Baculovirus and plasmid vector-mediated transgenic experiments in the embryonic cell cultures developed from the freshwater crustacean Daphnia magna

Baculovirus and plasmid vector-mediated transgenic experiments in the embryonic cell cultures developed from the freshwater crustacean Daphnia magna | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Baculovirus and plasmid vector-mediated transgenic experiments in the embryonic cell cultures developed from the freshwater crustacean Daphnia magna Sreevidya CP, Soumya Balakrishnan, Jayesh Puthumana This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3841832/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cell culture represents an indispensable tool for investigating fundamental biological processes. Nevertheless, technical challenges such as low cell yield, sub-optimal cell differentiation, and inadequate attachment to the growth substrate have restricted the application of this tool in many studies. Here, we introduce an easy protocol for the preparation of primary cell cultures from Daphnia magna embryos, offering a versatile approach to address cell biological questions in conjunction with the robust in vivo model of D. magna . The development of transgenic cells is an emerging interdisciplinary field that can be used for the fundamental understanding of normal and pathological responses of cells and the improvement of tissue functionality. The application of this technology to primary cells is still in its infancy but promises to accelerate research. In this work, embryonic cell culture is developed from D. magna ; and is used to standardize viral (BacIe1- GFP ) and plasmid vector (pCS- EF1α1-DSRed2 )-mediated transgenic experiments. The standardized conditions methodology for developing embryonic cell culture, Cellfectin-mediated transfection and baculovirus-mediated transduction methods envisage strengthening the crustacean cell line research and bringing forth the Daphnia cell culture system as a 'model' in vitro system for crustaceans. Additionally, the simplicity and flexibility of the methodology described are expected to lead to widespread use in many biological research areas, including their wide application to ecotoxicological and epigenetic studies which are currently limited to in vivo studies. This is the first report on the optimization of cell culture medium for freshwater crustaceans and the use of baculovirus for transduction studies in D. magna embryonic cell culture. Daphnia magna Primary cell culture BrdU assay XTT assay Transformation Transduction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 INTRODUCTION Crustaceans, a diverse group within the phylum Arthropoda, comprise approximately 67,000 species (Zhang, 2011 ). Daphnia , among these, holds significant importance in research as a model organism with its fully sequenced genome (Colbourne et al., 2005 ). Functioning as filter feeders, Daphnia species play a crucial role as keystone species in freshwater habitats globally. Notably, Daphnia spp. demonstrates heightened sensitivity to ecological and habitat changes compared to other freshwater species (Schindler, 1987 ). Various indicators of this sensitivity include alterations in reproduction for stress assessment, the formation of ephippia (Hebert and Crease, 1983 ), changes in vertical migration within the water column (Dawidowicz and Loose, 1992 ; Jeswin et al., 2016 ), the impact of phenoplast (Tollrian, 1993 ), and behavioral shifts (Gerhardt et al., 2005 ). Consequently, Daphnia species were preferred subjects for toxicity studies and assessments of environmental changes. The availability of its fully sequenced genome makes Daphnia a suitable model for comprehending the genomic implications of ecological changes (Shaw et al., 2008 ). Daphnia spp. exhibits a reproductive strategy of asexual parthenogenesis during favorable conditions and switches to sexual reproduction during unfavorable conditions (Hebert and Ward, 1972 ). So the genetic purity of Daphnia spp. can be effectively maintained in laboratory settings by providing suitable environmental conditions. Numerous aspects of Daphnia spp. have undergone comprehensive study, including ecology, phylogeny, reproduction, and life cycle (Koivisto, 1995 ; Colbourne, 1996 ; Decaestecker et al., 2005 ; Harris et al., 2012 ). The combination of Daphnia 's short lifespan and the availability of various genomic tools for molecular studies renders it an ideal model for research purposes (Altshuler et al., 2011 ). The initiation of primary cell culture development from crustaceans dates back to 1960 (Claydon, 2009 ). However, these experiments have not always yielded success mainly because of poor cell survival under in vitro conditions (Claydon, 2009 ; Jayesh et al., 2013 ). Recently, crustaceans have acquired greater attention than any other invertebrates since many economically important species are under this sub-phylum (Cai and Zhang, 2014 ), So several attempts have been made to develop cell lines from crustaceans (Claydon, 2009 ; Jayesh 2013 ) including the standardization of the biological and physiological factors crucial for the in vitro survival of crustacean cells, encompassing parameters such as pH, osmolarity, nutrients, and vitamins (Goswami et al., 2010 ; George et al., 2011 ; Jiang et al., 2011 ; Hong et al., 2013 ; Jayesh et al., 2013 , 2015; Jeswin et al., 2016 ). Despite available literature on the development of primary cell culture, achieving the permanent immortalization of cells has proven elusive, with an exception being a recent publication on the immortalization of a shrimp lymphoid organ cell line ( PmLyO-Sf9 ) through hybridization with an insect (Sf9) cell line (Anoop et al., 2021 ; Sathyabhama et al., 2021). Once a cell culture has been established from Daphnia spp., it becomes a valuable research tool for various applications. This includes the isolation and cultivation of pathogens, drug screening, exploration of metabolic pathways, production of antibodies, as well as studying the regulation of gene expression and evolutionary early mechanisms of cell-mediated cytotoxicity, apoptosis, and cell ageing. The development of immortalized cell lines, a crucial aspect of sustained cellular studies, often requires proper transgene studies. Transgenic research plays a pivotal role in bridging the gap between basic and applied molecular biology. Advancements in transgenic technology have provided a potent tool for targeted genetic manipulations. The ability to introduce genetic alterations at the cellular level allows researchers to bypass normal cellular senescence, enabling the generation of continuous, infinite cell lines (Brenin et al., 1997 ). Transgenesis is a method employed to introduce foreign nucleic acids into eukaryotic cells, thereby altering the genetic composition of the host cells (Kim & Eberwine, 2010 ). Vector constructs carrying the specific nucleic acids to be transfected can be broadly categorized as either viral or plasmid vectors. Viruses and plasmids, facilitated by an appropriate eukaryotic promoter, allow for the production of a foreign transgene (Colosimo et al., 2000 ). When selecting a gene delivery method, three crucial factors should be considered: 1) cost-effectiveness and ease of use, 2) absence of toxicity/immunogenicity, and 3) efficiency of transfection and gene expression. While existing delivery systems do not fully meet all three criteria, viral vectors, though efficient in vivo, face limitations due to immunogenic responses and cytotoxicity. Non-viral vector delivery systems, while less efficient, are minimally immunogenic, cost-effective, and easier to control for quality (Abul hassan et al., 2000; Zhang et al., 2010 ). In addressing the challenges of gene transfer, a crucial need for a clearer and more consistent approach is acknowledged. Consequently, here an attempt was made to achieve maximum transgenic success with minimal side effects through the thorough optimization of transduction using the baculovirus system and transfection using the Cellfectin reagent. The intent of the attempt was not to compare the efficiency of both transgenic techniques but to standardize a protocol suitable for Daphnia primary culture. In the context of D. magna cell culture, designing and standardizing transfection/transduction experiments can be challenging, especially when dealing with a novel cell culture and experiencing low cell numbers and attachment efficiency. In this study, efforts were made to address these challenges by standardizing the transgenic experiments, along with optimizing the cell culture medium for cell maintenance. Remarkably, the cells were successfully maintained for over two months in vitro, with their metabolic activity confirmed through BrdU and XTT assays. These findings highlight the potential of the standardized approach for sustained and functional D. magna primary cell cultures, offering a valuable platform for various research applications. 