytochrome c oxidase I (CoI) and Cytochrome b (Cytb). Mitochondrial genetic markers are excellent tools for phylogenetic studies because they are maternally inherited. Successfully identified haplotypes were aligned and with reference sequences in nucleotide databases (i.e., NCBI-BLAST: National Centre for Biotechnology Information and BOLD: Barcode of Life Data System). The phylogenetic relationships among gibel carp populations were then analyzed together with the reference sequences to understand the relationship and the level of hybridization with the species in CAC. RESULTS: Among the 132 aligned D-loop sequences 22 haplotypes were identified. Further examination of representative individuals of the 22 haplotypes, six Cytb and four CoI sequences were detected. The largest number of haplotypes of all three loci were found in Lake Balaton, the largest shallow lake in Central Europe. Based on the NCBI-BLAST alignment of the D-loop, haplotypes of Carassius auratus auratus and Carassius a. buergeri in CAC were identified in the C. gibelio samples. Further analysis of haplotypes with the other two mitochondrial markers confirmed the occurrence of intragenus hybridization of C. gibelio in the Hungarian waters. CONCLUSION: By using three mitochondrial markers (D-loop, Cytb, CoI), we genomically characterized a gibel carp-complex in Hungarian waters and assessed the C. gibelio phylogenetic status between them. Hybrid origin of locally invasive Carassius taxon was detected in Hungary. It points out that invasive species are not only present in Hungary but reproduce with each other in the waters, further accelerating their spread.

Does the Kis-Balaton Water Protection System (KBWPS) Effectively Safeguard Lake Balaton from Toxic Cyanobacterial Blooms?

Lake Balaton is the largest shallow lake in Central Europe. Its water quality is affected by its biggest inflow, the Zala River. During late 20th century, a wetland area named the Kis-Balaton Water Protection System (KBWPS) was constructed in the hopes that it would act as a filter zone and thus ameliorate the water quality of Lake Balaton. The aim of the present study was to test whether the KBWPS effectively safeguards Lake Balaton against toxic cyanobacterial blooms. During April, May, July and September 2018, severe cyanobacterial blooming was observed in the KBWPS with numbers reaching up to 13 million cells/mL at the peak of the bloom (July 2018). MC- and STX-coding genes were detected in the cyanobacterial biomass. Five out of nine tested microcystin congeners were detected at the peak of the bloom with the concentrations of MC-LR reaching 1.29 µg/L; however, accumulation of MCs was not detected in fish tissues. Histopathological analyses displayed severe hepatopancreas, kidney and gill alterations in fish obtained throughout the investigated period. In Lake Balaton, on the other hand, cyanobacterial numbers were much lower; more than 400-fold fewer cells/mL were detected during June 2018 and cyanotoxins were not detected in the water. Hepatic, kidney and gill tissue displayed few alterations and resembled the structure of control fish. We can conclude that the KBWPS acts as a significant buffering zone, thus protecting the water quality of Lake Balaton. However, as MC- and STX-coding genes in the cyanobacterial biomass were detected at both sites, regular monitoring of this valuable ecosystem for the presence of cyanobacteria and cyanotoxins is of paramount importance.

Embryonic exposure to low concentrations of aflatoxin B1 triggers global transcriptomic changes, defective yolk lipid mobilization, abnormal gastrointestinal tract development and inflammation in zebrafish.

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.

Effects of pharmaceutically active compounds (PhACs) on fish body and scale shape in natural waters.

