A cytological revisit on parthenogenetic Artemia and the deficiency of a meiosis-specific recombinase DMC1 in the possible transition from bisexuality to parthenogenesis.

Although parthenogenesis is widespread in nature and known to have close relationships with bisexuality, the transitional mechanism is poorly understood. Artemia is an ideal model to address this issue because bisexuality and "contagious" obligate parthenogenesis independently exist in its congeneric members. In the present study, we first performed chromosome spreading and immunofluorescence to compare meiotic processes of Artemia adopting two distinct reproductive ways. The results showed that, unlike conventional meiosis in bisexual Artemia, meiosis II in parthenogenic Artemia is entirely absent and anaphase I is followed by a single mitosis-like equational division. Interspecific comparative transcriptomics showed that two central molecules in homologous recombination (HR), Dmc1 and Rad51, exhibited significantly higher expression in bisexual versus parthenogenetic Artemia. qRT-PCR indicated that the expression of both genes peaked at the early oogenesis and gradually decreased afterward. Knocking-down by RNAi of Dmc1 in unfertilized females of bisexual Artemia resulted in a severe deficiency of homologous chromosome pairing and produced univalents at the middle oogenesis stage, which was similar to that of parthenogenic Artemia, while in contrast, silencing Rad51 led to no significant chromosome morphological change. Our results indicated that Dmc1 is vital for HR in bisexual Artemia, and the deficiency of Dmc1 may be correlated with or even possibly one of core factors in the transition from bisexuality to parthenogenesis.

Signaling Transduction Pathways and G-Protein-Coupled Receptors in Different Stages of the Embryonic Diapause Termination Process in Artemia.

Artemia is a widely distributed small aquatic crustacean, renowned for its ability to enter a state of embryonic diapause. The embryonic diapause termination (EDT) is closely linked to environmental cues, but the precise underlying mechanisms remain elusive. In this study, ATAC-seq and RNA-seq sequencing techniques were employed to explore the gene expression profiles in Artemia cysts 30 min after EDT. These profiles were compared with those during diapause and 5 h after EDT. The regulatory mechanisms governing the EDT process were analyzed through Gene Ontology (GO) enrichment analysis of differentially expressed genes. Furthermore, the active G-protein-coupled receptors (GPCRs) were identified through structural analysis. The results unveiled that the signaling transduction during EDT primarily hinges on GPCRs and the cell surface receptor signaling pathway, but distinct genes are involved across different stages. Hormone-mediated signaling pathways and the tachykinin receptor signaling pathway exhibited heightened activity in the '0-30 min' group, whereas the Wnt signaling pathway manifested its function solely in the '30 min-5 h' group. These results imply a complete divergence in the mechanisms of signal regulation during these two stages. Moreover, through structural analysis, five GPCRs operating at different stages of EDT were identified. These findings provide valuable insights into the signal regulation mechanisms governing Artemia diapause.

Eco-friendly green synthesis of AgNPs from Elaeocarpus serratus fruit extract: potential to antibacterial, antioxidant, cytotoxic effects of colon cancerous cells (HT-29) and its toxicity assessments of marine microcrustacean Artemia nauplii.

BACKGROUND: The present work demonstrated the green synthesis and characterization of silver nanoparticles (AgNPs) utilizing Elaeocarpus serratus fruit extract. The study examined the effectiveness of phytocompounds in fruit extract in reducing Ag+ to Ag° ions. METHODS: The water-soluble biobased substance production from silver ions to AgNPs in 45 min at room temperature. Surface plasmon resonance (SPR) peak was seen in the UV-visible absorption spectrum of the biologically altered response mixture. Examination with X-ray diffraction (XRD) showed that AgNPs are strong and have a face-centered cubic shape. Scanning electron microscope (SEM) investigation proved the production of AgNPs in a cuboidal shape. RESULTS: The AgNPs demonstrated remarkable antibacterial activity and a potent capacity to neutralize DPPH (2,2-Diphenyl-1-picrylhydrazyl) radicals. The highest growth inhibition was found for E. serratus against S. dysenteriae (18.5 ± 1.0 mm) and S. aureus (18 ± 1.2 mm). These nanoparticles exhibited robust antiradical efficacy even at low concentrations. The AgNPs additionally exhibited cytotoxic effects on (HT-29) human colon adenocarcinoma cancer cells. The MTT assay (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) indicated an inhibitory concentration (IC50) value of 49.1 ± 2.33 µg/mL for AgNPs, contrasting with the untreated cells of the negative control. The biotoxicity assessment using A. salina displayed mortality rates ranging from 8 to 69.33%, attributable to the E. serratus synthesized AgNPs. CONCLUSIONS: In our results concluded that simply first-hand information on that E. serattus fruit extract synthesized AgNPs were efficiently synthesized without the addition of any hazardous substances, and that they may be a strong antibacterial, antioxidant, and potential cytotoxic effects for the treatment of colon carcinoma cell lines.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

