Maize mutant screens: from classical methods to new CRISPR-based approaches.

Mutations play a pivotal role in shaping the trajectory and outcomes of a species evolution and domestication. Maize (Zea mays) has been a major staple crop and model for genetic research for more than 100 yr. With the arrival of site-directed mutagenesis and genome editing (GE) driven by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), maize mutational research is once again in the spotlight. If we combine the powerful physiological and genetic characteristics of maize with the already available and ever increasing toolbox of CRISPR-Cas, prospects for its future trait engineering are very promising. This review aimed to give an overview of the progression and learnings of maize screening studies analyzing forward genetics, natural variation and reverse genetics to focus on recent GE approaches. We will highlight how each strategy and resource has contributed to our understanding of maize natural and induced trait variability and how this information could be used to design the next generation of mutational screenings.

Sperm quality assessment in Ficopomatus enigmaticus (Fauvel, 1923): Effects of selected organic and inorganic chemicals across salinity levels.

Contamination by organic and inorganic compounds remains one of the most complex problems in both brackish and marine environments, causing potential implications for the reproductive success and survival of several broadcast spawners. Ficopomatus enigmaticus is a tubeworm polychaete that has previously been used as a model organism for ecotoxicological analysis, due to its sensitivity and ecological relevance. In the present study, the effects of five trace elements (zinc, copper, cadmium, arsenic and lead), one surfactant (sodium dodecyl sulfate, SDS) and one polycyclic aromatic hydrocarbon (benzo(a)pyrene, B(a)P) on the sperm quality of F. enigmaticus were investigated. Sperm suspensions were exposed in vitro to different concentrations of each selected contaminant under four salinity conditions (10, 20, 30, 35). Possible adverse effects on sperm function were assessed by measuring oxidative stress, membrane integrity, viability and DNA damage. Sperm quality impairments induced by organic contaminants were more evident than those induced by inorganic compounds. SDS exerted the largest effect on sperm. In addition, F. enigmaticus sperm showed high tolerance to salinity variation, supporting the wide use of this species as a promising model organism for ecotoxicological assays. Easy and rapid methods on polychaete spermatozoids were shown to be effective as integrated sperm quality parameters or as an alternative analysis for early assessment of marine and brackish water pollution.

Relationship between wild-caught organisms for bioassays and sampling areas: Widespread serpulid early-development comparison between two distinct populations after trace element exposure.

Previous studies suggested the suitability of the brackish-water serpulid (Ficopomatus enigmaticus) to be used as model organism for both marine and brackish waters monitoring, by the performance of sperm toxicity and larval development assays. The present study focused on larval development after the exposure of two F. enigmaticus populations (Mediterranean and Atlantic, collected in Italy and Portugal, respectively) to different trace elements (copper, mercury, arsenic, cadmium, and lead) at different concentrations. Results of larval development assays were presented as the percentage of abnormal developed larvae. The effect, measured in terms of EC50 for all toxicants tested, showed that mercury was the most toxic metal for larvae of both populations. Specifically, the tested trace elements may be racked in the following order from the highest to the lowest toxicity: Mediterranean: mercury > copper > lead > arsenic > cadmium; Atlantic: mercury > copper > cadmium > arsenic > lead. Responses of both populations were similar for arsenic. Lead was the least toxic element for the Atlantic population, while cadmium showed the least toxicity for the Mediterranean population. These preliminary results demonstrate the sensitivity and suitability of the organisms to be used in ecotoxicological bioassays and monitoring protocols. Moreover, chemical analyses on soft tissues and calcareous tubes of collected test organisms and their sampling site water were performed, to identify and quantify the concentration of the tested trace elements in these 3 matrices. Populations exhibited less sensitivity to a certain element together with a relevantly higher concentration of the same element in soft tissues. This may indicate a certain resistance to particular contaminant toxic effects by organisms that tend to accumulate the same toxicant. This highlights the potential correlation between wild-caught test organisms' responses and a deep characterization of the sampling site to identify putative abnormalities or differences in model organism response during bioassay execution.