2 MATERIALS AND METHODS The experimental animals, D. magna , were obtained as embryo capsules from a local vendor. The embryos were hatched in Adam medium (Aachener Daphnien medium) supplemented with vitamins. Species identity was confirmed through morphometric analysis, followed by molecular characterization of the mitochondrial genome ( Suppl. Figure 1 ). For the development of primary cell culture, entire animals and embryos were used to identify suitable tissue sources. The protocol, based on Robinson et al. (2006) with slight modifications, involved collecting 30–60 animals containing embryos in various developmental stages in a 1.5 mL microcentrifuge tube (MCT). The animals underwent surface sterilization through a series of washing steps, including 0.05% sodium hypochlorite (30s), PBS (50s), 70% ethanol (30s), PBS (50s), serum-free cell culture medium (1 min), and final washing with PBS (10s). For embryonic primary cell culture development, embryos were aseptically removed from the brood chamber of D. magna with sterile syringe needles, ensuring the integrity of the animal's gut to prevent contamination. After transferring the embryos to another MCT, the same washing steps were followed. The embryos were then transferred to a fresh cell culture medium in an MCT, gently macerated with a sterile homogenizer, and the suspension was centrifuged at 1000Xg for 5 min. The pellet containing cells were resuspended in fresh cell culture medium (Modified Schneider's insect medium, pH 6.8), seeded onto a 96-well plate, and maintained at 26˚C. Fresh cell culture medium was supplied upon any change in its color. To develop a primary cell culture from adult D. magna , 30–60 animals without embryos were placed in a fresh rearing medium and starved for 24 h. Following sterilization through the same series of washing steps, the animals were homogenized with minimal pressure. The suspension was centrifuged at 1000Xg for 5 min, and the pellet was resuspended in fresh cell culture medium containing antibiotics (penicillin 100 IU/mL, amphotericin B 50 µg/mL, and streptomycin 100 µg/mL). The suspension was then seeded on a 96-well plate and maintained at 26˚C. 2.1 Screening of suitable media for the primary cell culture developed from D. magna Seven commercial media (all purchased from Sigma-Aldrich, US) − 1X Leibovitz's L-15 Medium (1X L-15), Schneider's insect medium, TNM-FH insect medium (TNMFH), Grace's insect medium (Grace's), Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute (RPMI) medium were compared. These media were supplemented with 20% fetal bovine serum (Himedia, India), antibiotics (100 µg/mL streptomycin and 100 IU/mL penicillin), pH adjusted to 6.8, and an osmolarity of 380 ± 10 mOsm/kg. Cell viability and proliferation were assessed by observing the cells using an inverted phase-contrast microscope (Leica, Switzerland). Further modifications experimented with the selected Schneider's medium. In all experiments, the cells were seeded in 96-well plates with different concentrations of testing supplements and the control. The effects of the supplements were determined based on cell viability, proliferation, the percentage of cell attachment and monolayer formation which was observed daily for one month using an inverted phase-contrast microscope (Leica, Switzerland). The experiments were conducted in four distinct phases. In the first phase, the effects of FBS were tested (5–20%). As, heat-inactivated FBS is recommended by many researchers (Frerichs 1996 ; Mulford and Austin 1998 ; Fan and Wang 2002 ; Wenli and Shields 2007 ) an experiment was conducted to compare the effect of heat-inactivated FBS and non-heat-inactivated FBS (Himedia, India) on the cells when applied at different concentrations ranging from 5–20%. In the second phase, organic supplements such as MEM Vitamins (Gibco, US), Amino acids, Glucose, Trehalose, and Sucrose were tested (0.1, 0.5, and 1% concentrations). Similarly, the effects of cholesterol and selenium (0.01, 0.1, and 1ppb) were tested in the third and fourth phases, respectively. 2.2 Cellular and molecular responses of primary embryonic cell culture The cell viability was tested by acridine orange and ethidium bromide staining. Acridine orange is a vital dye that stains live and dead cells, while ethidium bromide stains only cells that lose cellular integrity. When the cells are exposed to both these stains simultaneously, the nuclei of 'live' cells will stain green, whereas the nuclei of 'dead' cells will stain orange-red color. Ten µL of modified Schneider's medium containing cells were mixed with 10 µL of 1X staining solution containing acridine orange and ethidium bromide (100 µg/mL of acridine orange and 100 µg /mL of ethidium bromide prepared in PBS and mixed gently), incubated for 2 min under room temperature (RT) in the dark. Then 500 µL of 1X PBS was added and centrifuged at 1000 g for 10min. The supernatant was discarded, and the pellet was resuspended in 20 µL of 1X PBS. 10 µL of the solution was loaded on a microscopic slide, and a coverslip was kept carefully without air bubbles (Kasibhatla 2006). The slide was then observed under a fluorescent microscope (Leica, Switzerland). DNA synthesis was analyzed using the BrdU (5-Bromo-2'-deoxyuridine) incorporation assay in primary embryonic cells grown in vitro for 6–8 h in 96-well plates with a 200 µL growth medium (Jose et al., 2011 ; Puthumana et al., 2015 ). To each well, 20 µL of a 10 mM BrdU solution was added, and one well without the addition of BrdU served as a control. After 24 h of incubation, the medium was removed, and the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and washed again with PBS. Subsequently, 2 M HCl was added to each well and incubated for 20 min. The acid was then neutralized with 0.1 M sodium borate (pH 8.5) for 2 min, followed by another wash with PBS. The cells were permeabilized with PBS containing 0.2% Triton X-100 and 3% BSA for 5 min. After blocking with 3% BSA in PBS for 1 h, a mouse monoclonal anti-BrdU antibody (Sigma, USA) at a 1:1000 dilution in 3% BSA was added and incubated for 1 h. The cells were washed thrice with PBS for 5 min each and then incubated for an additional hour with goat anti-mouse FITC conjugate at a 1:40 dilution (Sigma, USA). After washing with PBS, the wells were stained with DAPI (0.2 µg/mL) and observed under an inverted fluorescence microscope (Leica, Switzerland). DAPI and FITC signals were viewed under filters with 360–370 nm and 470–490 nm excitation wavelengths. The images were processed and merged using the "Leica Application Suite" (Leica Microsystems, Switzerland). The XTT assay assessed the cells' metabolic activity at 24 h and 5 days after seeding, i.e., 24 h, 48 h, 72 h, 96 h, and days 0, 5, 10, and 15. The change in cell density during this period was determined through XTT. The basic principle of this assay relies on the cleavage of a yellow tetrazolium salt into an orange-colored formazan by the mitochondrial enzyme, which functions in only metabolically active cells (Roche Applied Science, 2003). For the assay, 5 mL of XTT (sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitrobenzene sulfonic acid hydrate) and 100 µL of PMS (N-methyl dibenzopyrazine methyl sulfate) were mixed thoroughly. A 96-well plate containing 100 µL of culture medium and cells was treated with 50 µL of XTT/PMS mix and incubated at 26°C for 5 h. After incubation at 26°C, the absorbance was measured at 490 nm in a microplate reader (Tecan Infinite Tm, Austria) with a reference at 655 nm (Van Der Merwe et al., 2010 ; Puthumana et al., 2015 ). The subculture of attached cells was assessed using both enzymatic (trypsin-based) and mechanical (gentle pipetting) methods. The cells were gently washed with PBS and treated with a 0.05% trypsin-EDTA solution. After a brief incubation (1 min) at RT, the detachment of cells was observed under an inverted phase-contrast microscope. Upon detachment, the 0.05% trypsin-EDTA solution was discarded, and modified Schneider's medium with 10% FBS was added to inhibit trypsin activity. Subsequently, the cells were subcultured and maintained at 26°C. Periodic observations were made for any changes in media color (Jayesh et al., 2013 ). In the second method, the cell culture medium was discarded, and the cells were washed with PBS. Dissociation was achieved through gentle trituration using a medium containing FBS. The detached cells were then resuspended in a fresh medium without antibiotics and kept at 26°C. 2.3 Transgenic experiments in primary embryonic cell culture 2.3.1 Standardisation of transfection The concentration of Cellfectin ( Cellfectin™ II-Invitrogen) and plasmid along with the incubation time required for successful transfection were standardized. Therefore, different concentrations of Cellfectin reagent (3 µL- 10 µL) and plasmid (1 µg- 5 µg) were used for the experiments. The cells were seeded in 24-well plates, and the medium was changed after 24 h of incubation. Cellfectin reagent was diluted in 100 µL serum-free medium, mixed, and kept at RT for 30 min. Kato developed a Daphnia -specific vector, pCS- EF1α1-DSRed2 , which has been successfully introduced into D. magna embryonic eggs through microinjection (Kato et al., 2012). The same plasmid was kindly obtained from Dr. Hajime Watanabe, Osaka University, Japan. This plasmid contains EF1α1 as promoter, and expression was marked by the DSRed2 gene fused with the D. magna histone H2B gene. Different concentrations of plasmid (1–5 µg) were diluted in 100 µL serum-free medium, and a Cellfectin reagent was mixed (1:1) and incubated for 15min. Then, the mixture was added dropwise to the cell culture and incubated at 26 o C for 3 to 8 h (different incubation time). Then the medium was replaced with FBS containing modified medium for 72 h and observed under an inverted phase-contrast microscope (Leica, Switzerland) under the excitation wavelength of 470-490nm (Jayesh et al., 2013 ). 2.3.2 Standardisation of transduction Recombinant baculovirus-mediated transduction using shrimp-specific promoter (WSSV-Ie1 and IHHNV-P2) having green fluorescent protein as the marker (BacIe1-GFP and BacP2-GFP) was developed for transgene expression in P. monodon lymphoid cell by Puthumana et. al (Puthumana et al., 2016 ). Briefly, Ie1 promoter from WSSV and P2 promoter from IHHNV were isolated, amplified, and ligated into the pFASTBac 1™ transfer vector containing PH promoter (polyhedrin gene). These two vectors were used to standardise transduction in Daphnia primary cell culture. For that, Daphnia cells were seeded in 96-well plates and incubated in the modified Schneider’s medium for 24 h at 26ºC. Then the medium was replaced, and 100 \({\mu }\text{L}\) of supernatant containing recombinant baculovirus was added to100 \(\mu\) L medium and observed every 6 h for 24 h and subsequently observed every 24 h for a week under a UV microscope for the GFP signals. Daphnia cells without baculovirus were used as a negative control, and viruses with SF9 cells were used as the positive control. 