Degradation of multiple PAHs and co-contaminants by microbial consortia and their toxicity assessment.

The anthropogenic activities toward meeting the energy requirements have resulted in an alarming rise in environmental pollution levels. Among pollutants, polycyclic aromatic hydrocarbons (PAHs) are the most predominant due to their persistent and toxic nature. Amidst the several pollutants depuration methods, bioremediation utilizing biodegradation is the most viable alternative. This study investigated the biodegradation efficacy using developed microbial consortium PBR-21 for 2-4 ringed PAHs named naphthalene (NAP), anthracene (ANT), fluorene (FLU), and pyrene (PYR). The removal efficiency was observed up to 100 ± 0.0%, 70.26 ± 4.2%, 64.23 ± 2.3%, and 61.50 ± 2.6%, respectively, for initial concentrations of 400 mg L-1 for NAP, ANT, FLU, and PYR respectively. Degradation followed first-order kinetics with rate constants of 0.39 d-1, 0.10 d-1, 0.08 d-1, and 0.07 d-1 and half-life t 1 / 2  of 1.8 h, 7.2 h, 8.5 h, and 10 h, respectively. The microbial consortia were found to be efficient towards the co-contaminants with 1 mM concentration. Toxicity examination indicated that microbial-treated PAHs resulted in lesser toxicity in aquatic crustaceans (Artemia salina) than untreated PAHs. Also, the study suggests that indigenous microbial consortia PBR-21 has the potential to be used in the bioremediation of PAH-contaminated environment.

Microencapsulation of sunscreen reduces toxicity of its components to A. salina: Biochemical, behavioral and morphological studies.

Sunscreens contain several substances that cause damage to species where they are disposed. New formulations have been created to prevent such marine environmental damages. One promising formulation is the microencapsulated sunscreen. The objective of this study was to evaluate the possible safety to marine environment of one microencapsulated sunscreen formulation. The animal model Artemia salina (cists and nauplii) was tested with two sunscreen formulations (microencapsulated and non-microencapsulated) and toxicological, behavioral, morphological parameters as well as biochemical assays (lipoperoxidation and carbonylation tests) were analyzed. Results showed that microencapsulated sunscreen impeded some toxic effects caused by the release of the substances within the microcapsule in the highest concentration, reestablishing the mortality and hatching rates to control levels, while removing the sunscreen microcapsule by adding 1 % DMSO reduced the cyst hatching rate, increasing the nauplii mortality rate and decreased locomotor activity in higher concentrations. Finally, nauplii with 24 hours of life and exposed to sunscreen without the microcapsule showed an increase in mitochondrial activity (assessed at 48 hours after exposure) and presented malformations when exposed to the highest concentration non-microencapsulated concentration (assessed by SEM at 72 hours after exposure), when compared to the control group. These results together allow us to conclude that the microencapsulation process of a sunscreen helps protecting A. salina from the harmful effects of higher concentrations of said sunscreens. However, long-term studies must be carried out as it is not known how long a microencapsulated sunscreen can remain in the environment without causing harmful effects to the marine ecosystem and becoming an ecologically relevant pollutant.

Research on the Artemia nauplii microorganism compositions based on metagenomics next-generation sequencing.