BACKGROUND: In recent years, there are growing concerns about pharmaceutically active compounds (PhACs) in natural ecosystems. These compounds have been found in natural waters and in fish tissues worldwide. Regarding their growing distribution and abundance, it is becoming clear that traditionally used risk assessment methodologies and ecotoxicological studies have limitations in several respects. In our study a new, combined approach of environmental impact assesment of PhACs has been used. METHODS: In this study, the constant watercourses of the suburban region of the Hungarian capital (Budapest) were sampled, and the body shape and scale shape of three fish species (roach Rutilus rutilus, chub Squalius cephalus, gibel carp Carassius gibelio) found in these waters were analyzed, based on landmark-based geometric morphometric methods. Possible connections were made between the differences in body shape and scale shape, and abiotic environmental variables (local- and landscape-scale) and measured PhACs. RESULTS: Significant connections were found between shape and PhACs concentrations in several cases. Despite the relatively large number of compounds (54) detected, citalopram, propranolol, codeine and trimetazidine significantly affected only fish body and scale shape, based on their concentrations. These four PhACs were shown to be high (citalopram), medium (propranolol and codeine), and low (trimetazidine) risk levels during the environmental risk assessment, which were based on Risk Quotient calculation. Furthermore, seven PhACs (diclofenac, Estrone (E1), tramadol, caffeine 17α-Ethinylestradiol (EE2), 17α-Estradiol (aE2), Estriol (E3)) were also categorized with a high risk level. However, our morphological studies indicated that only citalopram was found to affect fish phenotype amongst the PhACs posing high risk. Therefore, our results revealed that the output of (traditional) environmental/ecological risk assessment based on ecotoxicological data of different aquatic organisms not necessarily show consistency with a "real-life" situation; furthermore, the morphological investigations may also be a good sub-lethal endpoint in ecotoxicological assessments.

Comparison of single- and multi-trait approaches to identify best wild candidates for aquaculture shows that the simple way fails.

In agriculture, diversifying production implies picking up, in the wild biodiversity, species or populations that can be domesticated and fruitfully produced. Two alternative approaches are available to highlight wild candidate(s) with high suitability for aquaculture: the single-trait (i.e. considering a single phenotypic trait and, thus, a single biological function) and multi-trait (i.e. considering multiple phenotypic traits involved in several biological functions) approaches. Although the former is the traditional and the simplest method, the latter could be theoretically more efficient. However, an explicit comparison of advantages and pitfalls between these approaches is lacking to date in aquaculture. Here, we compared the two approaches to identify best candidate(s) between four wild allopatric populations of Perca fluviatilis in standardised aquaculture conditions. Our results showed that the single-trait approach can (1) miss key divergences between populations and (2) highlight different best candidate(s) depending on the trait considered. In contrast, the multi-trait approach allowed identifying the population with the highest domestication potential thanks to several congruent lines of evidence. Nevertheless, such an integrative assessment is achieved with a far more time-consuming and expensive study. Therefore, improvements and rationalisations will be needed to make the multi-trait approach a promising way in the aquaculture development.

Thermal baths as sources of pharmaceutical and illicit drug contamination.

Despite the fact that there are tens of thousands of thermal baths in existence, knowledge about the occurrence of pharmaceutically active compounds (PhACs) in untreated thermal wastewater is very limited. Because used thermal water is typically legally discharged into surface waters without any treatment, the effluent poses environmental risks for the receiving water bodies. The aim of this study was to show the occurrence patterns and spatiotemporal characteristics of 111 PhACs in thermal wastewater. Six thermal water outflows of different thermal baths were tested in different seasons in the Budapest metropolitan region (Hungary), and diurnal analysis was performed. After solid-phase extraction, the samples were analysed and quantified by coupling supercritical fluid chromatography and mass spectrometry to perform simultaneous multi-residue drug analysis. The results confirm that water discharge pipes directly transport pharmaceuticals into surface water bodies; 34 PhACs were measured to be over the limit of quantification at least once, and 21 of them were found in more than one water sample. The local anaesthetic drug lidocaine, antiepileptic carbamazepine, analgesic derivative tramadol and illicit drug cocaine were detected in more than half of the samples. Caffeine, metoprolol and bisoprolol (cardiovascular drugs), benzoylecgonine (cocaine metabolite), diclofenac (NSAID), citalopram (antidepressant) and certain types of hormones also have a significant frequency of 30-50%. However, the occurrence and concentrations of PhACs vary according to the season and number/types of visitors. As demonstrated by the diurnal fluctuation, drug contamination of thermal waters can significantly vary, even for similar types of baths; furthermore, the quantity and types of some pollutants rapidly change in the discharged thermal wastewater.

The comparison of two different extenders for the improvement of large-scale sperm cryopreservation in common carp (Cyprinus carpio).

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.

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.

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.

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.

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.