2.4. Statistical analysis The results are the average of three sample replicates with a standard deviation. Onaway analysis of variance (ANOVA) was performed using SPSS ® software (version 21; SPSS Inc., Chicago, IL, USA) to confirm the data validity, and significant differences in all samples were tested by Tukey's post hoc test (Tukey's, p < 0.05). 3 RESULTS Development of primary cell culture from D. magna The embryos proved to be a superior source for cell culture from D. magna compared to adult animals, as it was possible to repeatedly develop axenic cell culture from the former. The cells maintained a healthy condition for over two months and exhibited both single and aggregate forms. Clustered cells were more abundant than individual cells and demonstrated better cell attachment (Fig. 1 ). Cells were observed to be healthy when in close contact with each other. Seeding cells in 96-well plates proved to be more efficient than culturing in tissue culture flasks. Fresh medium was supplied whenever a color change was observed in the medium. The cells mostly remained in suspension due to the slow rate of attachment, so medium changes were performed by exchanging half of the medium from the well with fresh medium. After two weeks of seeding, the first subculture was carried out. The comparison of cell subculturing methods involving pipetting and trypsinization revealed a substantial difference in cellular viability, with the first protocol (gentle pipetting) proving significantly less detrimental compared to the second protocol (trypsin treatment with pipetting). This observation leads to the hypothesis that Daphnia cells exhibit characteristics of "social cells," necessitating cooperative interactions and intimate physical contact. Consequently, maintaining dissociated cells in cellular clumps, rather than as single-cell suspensions during passaging, may be crucial. Upon initial passaging, an increased presence of cellular clumps is evident with gentle pipetting rather than trypsin dissociation. This disparity may contribute to the observed healthier state of pipetted cells compared to trypsinized cells. However, it is important to note that the cells are not reattached, and no sign of division after the passaging. After that, the cells remained in suspension, and the number of viable cells decreased gradually, and a complete monolayer could never be obtained. 3.1 Screening of suitable media for the primary cell culture of embryos from D. magna All the tested media supported cell survival for at least three hours. Schneider's insect medium was selected for further modification as it showed a suitable cell culture medium in terms of enhanced cell viability and attachment compared to other media. Subsequent tests to determine the optimal fetal bovine serum (FBS) concentration indicated that 10% was sufficient to support cell growth, and there were no additional benefits observed with 15% FBS (Table 1 ). Table 1 Screening and optimization media for the development of Daphnia magna primary cell cultures. I) Comparison of seven different commercially available cell culture media (with 20% FBS) to evaluate the most suitable medium that supports maximum cell viability and proliferation. II) Comparative effect of heat-inactivated and non-inactivated FBS. III) Effects of organic supplements such as glucose, trehalose, and sucrose in the selected medium. (IV) Effects of vitamins, amino acids, and cholesterol. V) Effects of selenium. The effect was scored as; – nil, + very low survival rate or few hours survival, ++ low rate or few days survival, +++ medium rate or few weeks survival, ++++ high rate or one-month survival, +++++ very high rate or more than one-month survival. Exp. No. Medium modifications Endpoints Cell attachment Survival Cell division Monolayer Formation I Schneider’s insect medium + ++ - - TNMFH Medium - + - - Grace insect medium - + - - L-15 + + - - RPMI - + - - MEM - + - - DMEM - + - - II Schneider’s insect medium + Heat inactivated FBS (5%) + ++ + - Schneider’s insect medium + Heat inactivated FBS (10%) + +++ + - Schneider’s insect medium + Heat inactivated FBS (15%) + +++ + - Schneider’s insect medium + Heat inactivated FBS (20%) + +++ + - Schneider’s insect medium + Normal FBS (5%) + ++ + - Schneider’s insect medium + Normal FBS (10%) + +++ + - Schneider’s insect medium + Normal FBS (15%) + +++ + - Schneider’s insect medium + Normal FBS (20%) + +++ + - III Schneider’s insect medium + 10%FBS + Glucose(C) Schneider’s insect medium + 10%FBS + Glucose (0.1%) + +++ ++ + Schneider’s insect medium + 10%FBS + Glucose (0.5%) + +++ ++ + Schneider’s insect medium + 10%FBS + Glucose (1%) + +++ ++ + Schneider’s insect medium + 10%FBS + Trehalose (C) Schneider’s insect medium + 10%FBS + Trehalose (0.1%) + +++ + - Schneider’s insect medium + 10%FBS + Trehalose (0.5%) + +++ + - Schneider’s insect medium + 10%FBS + Trehalose (1%) + +++ + - Schneider’s insect medium + 10%FBS + Sucrose (C) Schneider’s insect medium + 10%FBS + Sucrose (0.1%) + +++ + - Schneider’s insect medium + 10%FBS + Sucrose (0.5%) + +++ + - Schneider’s insect medium + 10%FBS + Sucrose (1%) + +++ + - VI Schneider’s insect medium + 10%FBS + 0.1%Glucose + Vitamins (C) Schneider’s insect medium + 10%FBS + 0.1%Glucose + Vitamins (0.1%) + ++++ ++ + Schneider’s insect medium + 10%FBS + 0.1%Glucose + Vitamins (0.5%) + ++++ ++ + Schneider’s insect medium + 10%FBS + 0.1%Glucose + Vitamins (1%) + ++++ ++ + V Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Aminoacids(C) Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Aminoacids (0.1%) + ++++ ++ + Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Aminoacids (0.5%) + ++++ ++ + Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Aminoacids (1%) + ++++ ++ + Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Cholesterol (C) Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Cholesterol (0.1%) + ++++ ++ + Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Cholesterol (0.5%) + ++++ ++ + Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Cholesterol (1%) + ++++ ++ + VI Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Selenium(0.01ppb) + ++++ ++ + Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Selenium(0.1ppb) ++ ++++ ++ ++ Schneider’s insect medium + 10%FBS + 0.1%Glucose + 0.1%Vitamins + Selenium(1ppb) + ++ ++ - Interestingly, when FBS was heat-inactivated, the cells exhibited similar results, suggesting that heat inactivation might not be necessary for Daphnia cells. Additionally, the introduction of glucose was found to enhance cell division during the initial stages (Table 1 ), but subcultured cells displayed no significant changes in response to the continued presence of glucose. Furthermore, supplementing the medium with glucose at regular intervals did not yield any further effects. The addition of amino acids and cholesterol proved to be ineffective, while vitamins demonstrated efficacy in enhancing cell attachment and survival at a concentration of 0.1% (Table 1 ). Both essential and non-essential amino acids did not exhibit observable effects on the cells. Incorporating selenium into Schneider's insect medium notably improved cell attachment. Cells attached within 24 h of seeding, a significant improvement compared to the control, where attachment occurred over three days. Moreover, the number of attached cells was higher at a concentration of 0.1 ng/mL (Table 1 ). This result suggests that cells require selenium for cell division at a critical concentration of 0.1 ng/mL, beyond which cytotoxicity and cell death occur. Based on these findings, the selected medium for cell culture was Schneider's insect medium (Table 1 ), modified by the addition of 0.1% glucose, 0.1% MEM vitamin mix, 0.1 ng/mL selenium, and 10% FBS. This modification resulted in enhanced cell growth, moderate proliferation, and good survival. The primary cell culture of D. magna exhibited cells with enlarged nuclei, producing FITC fluorescence signals within 24 h of incubation ( Fig. 3 ). These findings indicate that the in vitro microenvironment supports active DNA replication in Daphnia cells. After 24 h, only 2-4% of cells showed a positive response, predominantly during the washing steps of the assay; it is common for some attached cells to be washed off. Due to the lower number of attached cells within the initial 24 h, most were washed away, resulting in minimal positive signals. Utilizing a dual staining method facilitated the discrimination between live and dead cells. Live cells emitted green fluorescence under UV light, while dead cells exhibited an orange-red coloration (Fig. 2 ). Approximately 90% of cells displayed green fluorescence, signifying a predominance of viable cells after 24 h of growth. It is noteworthy that all experiments were conducted using 24 h old cells, and the assay confirmed a sufficient number of viable cells at this time point. To further assess cell viability and division in the Daphnia culture, an XTT assay was conducted. The results revealed a gradual increase in cell number (cell density) when cultured using the modified Schneider's insect medium supplemented with glucose, vitamins, and selenium. Both analyses over 24 h and five-day intervals demonstrated in vitro cell division (Fig. 4 ). A 17.74% increase in growth was observed in a 48 h old plate compared to cells at 24 h. Additionally, 24.47% and 31.80% growth were noted in the subsequent 72- and 96-h plates compared to 24 h cells. After five days, a 22.06% increment was observed. Moreover, a 49.9% increase occurred after ten days, with 54.3% and 65.5% growth observed after 15 and 20 days, respectively, compared to the first day of seeding. 