Vibrio species represent the predominant and significant pathogen in global marine fish and shellfish aquaculture. Vibrio species are ubiquitously presented in Artemia cyst hatcheries, and their notable colonization in live prey, particularly Artemia nauplii, leads to the transmission of these pathogens into the digestive system of larval organisms, causing serious problems in Vibriosis in marine aquaculture. To eliminate the Vibriospecies in Artemia nauplii, trichloroisocyanuric acid (TCCA) was used for sterilization of the nauplii. In this study, 3 different concentrations, including 0.5 ppm (FA group), 1.0 ppm (FB group) and 1.5 ppm (FC group) of TCCA were used to treat nauplii for 25 min, and then genomic DNA of the different groups were extracted followed by metagenomic next-generation sequencing (NGS). Bioinformatics analysis was applied and the results indicated that Proteobacter constituted the predominant component within each group at the phylum level, albeit accounting for only 58.68% in the FB group, which was significantly lower than in other groups (>86%). The relative abundance of Vibrio species at genus level showed that when compared with the control group, the FB group (15.8%) was reduced by 25.5%. Beta diversity showed differences between the FB group and the other groups, suggesting that treatment with 1.0ppm TCCA for 25 min would obviously reduce the Vibrio in Artemia nauplii. In conclusion, the Vibrio species were significantly reduced after treatment with TCCA, indicating that TCCA might be an alternative to antibiotics used for live food sterilization in marine aquaculture.

Combined effects of temperature and diet on the performance of larvae produced by young and old Palaemon serratus females.

Seasonal variations in environmental conditions determine the success of decapod larval development, and females transmit more energy in sub-optimal conditions to maximise the fitness of their offspring. The objective of this study was to focus on the combined effects of temperature (14, 18 and 22 °C) and food quality on the performance of larvae produced by 5 young (0+) and 5 old (I+) Palaemon serratus females. We prepared 3 diets based on Artemia, in decreasing order of total fatty acid content: freshly hatched nauplii (N), unenriched metanauplii (M) and metanauplii enriched with a mixture of microalgae (ME). At hatching, the larvae produced by I+ females had a higher biomass but a similar fatty acid concentration to those produced by 0+ females. Larvae survived better and developed relatively faster as temperature increased, and the longer they waited to metamorphose, the greater their weight at metamorphosis. These performances were diet-dependent, with more survival and more growth in less time with diet N than with the other two. Larvae from I+ females performed better than those from 0+ females, especially under the most stressful conditions. The greater biomass of the larvae of I+ females seems to have enabled them to follow a shorter, and therefore faster, development path than those of 0+ females. The larvae's diet also had an impact on post-metamorphic composition: larvae eating a diet richer in fatty acids produced richer juveniles and those eating a poorer diet produced juveniles with slightly more essential fatty acids. This study supports the high plasticity of caridean shrimp larval development and the importance of maternal effects on the fitness of offspring.

Acute Toxicity Assays with the Artemia salina Model: Assessment of Variables.

The use of the brine shrimp Artemia salina (Leach) in acute toxicity assays has great potential due to its simplicity, low cost and reproducibility. In the current study, some of the variables that can influence the reliability of the assay in terms of test organism survival, were evaluated as part of its implementation in our laboratory. The quality and type of water used, the buffer components and other parameters (salinity, pH and dissolved oxygen level), were all evaluated for optimisation purposes. DMSO (dimethyl sulphoxide) was used as the test substance in the toxicity assay, to evaluate the concentration limits as a solvent in sample preparation. Regarding the buffer salinity, pH and dissolved oxygen level, we found that a 25% to 30% deviation from the standard values did not affect the survival of the nauplii (the first-instar larval stage) under assay conditions. In summary, we corroborate the potential use of this model for the prediction of the toxic potential of substances, to inform future testing strategies.

Chemical Composition of Piper nigrum L. Cultivar Guajarina Essential Oils and Their Biological Activity.

