Evaluating the In Vivo Virulence of Environmental Pseudomonas aeruginosa Using Microinjection Model of Zebrafish (Danio rerio).

(1) Background: Microinjection of zebrafish (Danio rerio) embryos offers a promising model for studying the virulence and potential environmental risks associated with Pseudomonas aeruginosa. (2) Methods: This work aimed to develop a P. aeruginosa infection model using two parallel exposition pathways on zebrafish larvae with microinjection into the yolk and the perivitelline space to simultaneously detect the invasive and cytotoxic features of the examined strains. The microinjection infection model was validated with 15 environmental and clinical strains of P. aeruginosa of various origins, antibiotic resistance profiles, genotypes and phenotypes: both exposition pathways were optimized with a series of bacterial dilutions, different drop sizes (injection volumes) and incubation periods. Besides mortality, sublethal symptoms of the treated embryos were detected and analyzed. (3) Results: According to the statistical evaluation of our results, the optimal parameters (dilution, drop size and incubation period) were determined. (4) Conclusions: The tested zebrafish embryo microinjection infection model is now ready for use to determine the in vivo virulence and ecological risk of environmental P. aeruginosa.

The growth performance of pond-reared common carp (Cyprinus carpio) larvae propagated using cryopreserved sperm.

The aim of our study was to determine the efficacy of utilizing cryopreserved common carp sperm (in comparison to fresh sperm) for propagation at a Hungarian aquaculture facility. The sperm was frozen in 5 mL straws using an extender method that was previously tested in common carp. Sperm motility was monitored using a computer-assisted sperm analysis system. The hatching and malformation rates among the specimens were recorded before the stocking of larvae in both groups. The growth (body weight, total length) and survival rates of the fish were measured during the pre-nursing (from May to June: between 1 and 26 days post hatching) and grow-out periods (from June to October: between 26 and 105 days post hatching) of the same year. The fresh sperm, which was collected and pooled prior to fertilization, showed high MOT (97%), pMOT (92%), VCL (106 µm s-1), LIN (75%), and ALH (1.84 µm). Prior to the fertilization trial of the cryopreserved sperm, low MOT (34%), pMOT (14%), and VCL (61 µm s-1) values were observed in frozen-thawed sperm. A significantly higher hatching rate was measured in the fresh sperm group (87%) when compared to the cryopreserved sperm group (42%). No significant difference in the overall malformation rate was observed in larvae originating from either the fresh or frozen sperm. A significant difference between the two test groups was observed in the incidence of deformed tails (fresh: 20%, cryopreserved: 55%). Except for one sampling period, no significant difference in the body weight and total length of the fish larvae was found between the two groups throughout the pre-nursing and grow-out periods. A significantly higher larvae survival rate was noted in the fresh sperm (72%) as compared to the cryopreserved group (43%) by the end of the pre-nursing stage. However, no significant difference in survival rate was observed for the cryopreserved sperm (96%) in comparison to the fresh sperm (95%) by the end of the grow-out stage. The results of this study showed, for the first time in large-scale pond culturing, an equal growth and viability in larvae propagated from cryopreserved sperm when compared to fresh sperm (despite the limited available rearing ponds provided by the commercial company).

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.

Computer-Assisted Sperm Analysis to Test Environmental Toxicants.

Fish sperm show many measurable parameters which react sensitively in a dose- and time-dependent way to toxic exposure. Fish sperm is therefore used as an in vitro toxicology test system. One of the most sensitive and easily detectable parameters is progressive motility which can be measured by a computer-assisted sperm analysis (CASA) system. Here we describe a simple protocol to test the effect of environmental toxicants by using zebrafish (Danio rerio) sperm.

Mycotoxin Biodegradation Ability of the Cupriavidus Genus.

The biodegradation and biodetoxification ability of five prominent mycotoxins, namely aflatoxin B1 (AFB1), ochratoxin-A (OTA), zearalenone (ZON), T-2 toxin (T-2) and deoxynivalenol (DON) of Cupriavidus genus were investigated. Biological methods are the most appropriate approach to detoxify mycotoxins. The Cupriavidus genus has resistance to heavy metals and can be found in several niches such as root nodules and aquatic environments. The genus has 17 type strains, 16 of which have been investigated in the present study. According to the results, seven type strains can degrade OTA, four strains can degrade AFB1, four strains can degrade ZON and three strains can degrade T-2. None of the strains can degrade DON. The biodetoxification was measured using different biotests. SOS-chromotest was used for detecting the genotoxicity of AFB1, the BLYES test was used to evaluate the oestrogenicity of ZON, and the zebrafish embryo microinjection test was conducted to observe the teratogenicity of OTA, T-2 and their by-products. Two type strains, namely C. laharis CCUG 53908T and C. oxalaticus JCM 11285T reduced the genotoxicity of AFB1, whilst C. basilensis DSM 11853T decreased the oestrogenic of ZON. There were strains which were able to biodegrade more than two mycotoxins. Two strains degraded two mycotoxins, namely C. metalliduriens CCUG 13724T (AFB1, T-2) and C. oxalaticus (AFB1, ZON) whilst two strains C. pinatubonensis DSM 19553T and C. basilensis degraded three toxins (ZON, OTA, T-2) and C. numazuensis DSM 15562T degraded four mycotoxins (AFB1, ZON, OTA, T-2), which is unique a phenomenon amongst bacteria.