3.2 Transgenic expression 3.2.1 Transfection Figure 5 . Transfection experiment in D. magna primary embryonic cell cultures using Daphnia specific expression vector (pCS-EF1α1-DSRed2). Red fluorescent protein expression in D. magna primary embryonic cells shows that the DSRed2 proteins are expressed under the control of Daphnia specific promoter (EF1α1) in the expression vector pCS-EF 1α1-DSRed2. Scale bar: 50 µm In the initial screening protocol, a dose-dependent increase in fluorescence activity, highest in terms of the number of expressed cells, was observed with 3 µg of DNA during a four-hour exposure. Extending the transfection time by an additional hour revealed a dose-dependent increase in expression, which decreased upon further incubation. While an increase in plasmid concentration had a slightly positive effect on the number of expressed cells, concentrations of Cellfectin exceeding 7 µL showed no additional benefits. Notably, cells did not exhibit signs of stress even with an increased Cellfectin concentration of up to 10 µL. Fluorescence in cells was started within 3 h of incubation but initially, a few cells showed positive results. The number of expressed cells gradually increased, reaching a maximum after 6 h of incubation. In summary, the experiments concluded that transfection of cells using 7 µL of Cellfectin reagent and 3 µg of plasmid for 6 h incubation resulted in robust fluorescence (Fig. 5 ). This outcome indicates the successful transfection and expression of a Daphnia -specific expression vector in the developed primary cell culture. 3.2.2 Baculovirus transduction For this study, Baculovirus modified with Ie1 and P2 was employed. Notably, only PH-Ie1 demonstrated successful transfection into the Daphnia primary culture. This outcome successfully demonstrates the efficiency of the hybrid promoter baculovirus for transduction in Daphnia primary culture. GFP expression was detected on the third day in sf9 cells and the fourth day in Daphnia cells . Approximately 20% of cells expressed GFP, with a slight increase up to 30% after one week (Fig. 6 ). Importantly, no signs of expression were observed in the negative control, confirming that the recombinant virus can effectively infect Daphnia cells using a modified promoter system. 4 DISCUSSION Daphnia spp. serves as a highly relevant model organism extensively studied in various fields such as ecology, predator-induced polyphenisms, evolution, ecotoxicology, and genomics (Hebert and Ward 1972 ; Koivisto 1995 ; Decaestecker et al. 2005 ). To further expand research on Daphnia spp. at the cellular and molecular levels, the establishment of a cell line from Daphnia species is imperative. Notably, there has been only one previous report on the development of primary cell culture from D. magna , which was utilized for propagating viral particles (VSV) in Schneider's insect medium supplemented with 10% heat-inactivated FBS (Robinson et al., 2006). The cells in that study could be maintained for up to one week. In our current investigation, we aimed to enhance the Daphnia spp. cell culture system, focusing on improving the culture medium and conducting expression studies. Remarkably, our efforts led to the establishment of a cell culture system that could be maintained for over two months in vitro with medium modifications. The ongoing debate regarding the necessity of heat inactivation of FBS remains a question, but our findings suggest that heat inactivation is unnecessary and has neutral effects on Daphnia spp. cell culture. These advancements contribute to the potential for further exploration of cellular and molecular aspects in Daphnia research. The choice of tissue for cell culture is typically influenced by the nature of the studies being conducted. While results may vary depending on the body part and medium used for analysis, a general trend suggests that using younger animals as a tissue source tends to yield more successful cell culture initiation. In our studies, we utilized both the entire Daphnia body and eggs for cell culture. The cell yield was higher when the whole body was employed, accompanied by an increased rate of attachment and predominantly healthy cells. However, a significant challenge encountered was regular contamination. The filter-feeding nature of Daphnia , with associated microbes from the gut region, posed difficulties in overcoming such contaminations. Studies, such as one involving Artemia sinica embryos, have demonstrated an extended cell lifespan (200 days) compared to studies using other tissues (Jiang et al. 2011 ). This suggests that embryonic cells might hold promise for cell culture, particularly in terms of self-renewal capacity. Ovary cells are often preferred due to their totipotency, self-renewal capacity and the potential for continuously dividing cells. In primary cell culture, the isolated tissue from the animal is typically not sterile, necessitating surface sterilization before initiating cell culture. In our studies, the entire body of the Daphnia was sterilized before commencing tissue culture. The classical antibiotic combination of penicillin-streptomycin (Kasornchandra et al. 1999 ) was employed for this purpose along with ethanol and chlorine (Fraser and Hall, 1999 ) wash. Determining the optimal concentration of these agents to eliminate microbial contamination without causing harm to the desired cells is crucial and often requires empirical testing. Using a high concentration of antibiotics has been observed to adversely affect the health of Daphnia cells, leading to senescence. In contrast, eggs are generally free from microbes and can be ensured to be contamination-free through additional washing. The careful removal of eggs from the brood was the only step that required extra attention. The concentration of FBS (Fetal Bovine Serum) is a critical factor in cell culture, and concentrations exceeding 15% have been reported to inhibit cell attachment in various studies. For example, in the lymphoid organ of P. monodon , it was found that FBS concentrations greater than 15% hinder cell attachment (Hsu et al., 1995 ; Jayesh et al., 2013 ) clumping of cells in P. semisulcatus (Rosenthal and Diamant, 1990), Liocarcinus depurator and Carcinus maenas (Walton and Smith, 1999 ). Additionally, shorter cell survival was noted in Nephrops hematopoietic cultures (Mulford and Austin, 1998 ). In alignment with these findings, our study similarly indicates that increasing the FBS concentration to 15% has no further effects compared to 10%, suggesting that 10% FBS is sufficient for promoting cell growth. This underscores the importance of optimizing FBS concentrations in cell culture to achieve the desired outcomes. Several studies support the advantageous use of glucose supplements in cell culture medium, demonstrating a slight increase in cell attachment (Hsu et al., 1995 ; Goswami et al., 2010 ) and enhanced cell migration (Hsu et al., 1995 ; Mulford and Austin, 1998 ; Itami et al., 1999 ). In the context of our study, the division of Daphnia primary culture initially exhibited increased cell division when 0.1% glucose was added. The role of vitamins in the medium is critical, as invertebrates, including Daphnia , cannot synthesize carotenes, which are essential for their growth (Claydon and Owens, 2009). Schneider's insect medium also lacks biotin and riboflavin, considered crucial vitamins (Luedeman and Lightner, 1992 ). Additionally, crustaceans lack the ability for de novo synthesis of sterols and require a dietary source of cholesterol for growth, development, and survival (Fungwe et al., 1994 ). Surprisingly, our results indicate that the addition of cholesterol had no significant impact on cell culture, while survival was increased with vitamin supplementation. Selenium, essential for Daphnia reproduction and development (Keating and Dagbusan, 1984 ), demonstrated an improvement in cell attachment in our study at a concentration of 0.1 ppb, although gradual cell death was observed above this concentration. Initially, the attachment rate of Daphnia cells in primary cell culture was prolonged, but after the addition of selenium, cells exhibited attachment within 12–14 h. These findings contribute to understanding the specific requirements and responses of Daphnia cells in culture conditions. The efficiency of gene transcription significantly influences gene expression levels, and the interaction between the RNA polymerase complex and promoter sequences is crucial for initiating this process. The resulting RNA transcript separates from the gene at the transcription signal, ready for eventual translation (Khan, 2013 ). Successful gene expression in cells requires a suitable cell line and appropriate vectors. However, the lack of permanent cell lines and transgenic methods in crustaceans poses a challenge for such work. Transfection studies are commonly conducted in primary cultures with oncogenes to induce cell immortality (Ratner et al., 1985 ; Claydon and Owens, 2009; Puthumana et al., 2015 ). Various transgenic methods, including lipofection-mediated (Claydon and Owens, 2009; Xu et al., 2018 ), electroporation-mediated (Li et al., 2011; Shi et al., 2018 ), and microinjection-mediated (Preston et al., 2000 ; Hiruta et al., 2013 ; Xu et al., 2020 ), are steadily advancing. Nevertheless, transfecting suspension cell lines, a common challenge, is often hindered by decreased attachment of the transfection complex to the cell surface, leading to reduced uptake of the target DNA (Landauer et al., 2002 ; Montier et al., 2004 ; Praveen et al., 2011 ). In our study, an effective transfection of Daphnia cells was achieved using 7 µL of Cellfectin reagent and 3 µg plasmid DNA, incubated for 6 h before the medium change. This approach showed DsRed2 expression after 72 h of incubation, with nearly 60% of cells expressing DsRed2 . Our methodology provides a reliable, straightforward, and economically directed manipulation of suspension cells using Cellfectin. This advancement may contribute to the progress of genetic studies in Daphnia and other suspension cell systems. Baculovirus expression vector systems (BEVS) have been extensively employed since 1983 to produce recombinant proteins in insect cells, with the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) serving as the foundation for most BEVS (Smith et al., 1983 ). Typically, strong insect-virus gene promoters