The essential oils and aroma derived from the leaves (L), stems (St), and spikes (s) of Piper nigrum L. cv. Guajarina were extracted; the essential oils were extracted using hydrodistillation (HD), and steam distillation (SD), and the aroma was obtained by simultaneous distillation and extraction (SDE). Chemical constituents were identified and quantified using GC/MS and GC-FID. Preliminary biological activity was assessed by determining the toxicity against Artemia salina Leach larvae, calculating mortality rates, and determining lethal concentration values (LC50). The predominant compounds in essential oil samples included α-pinene (0-5.6%), ß-pinene (0-22.7%), limonene (0-19.3%), 35 linalool (0-5.3%), δ-elemene (0-10.1%), ß-caryophyllene (0.5-21.9%), γ-elemene (7.5-33.9%), and curzerene (6.9-31.7%). Multivariate analysis, employing principal component analysis (PCA) and hierarchical cluster analysis (HCA), revealed three groups among the identified classes and two groups among individual compounds. The highest antioxidant activity was found for essential oils derived from the leaves (167.9 41 mg TE mL-1). Larvicidal potential against A. salina was observed in essential oils obtained from the leaves (LC50 6.40 µg mL-1) and spikes (LC50 6.44 µg mL-1). The in silico studies demonstrated that the main compounds can interact with acetylcholinesterase, thus showing the potential molecular interaction responsible for the toxicity of the essential oil in A. salina.

Effects of carotenoid supplementation on colour, growth and physiological function of the endemic dwarf chameleon fish (Badis badis).

The global ornamental fish trade is expanding in response to increased demand for indigenous fish on the global market, while exogenous carotenoids can improve colouration. The 60-day trial investigated the effect of carotenoid supplementation, via Artemia, on colouration, growth and immunophysiology of Badis badis (dwarf chameleon fish). Carotenoid was enriched at 40 ppm (T1), 80 ppm (T2) and 120 ppm (T3) and compared with controls, C1 (unenriched) and C2 (oil-enriched). Fish larvae (average weight 0.12 g) were fed enriched-unenriched Artemia in triplicates (5 × 3) in aquarium tanks (15 L). C1 and T2 had better skin colour (lightness and whiteness) in the posterolateral and caudal fins respectively. The value of redness (a*) in the anterolateral region was higher in T2 and T3 (p < 0.05). The anterolateral red index was higher (p < 0.05) in T2 and T3, whereas in the posterolateral and caudal fins, T1 and T2 were higher (p < 0.05). Compared to C1 and C2, the hue angle in carotenoid groups was found to be low (p < 0.05). No significant change in the growth performance was noticed (p > 0.05). Immune scores such as lysozyme and alkaline protease were highest in T3 (p < 0.05), whereas protease activity was highest in T2 (80 ppm). Stress biomarkers, viz., superoxide dismutase, catalase and malondialdehyde were low in groups fed enriched Artemia (p < 0.05). The integrated biomarker response means and star plot area were lower in the enriched groups (T1-T3), while T2 was the lowest. Overall findings reveal that dietary carotenoid improves the colouration and immune status, but fail to promote growth. Furthermore, 80 ppm enrichment dose improves the overall performance. The findings can help fish keepers improve fish colour and health status through carotenoid supplementation.

Expression of Abdominal-B in the brine shrimp, Artemia franciscana, expands our evolutionary understanding of the crustacean abdomen.

The brine shrimp, Artemia franciscana, has a body plan composed of 11 thoracic segments, followed by 2 genital segments, and then 6 additional abdominal segments. Previous studies of Artemia reported that expression of the posterior-most Hox gene, Abdominal-B (Abd-B), is restricted to the genital segments and is not observed posteriorly in the abdomen at any developmental stage. This report was remarkable because it suggested that the Artemia abdomen posterior to the genital segments was a novel body region of 6 segments that bore no homology to any region in other crustaceans and was unique amongst arthropods in being a Hox-free segmented domain outside of the head. In this study, we used RT-PCR, antibody staining, and in situ hybridization on various stages of Artemia nauplii to show that Abd-B mRNA and protein are in fact expressed throughout the abdominal segments during Artemia development, but this expression later retracts to the two genital segments (G1, G2) and the T11 appendages. This suggests that Abd-B does play a role in specifying abdominal segment identity in all crustaceans that have been examined and suggests a common evolutionary origin for the crustacean abdomen.