Subcellular effects of imidazolium-based ionic liquids with varying anions on the marine bivalve Mytilus galloprovincialis.

Green Chemistry involves applying a set of principles aimed at minimizing the use of hazardous substances in the design, production, and application of chemical products. In recent decades, Ionic Liquids (ILs) have emerged as more environmentally friendly substitutes for traditional organic solvents. This preference is primarily due to their low vapor pressure, which results in minimal atmospheric pollution and enhanced industrial safety. However, existing literature highlights the toxicity of ILs towards aquatic invertebrates. Consequently, this study points to assess the biochemical effects of a selection of ILs through an in vitro approach. Specifically, digestive gland and gill cellular fractions (S9) of the marine bivalve Mytilus galloprovincialis were exposed to varying concentrations (0.05-2 µM) of three ILs featuring identical cations but different anions. The ILs tested were 1-ethyl-3-methylimidazolium octanoate ([EMIM][Oct]), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), and 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIM][EtSO4]). The results indicate that [EMIM][Oct] induces higher toxicity in both S9 tissues, highlighting a strong effect of the anion. Overall, antioxidant and biotransformation defenses were significantly altered for all three ILs assessed. While acetylcholinesterase activity was significantly inhibited of about half of control activity, indicating neurotoxic damage as part of the toxicity mode of action of these ILs, neither lipid peroxidation nor alterations to DNA integrity were observed (≥100 %). This study supports the use of in vitro techniques as important tools capable of generating reliable ecotoxicological data, which can be further considered as a screening before in vivo testing and used for in silico modeling.

In vitro screening of imidazolium and pyrrolidinium based ionic liquids toxicity on subcellular fractions of the Mediterranean mussel Mytilus galloprovincialis.

Ionic liquids (ILs) have been considered eco-friendly alternatives to conventional organic solvents. However, several studies have reported that ILs exert toxicity towards aquatic invertebrates. Applying in vitro methodology, the aim of the present study was to evaluate the potential effect of three ILs on the biochemical performance of exposed Mytilus galloprovincialis digestive gland and gills cellular fractions. Carboxylesterase might be involved in the derived toxicity mechanism of ILs as activity levels increased significantly in digestive gland exposed fractions. This group of ILs did not seem to induce genotoxicity, except in gills cellular fractions exposed to 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. In the literature, in vitro methodology has been suggested as an important complement to animal testing and in silico studies. The present research underlines its efficacy as a quick pre-screening before in vivo testing, particularly with heterogenic groups of substances with high variability in composition, such as ILs and deep eutectic solvents.

Toxicity of ionic liquids in marine and freshwater microorganisms and invertebrates: state of the art.

The variety of applications and expected growth in ionic liquid production are raising concerns about the release of these compounds into aquatic systems. Up to date, 103 studies have provided ecotoxicological data regarding the exposure effects of Ionic Liquids towards aquatic microorganisms and invertebrate species: 61 were devoted to freshwater species (n = 28), while marine species (n = 12) were mentioned in 42. The aim of this review, by gathering published studies on ionic liquids and model aquatic organisms, was to present the toxic effects described in distinct species and to understand which are the main factors influencing the toxicity of some ionic liquids. In accordance with the most recognized pattern, freshwater species were featured in a higher number of publications than marine ones. After literature analysis, algal species were the most represented organisms in aquatic toxicity assessments. Among tested compounds, the imidazolium cations in combination with long alkyl-chain anions, showed to be the most toxic one. In analytical terms, it is not straightforward to find the undissociated compound in a natural compartment, as ionic liquids are composed of ionic components, easily subjected to dissociation. Given the aforementioned, the present review paper points out the need of increasing the number of organisms being assessed in ionic liquids toxicity assays, in order to start defining monitoring procedures. Moreover, such would allow a better understanding of ionic liquids contamination status and, also, the opportunity to remark the effectiveness of new in silico methods for the ecotoxicity assessment of this kind of substances.

Are natural deep eutectic solvents always a sustainable option? A bioassay-based study.