such as polyhedrin or p10 are used to enhance the expression of recombinant proteins (Vaughn et al., 1977 ). Ensuring the synthesis of proteins in optimal quantity and quality is crucial. Various modifications to baculovirus vectors have been explored to enhance the quantity and quality of recombinant proteins produced by BEVS, as reviewed by Hitchman et al. ( 2011 ). In this context, we introduced a modified BEVS having the WSSV promoter gene Ie1 (Jayesh et al., 2015) to effectively transduce Daphnia cells. Almost 30% of cells were successfully transfected after one week of incubation indicating the efficiency of baculovirus in Daphnia cells. In conclusion, this study successfully modified and validated Schneider's insect-based cell culture medium for the development of a primary cell culture from D. magna , utilizing various tissues and embryos. The improvements observed in terms of extended longevity, enhanced DNA synthesis, metabolic activity, and increased transfection/transduction efficiency of the primary cell culture highlight the potential of Daphnia cell culture as a valuable 'model' in vitro system for crustaceans. The findings of this research are expected to contribute to the advancement of crustacean cell line studies. Furthermore, the methodologies and techniques outlined in this paper provide valuable insights that can assist researchers in developing and establishing cell lines from the crustacean model, particularly D. magna , for diverse applications. This work contributes to the broader field of cell culture research and underscores the potential of Daphnia as an effective model organism for further studies. Declarations Conflict of interests The authors declare that there are no conflicts of interest. Contributions SCP carried out the formal analysis and investigation. SCP and JP conducted the writing original draft and SB provided experimental support and made critical revisions. JP conceptualized, designed the methodology, and supervised SCP for all the experiments. Ethical approval All investigations were conducted as per guidelines provided by the Institutional Biosafety Committee (IBSC) and Ethical Committee of NCAAH, CUSAT in Kerala, India. Funding This study was supported by the Government of India: the Department of Biotechnology under the Innovative Young Biotechnologist Award (DBT-IYBA; BT/09/IYBA/2015/11). Author Contribution SCP carried out the formal analysis and investigation. SCP and JP conducted the writing original draft and SB provided experimental support and made critical revisions. JP conceptualized, designed the methodology, and supervised SCP for all the experiments. ACKNOWLEDGEMENTS The authors thank the Department of Biotechnology (DBT), Govt. of India, for the Innovative Young Biotechnologist Award (DBT-IYBA; BT/09/IYBA/2015/11) to Dr. Jayesh Puthumana and the funding for this research work. The first author thanks DBT for the fellowship. Data availability All data generated or analyzed during this study are included in this article. References Abul-Hassan K, Walmsley R, Boulton M (2000) Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells. Curr Eye Res 20(5):361–366 Altshuler I, Demiri B, Xu S et al (2011) An integrated multi-disciplinary approach for studying multiple stressors in freshwater ecosystems: Daphnia as a model organism. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3841832","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267385003,"identity":"c8feac5f-debf-4cd2-bee8-dba71be4a695","order_by":0,"name":"Sreevidya CP","email":"","orcid":"","institution":"Cochin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Sreevidya","middleName":"","lastName":"CP","suffix":""},{"id":267385004,"identity":"61508258-95a1-401c-b696-cf6b51611227","order_by":1,"name":"Soumya Balakrishnan","email":"","orcid":"","institution":"Cochin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Soumya","middleName":"","lastName":"Balakrishnan","suffix":""},{"id":267385005,"identity":"e94aa74a-96db-4a02-a0bb-3457ed9f93c7","order_by":2,"name":"Jayesh Puthumana","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACxgYwYYMQYSNSSxqcTVgLVN9hhBaCgLn9jOHDnzvOyxucP/z8AUONHQOfNAGdjD05xsa8Z24bbriRZtjAcCyZgU3mACE35ZhJM7bdTjC4wQDUwnaAgU0igYCW/jfmP3+2nUswOH/8YwPDP2K0zMgxY+BtO5BgcCDHsIGxjSgtz4qleduSDWfeyCmckdiXzENQi2F/8saPP9vs5PnOH9/w4cM3Ozn5GYS0NHAYIHhAxTz41QOBPAP7A4KKRsEoGAWjYIQDAKUMQ/KRSf+lAAAAAElFTkSuQmCC","orcid":"","institution":"Cochin University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jayesh","middleName":"","lastName":"Puthumana","suffix":""}],"badges":[],"createdAt":"2024-01-07 07:44:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3841832/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3841832/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49734639,"identity":"2dcb5dab-7b68-4b97-9492-b213b67fc3e5","added_by":"auto","created_at":"2024-01-17 07:01:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":322623,"visible":true,"origin":"","legend":"\u003cp\u003ePrimary embryonic cell culture developed from D. magna in optimized media. Proliferating round cells can be seen. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-3841832/v1/133ec7094d09bb3c90b13f52.png"},{"id":49734637,"identity":"b1d5c9b4-80b3-4a7c-bc61-b54a77c6b89f","added_by":"auto","created_at":"2024-01-17 07:01:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170521,"visible":true,"origin":"","legend":"\u003cp\u003eViability of D. magna primary embryonic cell cultures. Acridine orange and ethidium bromide-stained embryonic cells showing live (green) and dead cells (yellow/orange/red). Green: healthy cells; yellow/orange/red: Cell is early to late apoptosis or dead. Scale bar 20 µm.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-3841832/v1/3eea737451ee9e65abfee518.png"},{"id":49734638,"identity":"13246136-b56d-4549-977d-e6af8d0f8f34","added_by":"auto","created_at":"2024-01-17 07:01:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108311,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescent detection of BrdU incorporation in the DNA of D. magna embryonic cells (in vitro). BrdU incorporation confirmed that cells are at the S-Phase of the cell cycle. A: DAPI stained nucleus; B: FITC stained BrdU incorporated DNA; C: merged image (40 x magnifications). Scale bar: 20 µm.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-3841832/v1/676bb9769d7537acb2d013b3.png"},{"id":49734641,"identity":"80f73b9b-1026-4fd5-9f72-a31d465fd1ea","added_by":"auto","created_at":"2024-01-17 07:01:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18311,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability in terms of metabolic activity of primary embryonic cell culture developed from D. magna in the optimized media. A: XTT assay over 24h for a period of 92h for measuring the metabolic activity, and B: XTT assay over five days for 20 days for measuring the metabolic activity. All the values are mean of triplicates ± SD. ANOVA significant at (p\u0026lt;0.05). Different letters represent significant differences (p\u0026lt;0.05) in response to different viability after Tukey’s posthoc analysis.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-3841832/v1/204dc0dcc2081d246b04e2f4.png"},{"id":49734892,"identity":"a909d897-5803-4125-8cf9-65213521638e","added_by":"auto","created_at":"2024-01-17 07:09:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91784,"visible":true,"origin":"","legend":"\u003cp\u003eTransfection experiment in D. magna primary embryonic cell cultures using Daphnia specific expression vector (pCS-EF1α1-DSRed2). Red fluorescent protein expression in D. magna primary embryonic cells shows that the DSRed2 proteins are expressed under the control of Daphnia specific promoter (EF1α1) in the expression vector pCS-EF 1α1-DSRed2. Scale bar: 50 µm\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-3841832/v1/6efb451f085599768c1db2b7.png"},{"id":49734642,"identity":"fdaad511-65c3-4c5b-8f2e-c09bb97a560e","added_by":"auto","created_at":"2024-01-17 07:01:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":209361,"visible":true,"origin":"","legend":"\u003cp\u003eTransduction experiment in D. magna primary embryonic cell cultures using Baculovirus vector modified with WSSV Ie1 promoter and green fluorescent protein expression in D. magna primary embryonic cells shows that the GFP proteins are expressed under the control of WSSV-Ie1 promoter in the expression vector. Scale bar: 50 µm\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-3841832/v1/52630d3df2096d3f745816e3.png"},{"id":60664792,"identity":"8df7822f-a413-45ae-9272-0ffb34104100","added_by":"auto","created_at":"2024-07-19 09:12:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1685858,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3841832/v1/2574a0a1-6731-4da1-873e-f4f3984a2bc1.pdf"},{"id":49734643,"identity":"47dd215c-5f6f-43b6-ae5d-f78b1ed7c825","added_by":"auto","created_at":"2024-01-17 07:01:15","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":81567,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3841832/v1/7655a0c8d4cf660e7a85b9e7.