Biotransformation and detoxification of saxitoxin by Bacillus flexus in batch experiments.

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by some species of cyanobacteria. They are water soluble and relatively stable in the natural environment, and thereby represent a risk to animal and human health through a long-time exposure. STXs cannot be sufficiently removed by conventional water treatment methods. Therefore, this study investigates the potential STX biodegradation and detoxification by bacteria as a promising method for toxin removal. STX biodegradation experiments were conducted using Bacillus flexus SSZ01 strain in batch cultures. The results revealed that SSZ01 strain grew well and rapidly detoxified STX, with no lag phase observed. STX detoxification by SSZ01 strain was initial-toxin-concentration-dependent. The highest biotransformation rate (10 µg STX L-1 day-1) the pseudo-first-order kinetic constant (0.58 d-1) were obtained at the highest initial toxin concentration (50 µg L-1) and the lowest ones (0.06 µg STX L-1 day-1 and 0.14 d-1, respectively) were recorded at the lowest initial concentration (0.5 µg L-1). STX biotransformation rate increased with temperature, with highest occurred at 30 ºC. This rate was also influenced by pH, with highest obtained at pH8 and lowest at higher and lower pH values. HPLC chromatograms showed that STX biotransformation peak is corresponding to the least toxic STX analog (disulfated sulfocarbamoyl-C1 variant). The Artemia-based toxicity assay revealed that this biotransformation byproduct was nontoxic. This suggests the potential application of this bacterial strain in slow sand filters for cyanotoxin removal in water treatment plants. Being nontoxic, this byproduct needs to be assayed for its therapeutic effects toward neurodegenerative diseases.

In vitro biofilm inhibition efficacy of Aerva lanata flower extract against Gram negative and Gram-positive biofilm forming bacteria and toxicity analysis using Artemia salina.

A biofilm consists of Gram positive and Gram-negative bacteria enclosed in a matrix. Industrial biofouling is caused by biofilms, which can exhibit antimicrobial resistance during infections. Many biofilm studies find that nearly all biofilm communities consist of Gram positive and Gram-negative bacteria. It is therefore necessary to better understand the conserved themes in biofilm formation to develop therapeutics based on biofilm formation. Plant extracts can effectively combat pathogenic bacterial biofilms. This study evaluated the antibacterial and antibiofilm activity of Aerva lanata flower extract against Staphylococcus aureus and Pseudomonas aeruginosa. Methanol extract of dried A. lanata flower was tested against S. aureus and P. aeruginosa to determine the antibacterial activity (10, 25, 50, 75, 100 µg/mL) resulted in a maximum of 0.5-1 log reduction and 2 log reduction in comparison to the control or untreated bacterial cells respectively. A. lanata showed maximum biofilm inhibition up to 1.5-fold and 1-fold against P. aeruginosa and S. aureus. Light microscopic analysis of biofilm treated with A. lanata extract showed efficient distortion of the biofilm matrix. Further, the in vivo analysis of A. lanata in the Artemia salina brine shrimp model showed >50% survival and thus proving the efficacy of A. lanata extract in rescuing the brine shrimps against P. aeruginosa and S. aureus infection.

Prolonged ecological changes can affect morphometrics and gene expression profile? Focusing on Hsp-70 and NLHS-induced Hsp-70 of Artemia urmiana.

