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Polystyrene polymers cause severe toxicity to aquatic animals. However, the process and mechanisms of innate immunity of invertebrates living at the bottom of the food chain to these pollutants remain unclear. In this study, the blood system responses of zooplankton Artemia were assessed through in vivo and in vitro exposure to amino-modified polystyrene nanoplastics (PS-NH 2 NPs). The results indicated that the LC 50 values of PS-NH 2 NPs were 1.09 μg·mL -1 over 48 h and 0.42 μg·mL -1 over 7 d. Based on the five hemocyte subpopulations identified in Artemia, in vitro exposure assays revealed that phagocytosis was performed by plasmocytes and granulocytes with phagocytic rate of 22.64 %. TEM analysis further showed that PS-NH 2 NPs caused cytoplasm vacuolization, swollen mitochondria, and lipid processing disorder. Gene expression pattern results demonstrated that Spatzle, Tollip, Hsp70, Hsp90, Casp8, API5and Pxn were significantly upregulated upon acute and chronic exposure (p < 0.05), while chronic exposure could induce significantly upregulation of ProPO (p < 0.05). Moreover, PS-NH 2 NPs exposure remarkably varied the hemolymph microbiota and hemogram, particularly by increasing the proportion of adipohemocytes and phagocytes (p < 0.05) . Our findings suggest that PS-NH 2 NPs induce different responses in Artemia hemocyte, as primarily reflected by phagocytic processes, expression of immune and apoptosis relating genes, cell fates, hemogram and hemolymph microbiota variations. These findings support the possibility of using Artemia hemocytes as bioindicator to estimate nanoplastics pollution, thus contributing to hematological toxicity research in response to nanoplastics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Embryonic diapause is a common evolutionary adaptation observed across a wide range of organisms. Artemia is one of the classic animal models for diapause research. The current studies of Artemia diapause mainly focus on the induction and maintenance of the embryonic diapause, with little research on the molecular regulatory mechanism of Artemia embryonic reactivation. The first 5 h after embryonic diapause breaking has been proved to be most important for embryonic reactivation in Artemia . In this work, two high-throughput sequencing methods, ATAC-seq and RNA-seq, were integrated to study the signal regulation process in embryonic reactivation of Artemia at 5 h after diapause breaking. Through the GO and KEGG enrichment analysis of the high-throughput datasets, it was showed that after 5 h of diapause breaking, the metabolism and regulation of Artemia cyst were quite active. Several signal transduction pathways were identified in the embryonic reactivation process, such as G-protein-coupled receptor (GPCR) signaling pathway, cell surface receptor signaling pathway, hormone-mediated signaling pathway, Wnt, Notch, mTOR signaling pathways, etc. It indicates that embryonic reactivation is a complex process regulated by multiple signaling pathways. With the further protein structure analysis and RT-qPCR verification, 11 GPCR genes were identified, in which 5 genes function in the embryonic reactivation stage and the other 6 genes contribute to the diapause stage. The results of this work reveal the signal transduction pathways and GPCRs involved in the embryonic reactivation process of Artemia cysts. These findings offer significant clues for in-depth research on the signal regulatory mechanisms of the embryonic reactivation process and valuable insights into the mechanism of animal embryonic diapause.

The halophilic zooplankton brine shrimp Artemia has been used as an experimental animal in multidisciplinary studies. However, the reproductive patterns and its regulatory mechanisms in Artemia remain unclear. In this study, the ovarian development process of parthenogenetic Artemia ( A. parthenogenetica ) was divided into five stages, and oogenesis or egg formation was identified in six phases. The oogenesis mode was assumed to be polytrophic. We also traced the dynamic translocation of candidate germline stem cells (cGSCs) using EdU labelling and elucidated several key cytological events in oogenesis through haematoxylin and eosin staining and fluorescence imaging. Distinguished from the ovary structure of insects and crustaceans, Artemia germarium originated from ovariole buds and are located at the base of the ovarioles. RNA-seq based on five stages of ovarian development identified 2657 upregulated genes related to reproduction by pair-to-pair comparison. Gbb , Dpp , piwi , vasa , nanos , VgA and VgR genes associated with cGSCs recognition and reproductive development were screened and verified using qPCR. Silencing of the VgR gene in A. parthenogenetica ( Ap-VgR ) at ovarian development Stage II led to a low level of gene expression (less than 10%) within 5 days, which resulted in variations in oogenesis-related gene expression and significantly inhibited vitellogenesis, impeded oocyte maturation, and eventually decreased the number of offspring. In conclusion, we have illustrated the patterns of ovarian development, outlined the key spatio-temporal features of oogenesis and identified the negative impacts of VgR gene knockdown on oogenesis using A. parthenogenetica as an experimental animal. The findings of this study also lay a foundation for the further study of reproductive biology of invertebrates.

The cell line is an important experimental tool to investigate the mechanism of host-pathogen interactions and to diagnose and cure diseases. However, their application in aquatic invertebrates is limited because they lack immortalized cell lines. The brine shrimp Artemia produces encysted diapause gastrula embryos under stress conditions. The biological advantages of Artemia embryos, such as quick switching on of the cell cycle upon short-term hydration and easy control of microbial contamination via decapsulation, makes them a candidate material for cell culture in vitro. This study identified five subpopulations of embryonic cells at the gastrula stage based on morphological characteristics. After 3–4 h of hydration, embryonic cells were derived from the cysts and successfully cultured for 10 days in 1.2 × L-15 medium containing 15% fetal bovine serum, with a cell viability of more than 90%. Using this primary cell culture system, Vibrio anguillarum and V. parahaemolyticus strains were screened as possible infectious Vibrio strains using laser confocal microscopy observations. The pathological features of viable Vibrio -challenged embryonic cells were further studied by comparing formaldehyde-inactivated and autoclaved Vibrio and polystyrene beads using SEM observation. The results confirmed that both Vibrio strains could challenge embryonic cells and cause some cytopathological features, such as cell aggregation and concaves. Our study provides the first evidence that in vitro cell culture of Artemia embryos can be used to demonstrate the characteristics of Vibrio infection. The primary cell culture system established in this study will facilitate research on the mechanism of host-pathogen interactions in aquatic animals. • Artemia embryos are good experimental materials which make it a candidate for cell culture in vitro. • Embryonic cells of Artemia were successfully cultured for 10 days in a suitable cell culture system, with cell viability of more than 90%. • The result clarified that embryonic cell in vitro were caused some pathologic features in process of Vibrio challenged. • The established primary cell culture system will facilitate to the mechanism of host-pathogen interactions of aquatic animals and stem cell fates determination. [ABSTRACT FROM AUTHOR]