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Baculovirus and plasmid vector-mediated transgenic experiments in the embryonic cell cultures developed from the freshwater crustacean Daphnia magna","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003eCrustaceans, a diverse group within the phylum Arthropoda, comprise approximately 67,000 species (Zhang, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eDaphnia\u003c/em\u003e, among these, holds significant importance in research as a model organism with its fully sequenced genome (Colbourne et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Functioning as filter feeders, \u003cem\u003eDaphnia\u003c/em\u003e species play a crucial role as keystone species in freshwater habitats globally. Notably, \u003cem\u003eDaphnia\u003c/em\u003e spp. demonstrates heightened sensitivity to ecological and habitat changes compared to other freshwater species (Schindler, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Various indicators of this sensitivity include alterations in reproduction for stress assessment, the formation of ephippia (Hebert and Crease, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), changes in vertical migration within the water column (Dawidowicz and Loose, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Jeswin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the impact of phenoplast (Tollrian, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), and behavioral shifts (Gerhardt et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Consequently, \u003cem\u003eDaphnia\u003c/em\u003e species were preferred subjects for toxicity studies and assessments of environmental changes. The availability of its fully sequenced genome makes \u003cem\u003eDaphnia\u003c/em\u003e a suitable model for comprehending the genomic implications of ecological changes (Shaw et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDaphnia\u003c/em\u003e spp. exhibits a reproductive strategy of asexual parthenogenesis during favorable conditions and switches to sexual reproduction during unfavorable conditions (Hebert and Ward, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). So the genetic purity of \u003cem\u003eDaphnia\u003c/em\u003e spp. can be effectively maintained in laboratory settings by providing suitable environmental conditions. Numerous aspects of \u003cem\u003eDaphnia\u003c/em\u003e spp. have undergone comprehensive study, including ecology, phylogeny, reproduction, and life cycle (Koivisto, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Colbourne, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Decaestecker et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Harris et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The combination of \u003cem\u003eDaphnia\u003c/em\u003e's short lifespan and the availability of various genomic tools for molecular studies renders it an ideal model for research purposes (Altshuler et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe initiation of primary cell culture development from crustaceans dates back to 1960 (Claydon, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, these experiments have not always yielded success mainly because of poor cell survival under in vitro conditions (Claydon, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Jayesh et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Recently, crustaceans have acquired greater attention than any other invertebrates since many economically important species are under this sub-phylum (Cai and Zhang, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), So several attempts have been made to develop cell lines from crustaceans (Claydon, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Jayesh \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) including the standardization of the biological and physiological factors crucial for the in vitro survival of crustacean cells, encompassing parameters such as pH, osmolarity, nutrients, and vitamins (Goswami et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; George et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hong et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jayesh et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, 2015; Jeswin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Despite available literature on the development of primary cell culture, achieving the permanent immortalization of cells has proven elusive, with an exception being a recent publication on the immortalization of a shrimp lymphoid organ cell line (\u003cem\u003ePmLyO-Sf9\u003c/em\u003e) through hybridization with an insect (Sf9) cell line (Anoop et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sathyabhama et al., 2021).\u003c/p\u003e \u003cp\u003eOnce a cell culture has been established from \u003cem\u003eDaphnia\u003c/em\u003e spp., it becomes a valuable research tool for various applications. This includes the isolation and cultivation of pathogens, drug screening, exploration of metabolic pathways, production of antibodies, as well as studying the regulation of gene expression and evolutionary early mechanisms of cell-mediated cytotoxicity, apoptosis, and cell ageing.\u003c/p\u003e \u003cp\u003eThe development of immortalized cell lines, a crucial aspect of sustained cellular studies, often requires proper transgene studies. Transgenic research plays a pivotal role in bridging the gap between basic and applied molecular biology. Advancements in transgenic technology have provided a potent tool for targeted genetic manipulations. The ability to introduce genetic alterations at the cellular level allows researchers to bypass normal cellular senescence, enabling the generation of continuous, infinite cell lines (Brenin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTransgenesis is a method employed to introduce foreign nucleic acids into eukaryotic cells, thereby altering the genetic composition of the host cells (Kim \u0026amp; Eberwine, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Vector constructs carrying the specific nucleic acids to be transfected can be broadly categorized as either viral or plasmid vectors. Viruses and plasmids, facilitated by an appropriate eukaryotic promoter, allow for the production of a foreign transgene (Colosimo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen selecting a gene delivery method, three crucial factors should be considered: 1) cost-effectiveness and ease of use, 2) absence of toxicity/immunogenicity, and 3) efficiency of transfection and gene expression. While existing delivery systems do not fully meet all three criteria, viral vectors, though efficient in vivo, face limitations due to immunogenic responses and cytotoxicity. Non-viral vector delivery systems, while less efficient, are minimally immunogenic, cost-effective, and easier to control for quality (Abul hassan et al., 2000; Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addressing the challenges of gene transfer, a crucial need for a clearer and more consistent approach is acknowledged. Consequently, here an attempt was made to achieve maximum transgenic success with minimal side effects through the thorough optimization of transduction using the baculovirus system and transfection using the Cellfectin reagent. The intent of the attempt was not to compare the efficiency of both transgenic techniques but to standardize a protocol suitable for \u003cem\u003eDaphnia\u003c/em\u003e primary culture.\u003c/p\u003e \u003cp\u003eIn the context of \u003cem\u003eD. magna\u003c/em\u003e cell culture, designing and standardizing transfection/transduction experiments can be challenging, especially when dealing with a novel cell culture and experiencing low cell numbers and attachment efficiency. In this study, efforts were made to address these challenges by standardizing the transgenic experiments, along with optimizing the cell culture medium for cell maintenance. Remarkably, the cells were successfully maintained for over two months in vitro, with their metabolic activity confirmed through BrdU and XTT assays. These findings highlight the potential of the standardized approach for sustained and functional \u003cem\u003eD. magna\u003c/em\u003e primary cell cultures, offering a valuable platform for various research applications.\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cp\u003eThe experimental animals, \u003cem\u003eD. magna\u003c/em\u003e, were obtained as embryo capsules from a local vendor. The embryos were hatched in Adam medium (Aachener Daphnien medium) supplemented with vitamins. Species identity was confirmed through morphometric analysis, followed by molecular characterization of the mitochondrial genome (\u003cb\u003eSuppl. Figure\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFor the development of primary cell culture, entire animals and embryos were used to identify suitable tissue sources. The protocol, based on Robinson et al. (2006) with slight modifications, involved collecting 30\u0026ndash;60 animals containing embryos in various developmental stages in a 1.5 mL microcentrifuge tube (MCT). The animals underwent surface sterilization through a series of washing steps, including 0.05% sodium hypochlorite (30s), PBS (50s), 70% ethanol (30s), PBS (50s), serum-free cell culture medium (1 min), and final washing with PBS (10s).\u003c/p\u003e \u003cp\u003eFor embryonic primary cell culture development, embryos were aseptically removed from the brood chamber of \u003cem\u003eD. magna\u003c/em\u003e with sterile syringe needles, ensuring the integrity of the animal's gut to prevent contamination. After transferring the embryos to another MCT, the same washing steps were followed. The embryos were then transferred to a fresh cell culture medium in an MCT, gently macerated with a sterile homogenizer, and the suspension was centrifuged at 1000Xg for 5 min. The pellet containing cells were resuspended in fresh cell culture medium (Modified Schneider's insect medium, pH 6.8), seeded onto a 96-well plate, and maintained at 26˚C. Fresh cell culture medium was supplied upon any change in its color.\u003c/p\u003e \u003cp\u003eTo develop a primary cell culture from adult \u003cem\u003eD. magna\u003c/em\u003e, 30\u0026ndash;60 animals without embryos were placed in a fresh rearing medium and starved for 24 h. Following sterilization through the same series of washing steps, the animals were homogenized with minimal pressure. The suspension was centrifuged at 1000Xg for 5 min, and the pellet was resuspended in fresh cell culture medium containing antibiotics (penicillin 100 IU/mL, amphotericin B 50 \u0026micro;g/mL, and streptomycin 100 \u0026micro;g/mL). The suspension was then seeded on a 96-well plate and maintained at 26˚C.