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The protandric shrimp Hippolyte inermis is the only known marine invertebrate whose sex determination is strongly influenced by the composition of its food. In H. inermis, a sex reversal is triggered by the ingestion of diatoms of the genus Cocconeis associated with leaves of the seagrass Posidonia oceanica. These diatoms contain compounds that promote programmed cell death (PCD) in H. inermis and also in human cancer cells. Transcriptomic analyses suggested that ferroptosis is the primary trigger of the shrimp’s sex reversal, leading to the rapid destruction of the androgen gland (AG) followed by a chain of apoptotic events transforming the testes into ovaries. Here, we propose a molecular approach to detect the effects of compounds stimulating the PCD. An RNA extraction method, suitable for young shrimp post-larvae (five days after metamorphosis; PL5 stage), was established. In addition, six genes involved in apoptosis, four involved in ferroptosis, and seven involved in the AG switch were mined from the transcriptome, and their expression levels were followed using real-time qPCR in PL5 fed on Cocconeis spp., compared to PL5 fed on a basic control feed. Our molecular approach, which detected early signals of sex reversal, represents a powerful instrument for investigating physiological progression and patterns of PCD in marine invertebrates. It exemplifies the physiological changes that may start a few days after the settlement of post-larvae and determine the life destiny of an individual.

This study describes our attempts to generate a sustainable cell culture of Macrobrachium rosenbergii. We present here a continuous longitudinal study on the embryonic primary cell culture of freshwater prawn M. rosenbergii that was uniquely monitored for up to 90 days with regard to its morphology, metabolic activity, and cell-cycle parameters. The daily monitoring of cells’ wellbeing and morphology showed seeded cells to be changing from attached singular diverse-sized cells after days 4–10 to interconnected clusters of cells, which apparently increased in number as detected by their density in the well. Moreover, the cultures demonstrated an autonomous transition during days 7–10, from completely two-dimensional (2D) morphology to a combination of 2D and three-dimensional (3D) growing structures, leading to the formation of multilayered spheroid-like cell masses. The metabolic activity of cultures showed a non-linear elevated pattern peaking on day 26, demonstrating proliferation and increment in the number of cells, retaining statistically significant elevated metabolic activity up to 40 days, and thereafter gradually declining. In parallel, cell-cycle analyses performed through florescence-activated cell sorting (FACS) showed that the G0/G1 and S phases were inversely proportional to each other. Proliferation, based on metabolic activity, in the cultures was sustained by a significant increase in the portion of cells arrested in the S phase, from day 4 up to day 24, and then a decrease between days 45 and 90. Sorting the populations in the M. rosenbergii primary embryonic cell culture on days 3 and 24 revealed eight seeded populations, most of them expressing the putative proliferation markers MrMYC and MrPCNA, while six of them expressed also the putative stem-cell markers MrOct-4 and MrSox-2/3. Therefore, assuming the increment in cell density and metabolic activity and the reduction in G0/G1 distribution toward S, as well as the increment in G2/M, all pointing toward proliferation, we further hypothesized that splitting the cultures along the experiment at the high-proliferating mitotic ratio peaks would enable successful passages. Indeed, in prawn embryonic primary cell culture, we succeeded in executing two consecutive passages: the first after 8 days in culture and the second 4 days following the first passage. Cells after both passages expressed the species-specific Mr18S, along with the proliferative markers MrMYC and MrPCNA and the stem-cell markers MrOct-4 and MrSox-2/3. After several decades of research efforts to establish a crustacean cell line—with no published success—here, we present M. rosenbergii cultures composed of putative proliferating/stem-cell subpopulations or appearing like clones. These mix-population prawn embryonic primary cell cultures could serve as a basic platform for immortalization and contribute to the long-term goal of establishing sustainable cell-culture lines.