The traditional use of organic solvents in various branches of industry is being rethought as these compounds very often display high volatility, toxicity and lipophilicity (related to the ability to interact with biological membranes). More recently, developments in the field of Green Chemistry are focusing on the design of more sustainable and cost-effective solvent alternatives like Ionic Liquids (ILs), bio-based solvents and natural deep eutectic solvents (NADESs). The present study aimed at performing an ecotoxicological screening of 15 NADESs using an extensive set of marine and freshwater bioassays, based on different endpoints as the following: immobilization of the crustacean Daphnia magna, growth inhibition of Raphidocelis subcapitata and of Phaeodactylum tricornutum, larval development alterations on the serpulid Ficopomatus enigmaticus and bioluminescence inhibition of Aliivibrio fischeri. What emerged was a general absence of toxicity of all samples. However, both algal assays showed a certain degree of biostimulation, up to over 100% growth increase in respect to controls with 8 out of 15 compounds tested with Raphidocelis subcapitata. Despite NADESs-induced negligible toxicity effects to invertebrates, encouraging their labelling as "sustainable" solvents, the liability of their intentional or accidental release into aquatic systems may represent a serious risk in terms of ecosystem functioning impairments.

Biochemical response of Ficopomatus enigmaticus adults after exposure to organic and inorganic UV filters.

With the increase of UV filters usage and consequent release into aquatic environments, the concerns about their potential ecological risks are also increasing. According to this, in the present study, adult polychaetes of the species Ficopomatus enigmaticus were chronically exposed to three concentrations (0.01, 0.1 and 0.5 mg/L) of organic and inorganic filters (Ethylhexyl methoxycinnamate (EHMC) and nanoparticulate Zinc oxide (nZnO), respectively) in order to analyse biochemical responses related to cellular damage, antioxidant defence, biotransformation mechanisms and, lastly, neurotoxicity. Despite major lipid peroxidation caused by EHMC was observed, both UV filters have produced the same response patterns. In details, a clear concentration-dependent activation of glutathione S-transferases and a significant decrease of acetylcholinesterase levels defined an important neurotoxic effect was observed for both contaminants. These results become important to expand the limited scientific literature on biochemical responses of marine and brackish water invertebrates to organic and inorganic UV filters.

Ecotoxicological screening of UV-filters using a battery of marine bioassays.

The present study aimed to assess the toxicity of seven UV-filters: zinc oxide nanoparticles (nZnO, particle size <100 nm), titanium dioxide nanoparticles (nTiO2, primary particle size 21 nm), 2-ethylhexyl-4-methoxycinnamate (EHMC), 4-methylbenzylidene camphor (4-MBC), avobenzone (AVO), octocrylene (OCTO) and benzophenone-3 (BP-3) on three species: Aliivibrio fischeri (inhibition of bioluminescence), Phaeodactylum tricornutum (growth inhibition) and Ficopomatus enigmaticus (larval development success). Results showed nTiO2 to be the most toxic for P. tricornutum (EC50 0.043 mg L-1), while no effect was observed in A. fischeri and F. enigmaticus. EHMC was the most toxic to A. fischeri (EC50 0.868 mg L-1 (15 min) and 1.06 mg L-1 (30 min)) and the second most toxic to P. tricornutum. For F. enigmaticus, the lowest percentages of correct development resulted from 4-MBC exposure, with EC50 of 0.836 mg L-1. Overall, AVO induced low toxicity to every assessed species and OCTO was the least toxic for F. enigmaticus larvae. Considering the results obtained for F. enigmaticus, further larval development assays were performed with nZnO and EHMC under different light (light vs darkness) and temperature (20 and 25 °C) conditions, showing higher percentages of correct development at 25 °C, independently on light/darkness conditions. Under different temperature and photoperiod conditions, nZnO was more toxic than EHMC. Overall, nZnO and EHMC were among the most toxic UV filters tested and, when testing the effects of these UV-filters with temperature the results highlight that the impacts are liable to be lessened at higher temperatures (25 °C compared with 20 °C), in the case of this estuarine polychaete species. Nevertheless, further experiments are necessary to describe the effects of these two UV-filters at different organization levels, to study the toxicity of eventual degradation by-products and to provide more information on the combination of different stressors.