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Screening of suitable media for the primary cell culture developed from \u003cem\u003eD. magna\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSeven commercial media (all purchased from Sigma-Aldrich, US) \u0026minus;\u0026thinsp;1X Leibovitz's L-15 Medium (1X L-15), Schneider's insect medium, TNM-FH insect medium (TNMFH), Grace's insect medium (Grace's), Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute (RPMI) medium were compared. These media were supplemented with 20% fetal bovine serum (Himedia, India), antibiotics (100 \u0026micro;g/mL streptomycin and 100 IU/mL penicillin), pH adjusted to 6.8, and an osmolarity of 380\u0026thinsp;\u0026plusmn;\u0026thinsp;10 mOsm/kg. Cell viability and proliferation were assessed by observing the cells using an inverted phase-contrast microscope (Leica, Switzerland).\u003c/p\u003e \u003cp\u003eFurther modifications experimented with the selected Schneider's medium. In all experiments, the cells were seeded in 96-well plates with different concentrations of testing supplements and the control. The effects of the supplements were determined based on cell viability, proliferation, the percentage of cell attachment and monolayer formation which was observed daily for one month using an inverted phase-contrast microscope (Leica, Switzerland).\u003c/p\u003e \u003cp\u003eThe experiments were conducted in four distinct phases. In the first phase, the effects of FBS were tested (5\u0026ndash;20%). As, heat-inactivated FBS is recommended by many researchers (Frerichs \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Mulford and Austin \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Fan and Wang \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wenli and Shields \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) an experiment was conducted to compare the effect of heat-inactivated FBS and non-heat-inactivated FBS (Himedia, India) on the cells when applied at different concentrations ranging from 5\u0026ndash;20%. In the second phase, organic supplements such as MEM Vitamins (Gibco, US), Amino acids, Glucose, Trehalose, and Sucrose were tested (0.1, 0.5, and 1% concentrations). Similarly, the effects of cholesterol and selenium (0.01, 0.1, and 1ppb) were tested in the third and fourth phases, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cellular and molecular responses of primary embryonic cell culture\u003c/h2\u003e \u003cp\u003eThe cell viability was tested by acridine orange and ethidium bromide staining. Acridine orange is a vital dye that stains live and dead cells, while ethidium bromide stains only cells that lose cellular integrity. When the cells are exposed to both these stains simultaneously, the nuclei of 'live' cells will stain green, whereas the nuclei of 'dead' cells will stain orange-red color. Ten \u0026micro;L of modified Schneider's medium containing cells were mixed with 10 \u0026micro;L of 1X staining solution containing acridine orange and ethidium bromide (100 \u0026micro;g/mL of acridine orange and 100 \u0026micro;g /mL of ethidium bromide prepared in PBS and mixed gently), incubated for 2 min under room temperature (RT) in the dark. Then 500 \u0026micro;L of 1X PBS was added and centrifuged at 1000 g for 10min. The supernatant was discarded, and the pellet was resuspended in 20 \u0026micro;L of 1X PBS. 10 \u0026micro;L of the solution was loaded on a microscopic slide, and a coverslip was kept carefully without air bubbles (Kasibhatla 2006). The slide was then observed under a fluorescent microscope (Leica, Switzerland).\u003c/p\u003e \u003cp\u003eDNA synthesis was analyzed using the BrdU (5-Bromo-2'-deoxyuridine) incorporation assay in primary embryonic cells grown in vitro for 6\u0026ndash;8 h in 96-well plates with a 200 \u0026micro;L growth medium (Jose et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Puthumana et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To each well, 20 \u0026micro;L of a 10 mM BrdU solution was added, and one well without the addition of BrdU served as a control. After 24 h of incubation, the medium was removed, and the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and washed again with PBS. Subsequently, 2 M HCl was added to each well and incubated for 20 min. The acid was then neutralized with 0.1 M sodium borate (pH 8.5) for 2 min, followed by another wash with PBS. The cells were permeabilized with PBS containing 0.2% Triton X-100 and 3% BSA for 5 min. After blocking with 3% BSA in PBS for 1 h, a mouse monoclonal anti-BrdU antibody (Sigma, USA) at a 1:1000 dilution in 3% BSA was added and incubated for 1 h. The cells were washed thrice with PBS for 5 min each and then incubated for an additional hour with goat anti-mouse FITC conjugate at a 1:40 dilution (Sigma, USA). After washing with PBS, the wells were stained with DAPI (0.2 \u0026micro;g/mL) and observed under an inverted fluorescence microscope (Leica, Switzerland). DAPI and FITC signals were viewed under filters with 360\u0026ndash;370 nm and 470\u0026ndash;490 nm excitation wavelengths. The images were processed and merged using the \"Leica Application Suite\" (Leica Microsystems, Switzerland).\u003c/p\u003e \u003cp\u003eThe XTT assay assessed the cells' metabolic activity at 24 h and 5 days after seeding, i.e., 24 h, 48 h, 72 h, 96 h, and days 0, 5, 10, and 15. The change in cell density during this period was determined through XTT. The basic principle of this assay relies on the cleavage of a yellow tetrazolium salt into an orange-colored formazan by the mitochondrial enzyme, which functions in only metabolically active cells (Roche Applied Science, 2003). For the assay, 5 mL of XTT (sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitrobenzene sulfonic acid hydrate) and 100 \u0026micro;L of PMS (N-methyl dibenzopyrazine methyl sulfate) were mixed thoroughly. A 96-well plate containing 100 \u0026micro;L of culture medium and cells was treated with 50 \u0026micro;L of XTT/PMS mix and incubated at 26\u0026deg;C for 5 h. After incubation at 26\u0026deg;C, the absorbance was measured at 490 nm in a microplate reader (Tecan Infinite Tm, Austria) with a reference at 655 nm (Van Der Merwe et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Puthumana et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe subculture of attached cells was assessed using both enzymatic (trypsin-based) and mechanical (gentle pipetting) methods. The cells were gently washed with PBS and treated with a 0.05% trypsin-EDTA solution. After a brief incubation (1 min) at RT, the detachment of cells was observed under an inverted phase-contrast microscope. Upon detachment, the 0.05% trypsin-EDTA solution was discarded, and modified Schneider's medium with 10% FBS was added to inhibit trypsin activity. Subsequently, the cells were subcultured and maintained at 26\u0026deg;C. Periodic observations were made for any changes in media color (Jayesh et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the second method, the cell culture medium was discarded, and the cells were washed with PBS. Dissociation was achieved through gentle trituration using a medium containing FBS. The detached cells were then resuspended in a fresh medium without antibiotics and kept at 26\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Transgenic experiments in primary embryonic cell culture\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Standardisation of transfection\u003c/h2\u003e \u003cp\u003eThe concentration of Cellfectin \u003cb\u003e(\u003c/b\u003eCellfectin\u0026trade; II-Invitrogen) and plasmid along with the incubation time required for successful transfection were standardized. Therefore, different concentrations of Cellfectin reagent (3 \u0026micro;L- 10 \u0026micro;L) and plasmid (1 \u0026micro;g- 5 \u0026micro;g) were used for the experiments. The cells were seeded in 24-well plates, and the medium was changed after 24 h of incubation. Cellfectin reagent was diluted in 100 \u0026micro;L serum-free medium, mixed, and kept at RT for 30 min. Kato developed a \u003cem\u003eDaphnia\u003c/em\u003e-specific vector, pCS-\u003cem\u003eEF1α1-DSRed2\u003c/em\u003e, which has been successfully introduced into \u003cem\u003eD. magna\u003c/em\u003e embryonic eggs through microinjection (Kato et al., 2012). The same plasmid was kindly obtained from Dr. Hajime Watanabe, Osaka University, Japan. This plasmid contains \u003cem\u003eEF1α1\u003c/em\u003e as promoter, and expression was marked by the \u003cem\u003eDSRed2\u003c/em\u003e gene fused with the \u003cem\u003eD. magna\u003c/em\u003e histone \u003cem\u003eH2B\u003c/em\u003e gene. Different concentrations of plasmid (1\u0026ndash;5 \u0026micro;g) were diluted in 100 \u0026micro;L serum-free medium, and a Cellfectin reagent was mixed (1:1) and incubated for 15min. Then, the mixture was added dropwise to the cell culture and incubated at 26\u003csup\u003eo\u003c/sup\u003eC for 3 to 8 h (different incubation time). Then the medium was replaced with FBS containing modified medium for 72 h and observed under an inverted phase-contrast microscope (Leica, Switzerland) under the excitation wavelength of 470-490nm (Jayesh et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Standardisation of transduction\u003c/h2\u003e \u003cp\u003eRecombinant baculovirus-mediated transduction using shrimp-specific promoter (WSSV-Ie1 and IHHNV-P2) having green fluorescent protein as the marker (BacIe1-GFP and BacP2-GFP) was developed for transgene expression in \u003cem\u003eP. monodon\u003c/em\u003e lymphoid cell by Puthumana et. al (Puthumana et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Briefly, Ie1 promoter from WSSV and P2 promoter from IHHNV were isolated, amplified, and ligated into the pFASTBac 1\u0026trade; transfer vector containing PH promoter (polyhedrin gene). These two vectors were used to standardise transduction in \u003cem\u003eDaphnia\u003c/em\u003e primary cell culture.