BACKGROUND: In recent years, many aquatic ecosystems, including Urmia Lake, have undergone severe ecological tensions. This lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated and its salinity has dramatically increased over the last three decades. In the face of the long period environmental stresses, understanding the adaptation and ecological plasticity mechanisms is the most interesting challenges in genetic and applied ecology. These mechanisms may probably be driven by inducing expression of some genes involved in adaptation such as Hsp-70 and also adjusting morphological parameters. But they are yet to be understood. Hence, the present work aimed to study the mechanisms, along with testing the hypothesis that non-lethal heat shocked nauplii originating from drought period can evoke Hsp-70 expression more than those from rainy period. METHODS: This study measured and analyzed morphometrical characters of adult male and female Artemia urmiana over three decades. Then, the influence of three-decade ecological crisis on Hsp-70 and non-lethal heat shock (NLHS)-induced Hsp-70 expression levels of nauplii of Artemia urmiana habiting Urmia Lake using Real-time PCR technique, based on cyst collections in 1994 (rainy period) to 2020 (drought period), was evaluated. RESULTS: The morphometrics results showed that the morphological characters were significantly shrunk in 2020 compared to 1994 (CI 95%, p < 0.05). Furthermore, our results depicted that, Hsp-70 expression level was significantly upregulated in response to the prolonged ecological crisis, (CI 95%, P < 0.0001), and also interestingly, the nauplii exposed to longe-term ecological crisis (belong to 2020) were able to increase Hsp-70 expression more than other ones in response to environmental stressors including heat. CONCLUSIONS: The present results showed the involvement of Hsp-70 in the adaptation of Artemia urmiana to long term ecological alteration at the cost of shrinking morphometric parameters.

Progress and promise of alternative animal and non-animal methods in biomedical research.

Cell culture and invertebrate animal models reflect a significant evolution in scientific research by providing reliable evidence on the physiopathology of diseases, screening for new drugs, and toxicological tests while reducing the need for mammals. In this review, we discuss the progress and promise of alternative animal and non-animal methods in biomedical research, with a special focus on drug toxicity.

Insights into the Microbicidal, Antibiofilm, Antioxidant and Toxicity Profile of New O-Aryl-Carbamoyl-Oxymino-Fluorene Derivatives.

The unprecedented increase in microbial resistance rates to all current drugs raises an acute need for the design of more effective antimicrobial strategies. Moreover, the importance of oxidative stress due to chronic inflammation in infections with resistant bacteria represents a key factor for the development of new antibacterial agents with potential antioxidant effects. Thus, the purpose of this study was to bioevaluate new O-aryl-carbamoyl-oxymino-fluorene derivatives for their potential use against infectious diseases. With this aim, their antimicrobial effect was evaluated using quantitative assays (minimum inhibitory/bactericidal/biofilms inhibitory concentrations) (MIC/MBC/MBIC), the obtained values being 0.156-10/0.312-10/0.009-1.25 mg/mL), while some of the involved mechanisms (i.e., membrane depolarization) were investigated by flow cytometry. The antioxidant activity was evaluated by studying the scavenger capacity of DPPH and ABTS•+ radicals and the toxicity was tested in vitro on three cell lines and in vivo on the crustacean Artemia franciscana Kellog. The four compounds derived from 9H-fluoren-9-one oxime proved to exhibit promising antimicrobial features and particularly, a significant antibiofilm activity. The presence of chlorine induced an electron-withdrawing effect, favoring the anti-Staphylococcus aureus and that of the methyl group exhibited a +I effect of enhancing the anti-Candida albicans activity. The IC50 values calculated in the two toxicity assays revealed similar values and the potential of these compounds to inhibit the proliferation of tumoral cells. Taken together, all these data demonstrate the potential of the tested compounds to be further used for the development of novel antimicrobial and anticancer agents.

Prepared microplastics interaction with Artemia salina under low pH conditions representing ocean acidification; a simulated environmental exposure.