Investigation of Fertilizing Capacity of Zebrafish (Danio rerio) Sperm Exposed to Heavy Metals.

The objective of our study was to investigate the effects of heavy metals on the fertilizing capacity of exposed zebrafish sperm, on embryonic survival, and on occurrence of embryonic deformities following fertilization with exposed sperm. It is important to test heavy metals because they are well-known pollutants. Sperm of externally fertilizing species can get in contact with pollutants found in aquatic environment. Zebrafish sperm, despite its advantages, has seldom been used in in vitro toxicological studies and no reports are available regarding the fertilizing capacity of exposed sperm. Zebrafish sperm was stripped and exposed to concentrations of the tested heavy metals (Zn2+, Cd2+, Cr3+, Cu2+, Ni2+, Hg2+, As3+) for 30 or 120 minutes. Calculated half-maximal effective concentration (EC50) values do not differ significantly from those calculated for motility for any of the tested heavy metals, which means fertilization rate can indicate the toxicity of the given substance following exposure of sperm. Thus, its application as in vitro toxicological end point is reasonable. The survival of embryos and embryonic development have not been affected by the exposure of spermatozoa, which means all alterations in spermatozoa caused by heavy metals have been expressed before 24 hours post fertilization.

An Easy Method for Cryopreservation of Zebrafish (Danio rerio) Sperm.

We developed an easy, efficient, and cheap protocol for zebrafish sperm cryopreservation carried out on dry ice (20 min) using simple composition solution (200 mM glucose, 40 mM KCl, 30 mM Tris, pH = 8.0). The average efficiency of the present cryopreserve method was between 10% and 20% (expressed as fertilization rate). The experiments were conducted and repeated at two different locations, in different countries, yielding very similar results, showing the reproducibility and applicability of the method.

In vitro toxicology test system based on common carp (Cyprinus carpio) sperm analysis.

The effect of heavy metals on the motility parameters of common carp sperm was investigated. In vitro test systems are widespread in ecotoxicology, and fish sperm can be a suitable model. For this reason, studies had been carried out in this topic; however, the published methods are not standard in several aspects (donor species, measured endpoint, etc.). In this study, a previously published toxicology-aimed sperm analysis protocol was tested to examine the effect of heavy metals (arsenic, cadmium, chromium, copper, mercury, nickel, zinc,) on common carp sperm. According to our results, PMOT is the most sensitive of the investigated parameters: dose-response was observed in case of each metal at low concentrations, already after 30 min of exposure. VCL was less sensitive: lower effects were observed at the same concentrations compared to PMOT. Among the examined parameters, LIN was the least affected: a dose-response was observed only in case of arsenic and mercury. The same sensitivity of motility parameters was observed on zebrafish sperm previously. Moreover, we found that PMOT, VCL, and LIN of common carp sperm were affected at the same concentrations as it had been observed in zebrafish, when the identical analytical protocol was applied. The only exception was As3+, where common carp sperm proved to be more sensitive: lower concentrations already reduced its motility parameters. Consequently, PMOT of common carp sperm is an accurate and fast bioindicator of aquatic pollution.

Development of an in vitro toxicological test system based on zebrafish (Danio rerio) sperm analysis.

The effect of seven heavy metals on the motility parameter of zebrafish sperm was tested in order to develop an in vitro toxicological test system as an alternative to live animal testing. In vitro test systems are currently preferred in ecotoxicology due to their practical and ethical advantages and fish sperm can be a suitable model. A number of studies had been carried out previously on this topic, but the described methods had not been standardized in numerous aspects (donor species, measured endpoint, etc.). In this study, heavy metals (mercury, arsenic, chromium, zinc, nickel, copper, cadmium) were used as reference toxicants with known toxicity to develop a standardized fish sperm in vitro assay. The tested concentrations were determined based on preliminary range finding tests. The endpoints were progressive motility (PMOT, %), curvilinear velocity (VCL, µm/s), and linearity (LIN, %) measured by a computer-assisted sperm analysis (CASA) system. According to our results, PMOT was the most sensitive of the three investigated parameters: dose-response curves were observed for each metal at relatively low concentrations. VCL values were less sensitive: higher concentrations were needed to observe changes. Of the three parameters, LIN was the least affected: dose-response relationship was observed only in the case of mercury (e.g., lowest observed effect concentration (LOEC) of Hg at 120 min: 1 mg/L for PMOT, 2.5 mg/L for VCL, 5 mg/L for LIN; LOEC of Cu at 120 min: 1 mg/L for PMOT, 5 mg/L for VCL, any for LIN). The order of toxicity as determined by PMOT was as follows: Hg2+ > As3+ > Cd2+ > Cu2+ > Zn2+ > Cr3+ > Ni2+. In conclusion, we found that PMOT of zebrafish sperm was an accurate and fast bioindicator of heavy metal load. Sperm analysis can be adopted to estimate the possible toxic effects of various chemicals in vitro. Future investigations should concentrate on the applicability of this assay to other contaminants (e.g., organic pollutants).

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos.

BACKGROUND: Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. METHODS: In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. RESULTS: Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90-120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. CONCLUSIONS: Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development.