Despite extensive research spanning several decades, a commercial cell line for prawns remains elusive, and we have yet to determine the ideal conditions required for successful in vitro cell cultures of crustaceans. This study marks the first application of mass spectrometry to analyze lipid content changes in prawn culture media, allowing us to investigate how specific metabolites in media correlate with cell cycle and proliferation status. To investigate these relationships, short-term cell culture experiments were conducted using three commercial media: Dulbecco's modified eagle medium (DMEM) high glucose, Opti-Minimal essential media (Opti-MEM), and DMEM:F12. We monitored metabolic activity, performed cell-cycle analysis, and used mass spectrometry to analyze lipid metabolic profiles of culture media. This comprehensive approach enables us to examine the interplay between media lipidomic content and cellular behavior. Our results revealed that all three media sustained cellular metabolic activity. DMEM high glucose and Opti-MEM showed peaks in metabolic activity at day 4, while DMEM:F12 exhibited a peak at day 7. Opti-MEM supported the shortest doubling time, making it ideal for short-term culture. Interestingly, DMEM high glucose retained more attached cells on day 1 and had the highest count on day 4, with a noticeable trend towards the G2/M cell-cycle phase. Moreover, our lipidomic analysis of Opti-MEM revealed significantly higher concentrations of 26 annotated lipids compared to the other media. Notably, lipids like phosphatidylcholine and phosphatidylserine were consistently abundant in all three media, and palmitate levels correlated with proliferation phase. These findings have significant implications for establishing sustainable crustacean cell culture lines. Understanding the lipidomic compositions and their associations with cell-cycle dynamics and proliferation allows for precise conditioning of culture media to promote sustainable growth and delay cellular entry into quiescence phases. This knowledge contributes to the long-term goal of maintaining robust prawn cell cultures for various research and biotechnological applications.

Crustacean cell line immortalization has gained a great deal of attention in recent decades for both scientific and applied reasons. Our goal in this study was to advance the state of art towards establishing an immortalized cell line by improving the proliferation rates of primary cells isolated from embryos of the giant freshwater prawn Macrobrachium rosenbergii by using a lentivirus expressing the Ras oncogene. The choice of Ras derived from its involvement in various cellular pathways, such as cell growth, differentiation, and survival, and its use as a tool for in-vitro immortalization, e.g., a specific mutated Ras (RasV12) was used to generate an arthropod cell line. Complementarily, in-silico screening of M. rosenbergii transcriptomic libraries for Ras expression indicated that Ras is already expressed at very early stages of embryo development. In the current study, we transduced primary M. rosenbergii embryonic cells with a lentivirus expressing RasV12 by using the white spot syndrome virus (WSSV IE1) promoter. Expression and sequencing (as followed by sequencing cDNA, confocal microscopy and FACS analysis) of the mutated Ras in the transduced cells confirmed that the lentivirus was successfully integrated into the genome. The lenti-MrRas transduction rate was 23% in the total primary cell population and more than 80% in a sub-population of cells with high granularity. Proliferation of lenti-MrRas transfected cells was enhanced to almost 1200% of the seeding density by the end of our experiment (18 days), which was double that of the control. We were thus successful in enhancing the longevity of embryonic primary cell cultures by ectopic expression of the mutated Ras protein, but the improvement was not sufficient for immortalization.

In vertebrate reproduction, metabolism, growth and development, essential roles are played by glycoprotein hormones, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH), all of which are heterodimers consisting of two subunits, a structurally identical alpha subunit, and a variable beta subunit, which provides specificity. A 'new' glycoprotein hormone heterodimer identified in both vertebrates and invertebrates, including decapod crustaceans, was shown to be composed of the glycoprotein alpha 2 (GPA2) and glycoprotein beta 5 (GPB5) subunits. The putative receptor for GPA2/GPB5 in invertebrates is the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1). In this study in the giant freshwater prawn, Macrobrachium rosenbergii, we identified and characterized the GPA2 (MrGPA2), GPB5 (MrGPB5) and LGR1 (MrLGR1) encoding genes and revealed their spatial expression patterns in female animals. Loss-of-function RNA interference (RNAi) experiments in M. rosenbergii females demonstrated a negative correlation between MrGPA2/MrGPB5 silencing and MrLGR1 transcript levels, suggesting a possible ligand–receptor interaction. The relative transcript levels of M. rosenbergii vitellogenin (MrVg) in the hepatopancreas were significantly reduced following MrGPA2/MrGPB5 knockdown. MrLGR1 loss-of-function induced MrVg receptor (MrVgR) transcript levels in the ovary and resulted in significantly larger oocytes in the silenced group compared to the control group. Our results provide insight into the possible role of GPA2/GPB5-LGR1 in female reproduction, as shown by its effect on MrVg and MrVgR expression and on the oocyte development. Here, we suggest that the GPA2/GPB5 heterodimer act as a gonad inhibiting factor in the eyestalk-hepatopancreas-ovary endocrine axis in M. rosenbergii.