Ocean acidification and microplastic pollution are two of the major ecological concerns. The distribution of large quantities of plastic debris and microplastics all across the oceans emphasises the need to determine the influence of microplastics in ocean acidification and to evaluate its concomitant toxicological effects on aquatic life forms. Studies on the combined impact of both the stressors are very limited, but much needed in the current scenario. Where most of the present-day research use purchased microplastics of defined size and morphology (microspheres, fibres, rods, etc.), the present study employs prepared "true to life microplastics" that resemble the environmental microplastic pollutants in morphology and size heterogeneity. The present study focusses on evaluating the fate and impact of oceanic microplastics on the physiology and development of Artemia salina (Brine shrimp), one among the most ecologically significant zooplankton species. Natural sea water was acidified by controlled perturbation of carbon dioxide using a valve system. The hatching rate of A. salina cysts receded significantly (p < 0.05) upon singular exposures to microplastics and low pH (7.80), whereas combined effect was insignificant. The reactive oxygen species (ROS) elevated as a result of individual exposures to microplastics and low pH. However, only in 0.5 mg mL-1 PE treatments at pH 7.80, an additive impact was reported for ROS activity (p < 0.05). The SOD activities increased significantly but it can be attributed as the individual responses towards exposure to both the stressors. A significant additive impact was not observed for SOD activity (p > 0.05). But during the development, significant morphological anomalies were observed. Changes in the appendages of nauplii and juveniles as a result of combined exposure to microplastics and low pH treatments are significant findings. Our observations suggest that coupled exposure to microplastics and low pH could induce significant oxidative stress in the marine zooplanktons and also adversely affect their normal development. Findings from the current study emphasise the need for further research to understand the coupled toxicological impacts of ocean acidification and predominant pollutants such as microplastics to other marine animals as well.

Chemical Composition, Preliminary Toxicity, and Antioxidant Potential of Piper marginatum Sensu Lato Essential Oils and Molecular Modeling Study.

The essential oils (OEs) of the leaves, stems, and spikes of P. marginatum were obtained by hydrodistillation, steam distillation, and simultaneous extraction. The chemical constituents were identified and quantified by GC/MS and GC-FID. The preliminary biological activity was determined by assessing the toxicity of the samples to Artemia salina Leach larvae and calculating the mortality rate and lethal concentration (LC50). The antioxidant activity of the EOs was determined by the DPPH radical scavenging method. Molecular modeling was performed using molecular docking and molecular dynamics, with acetylcholinesterase being the molecular target. The OES yields ranged from 1.49% to 1.83%. The EOs and aromatic constituents of P. marginatum are characterized by the high contents of (E)-isoosmorhizole (19.4-32.9%), 2-methoxy-4,5-methylenedioxypropiophenone (9.0-19.9%), isoosmorhizole (1.6-24.5%), and 2-methoxy-4,5-methylenedioxypropiophenone isomer (1.6-14.3%). The antioxidant potential was significant in the OE of the leaves and stems of P. marginatum extracted by SD in November (84.9 ± 4.0 mg TE·mL-1) and the OEs of the leaves extracted by HD in March (126.8 ± 12.3 mg TE·mL-1). Regarding the preliminary toxicity, the OEs of Pm-SD-L-St-Nov and Pm-HD-L-St-Nov had mortality higher than 80% in concentrations of 25 µg·mL-1. This in silico study on essential oils elucidated the potential mechanism of interaction of the main compounds, which may serve as a basis for advances in this line of research.

Chemical, Antioxidant and Biological Studies of Brassica incana subsp. raimondoi (Brassicaceae) Leaf Extract.

Brassica incana subsp. raimondoi is an endemic taxon present in a restricted area located on steep limestone cliffs at an altitude of about 500 m a.s.l. in eastern Sicily. In this research, for the first time, studies on the phytochemical profile, the antioxidant properties in cell-free and cell-based systems, the cytotoxicity on normal and cancer cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, and on Artemia salina Leach, were performed. The total phenolic, flavonoid, and condensed tannin contents of the leaf hydroalcoholic extract were spectrophotometrically determined. Ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) analysis highlighted the presence of several phenolic acids, flavonoids, and carotenoids, while High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) identified various kaempferol and isorhamnetin derivatives. The extract exhibited different antioxidant properties according to the five in vitro methods used. Cytotoxicity by MTT assay evidenced no impact on normal human fibroblasts (HFF-1) and prostate cancer cells (DU145), and cytotoxicity accompanied by necrotic cell death for colon cancer cells (CaCo-2) and hepatoma cells (HepG2), starting from 100 µg/mL and 500 µg/mL, respectively. No cytotoxic effects were detected by the A. salina lethality bioassay. In the H2O2-induced oxidative stress cell model, the extract counteracted cellular reactive oxygen species (ROS) production and preserved non-protein thiol groups (RSH) affected by H2O2 exposure in HepG2 cells. Results suggest the potential of B. incana subsp. raimondoi as a source of bioactive molecules.