The CRISPR/Cas genome editing approach in non-model organisms poses challenges that remain to be resolved. Here, we demonstrated a generalized roadmap for a de novo genome annotation approach applied to the non-model organism Macrobrachium rosenbergii. We also addressed the typical genome editing challenges arising from genetic variations, such as a high frequency of single nucleotide polymorphisms, differences in sex chromosomes, and repetitive sequences that can lead to off-target events. For the genome editing of M. rosenbergii, our laboratory recently adapted the CRISPR/Cas genome editing approach to embryos and the embryonic primary cell culture. In this continuation study, an annotation pipeline was trained to predict the gene models by leveraging the available genomic, transcriptomic, and proteomic data, and enabling accurate gene prediction and guide design for knock-outs. A next-generation sequencing analysis demonstrated a high frequency of genetic variations in genes on both autosomal and sex chromosomes, which have been shown to affect the accuracy of editing analyses. To enable future applications based on the CRISPR/Cas tool in non-model organisms, we also verified the reliability of editing efficiency and tracked off-target frequencies. Despite the lack of comprehensive information on non-model organisms, this study provides an example of the feasibility of selecting and editing specific genes with a high degree of certainty. [ABSTRACT FROM AUTHOR]

In vertebrate reproduction, metabolism, growth and development, essential roles are played by glycoprotein hormones, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH), all of which are heterodimers consisting of two subunits, a structurally identical alpha subunit, and a variable beta subunit, which provides specificity. A 'new' glycoprotein hormone heterodimer identified in both vertebrates and invertebrates, including decapod crustaceans, was shown to be composed of the glycoprotein alpha 2 (GPA2) and glycoprotein beta 5 (GPB5) subunits. The putative receptor for GPA2/GPB5 in invertebrates is the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1). In this study in the giant freshwater prawn, Macrobrachium rosenbergii , we identified and characterized the GPA2 (MrGPA2) , GPB5 (MrGPB5) and LGR1 (MrLGR1) encoding genes and revealed their spatial expression patterns in female animals. Loss-of-function RNA interference (RNAi) experiments in M. rosenbergii females demonstrated a negative correlation between MrGPA2/MrGPB5 silencing and MrLGR1 transcript levels, suggesting a possible ligand–receptor interaction. The relative transcript levels of M. rosenbergii vitellogenin (MrVg) in the hepatopancreas were significantly reduced following MrGPA2/MrGPB5 knockdown. MrLGR1 loss-of-function induced MrVg receptor (MrVgR) transcript levels in the ovary and resulted in significantly larger oocytes in the silenced group compared to the control group. Our results provide insight into the possible role of GPA2/GPB5-LGR1 in female reproduction, as shown by its effect on MrVg and MrVgR expression and on the oocyte development. Here, we suggest that the GPA2/GPB5 heterodimer act as a gonad inhibiting factor in the eyestalk-hepatopancreas-ovary endocrine axis in M. rosenbergii. [ABSTRACT FROM AUTHOR]

Agricultural lands are integrated into and interact with natural areas. Such is the case of Emek HaMa'ayanot, northern Israel, comprising a springs-rich area characterized by multiple land-uses, including spring-water-based aquaculture, recreational springs, and nature reserves. Aquacultural farms suffer from pest snails that carry fish disease; in the study region, these species are invasive (Thiara scabra, Tarebia granifera, Pseudosuccinea columella) and outbreak endemic (Melanoides tuberculata). Previous snail control efforts have focused on individual fishponds without considering management on larger environmental scales in the waterways from the source springs to the fish farms. To broaden our understanding of the status of the pest snail problem in the study area prior to suggesting environmental managerial solutions, we quantified changes in the community composition of snail species along the springs-to-fishponds gradients in a spatially explicit system. We found a remarkable increase in pest snail abundances along these gradients, indicating that pest snails might be invading upstream towards the springs. There were always nearly 100% pest snails in the endpoint sites for water tracks that ended in fishponds. Moreover, pest snails dominated the site when it was used as a fishpond, even though the site was also a spring. In contrast, in a water track that does not end in a fish farm, the relative abundances of non-pest snail species was similar between the source spring and the downstream endpoint, in spite of an increase in pest snail abundance at a midpoint site. These results suggest that invasive pest snails are actively moving upstream and that the fishponds have a marked upstream effect on the ability of non-pest snails to resist pest species invasions. We suggest further investigation of possible strategies for biocontrol of the observed invasion of the snails into natural areas as a basis for environmental management efforts. Finally, the observations made during this study could have practical global implications for snail management in aquaculture and agriculture, and for the control of snails and snail vectors implicated in animal and human diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sharon Moscovitz, Hanoch Glassner, Eliahu D. Aflalo report financial support was provided by Israel Ministry of Agriculture and Rural Development. Amir Sagi reports financial support was provided by Israel Ministry of Agriculture and Rural Development. Sharon Moscovitz, Hanoch Glassner, Eliahu D. Aflalo report financial support was provided by Keren Kayemeth LeIsrael - Jewish National Fund. Amir Sagi reports financial support was provided by Keren Kayemeth LeIsrael - Jewish National Fund., (Copyright © 2023. Published by Elsevier Ltd.)