\u003c/p\u003e \u003cp\u003eFor that, \u003cem\u003eDaphnia\u003c/em\u003e cells were seeded in 96-well plates and incubated in the modified Schneider\u0026rsquo;s medium for 24 h at 26\u0026ordm;C. Then the medium was replaced, and 100 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\text{L}\\)\u003c/span\u003e\u003c/span\u003e of supernatant containing recombinant baculovirus was added to100 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003eL medium and observed every 6 h for 24 h and subsequently observed every 24 h for a week under a UV microscope for the GFP signals. \u003cem\u003eDaphnia\u003c/em\u003e cells without baculovirus were used as a negative control, and viruses with SF9 cells were used as the positive control.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe results are the average of three sample replicates with a standard deviation. Onaway analysis of variance (ANOVA) was performed using SPSS\u003csup\u003e\u0026reg;\u003c/sup\u003e software (version 21; SPSS Inc., Chicago, IL, USA) to confirm the data validity, and significant differences in all samples were tested by Tukey's post hoc test (Tukey's, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cp\u003e \u003cb\u003eDevelopment of primary cell culture from\u003c/b\u003e \u003cb\u003eD. magna\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe embryos proved to be a superior source for cell culture from \u003cem\u003eD. magna\u003c/em\u003e compared to adult animals, as it was possible to repeatedly develop axenic cell culture from the former. The cells maintained a healthy condition for over two months and exhibited both single and aggregate forms. Clustered cells were more abundant than individual cells and demonstrated better cell attachment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cells were observed to be healthy when in close contact with each other. Seeding cells in 96-well plates proved to be more efficient than culturing in tissue culture flasks. Fresh medium was supplied whenever a color change was observed in the medium. The cells mostly remained in suspension due to the slow rate of attachment, so medium changes were performed by exchanging half of the medium from the well with fresh medium. After two weeks of seeding, the first subculture was carried out.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe comparison of cell subculturing methods involving pipetting and trypsinization revealed a substantial difference in cellular viability, with the first protocol (gentle pipetting) proving significantly less detrimental compared to the second protocol (trypsin treatment with pipetting). This observation leads to the hypothesis that \u003cem\u003eDaphnia\u003c/em\u003e cells exhibit characteristics of \"social cells,\" necessitating cooperative interactions and intimate physical contact. Consequently, maintaining dissociated cells in cellular clumps, rather than as single-cell suspensions during passaging, may be crucial.\u003c/p\u003e \u003cp\u003eUpon initial passaging, an increased presence of cellular clumps is evident with gentle pipetting rather than trypsin dissociation. This disparity may contribute to the observed healthier state of pipetted cells compared to trypsinized cells. However, it is important to note that the cells are not reattached, and no sign of division after the passaging. After that, the cells remained in suspension, and the number of viable cells decreased gradually, and a complete monolayer could never be obtained.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Screening of suitable media for the primary cell culture of embryos from \u003cem\u003eD. magna\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAll the tested media supported cell survival for at least three hours. Schneider's insect medium was selected for further modification as it showed a suitable cell culture medium in terms of enhanced cell viability and attachment compared to other media. Subsequent tests to determine the optimal fetal bovine serum (FBS) concentration indicated that 10% was sufficient to support cell growth, and there were no additional benefits observed with 15% FBS (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eScreening and optimization media for the development of Daphnia magna primary cell cultures. I) Comparison of seven different commercially available cell culture media (with 20% FBS) to evaluate the most suitable medium that supports maximum cell viability and proliferation. II) Comparative effect of heat-inactivated and non-inactivated FBS. III) Effects of organic supplements such as glucose, trehalose, and sucrose in the selected medium. (IV) Effects of vitamins, amino acids, and cholesterol. V) Effects of selenium. The effect was scored as; \u0026ndash; nil, + very low survival rate or few hours survival, ++ low rate or few days survival, +++ medium rate or few weeks survival, ++++ high rate or one-month survival, +++++ very high rate or more than one-month survival.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExp. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMedium modifications\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eEndpoints\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell attachment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSurvival\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCell division\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMonolayer Formation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTNMFH Medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrace insect medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL-15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRPMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDMEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eII\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;Heat inactivated FBS (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;Heat inactivated FBS (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;Heat inactivated FBS (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;Heat inactivated FBS (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;Normal FBS (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;Normal FBS (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;Normal FBS (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;Normal FBS (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIII\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Glucose(C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Glucose (0.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Glucose (0.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Glucose (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Trehalose (C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Trehalose (0.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Trehalose (0.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Trehalose (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Sucrose (C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Sucrose (0.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Sucrose (0.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;Sucrose (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;Vitamins (C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;Vitamins (0.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;Vitamins (0.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;Vitamins (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Aminoacids(C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Aminoacids (0.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Aminoacids (0.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Aminoacids (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Cholesterol (C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Cholesterol (0.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Cholesterol (0.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Cholesterol (1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Selenium(0.01ppb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Selenium(0.1ppb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchneider\u0026rsquo;s insect medium\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;0.1%Glucose\u0026thinsp;+\u0026thinsp;0.1%Vitamins\u0026thinsp;+\u0026thinsp;Selenium(1ppb)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eInterestingly, when FBS was heat-inactivated, the cells exhibited similar results, suggesting that heat inactivation might not be necessary for \u003cem\u003eDaphnia\u003c/em\u003e cells. Additionally, the introduction of glucose was found to enhance cell division during the initial stages (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), but subcultured cells displayed no significant changes in response to the continued presence of glucose. Furthermore, supplementing the medium with glucose at regular intervals did not yield any further effects.\u003c/p\u003e \u003cp\u003eThe addition of amino acids and cholesterol proved to be ineffective, while vitamins demonstrated efficacy in enhancing cell attachment and survival at a concentration of 0.1% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Both essential and non-essential amino acids did not exhibit observable effects on the cells.\u003c/p\u003e \u003cp\u003eIncorporating selenium into Schneider's insect medium notably improved cell attachment. Cells attached within 24 h of seeding, a significant improvement compared to the control, where attachment occurred over three days. Moreover, the number of attached cells was higher at a concentration of 0.1 ng/mL (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This result suggests that cells require selenium for cell division at a critical concentration of 0.1 ng/mL, beyond which cytotoxicity and cell death occur.\u003c/p\u003e \u003cp\u003eBased on these findings, the selected medium for cell culture was Schneider's insect medium (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), modified by the addition of 0.1% glucose, 0.1% MEM vitamin mix, 0.1 ng/mL selenium, and 10% FBS. This modification resulted in enhanced cell growth, moderate proliferation, and good survival.\u003c/p\u003e \u003cp\u003eThe primary cell culture of \u003cem\u003eD. magna\u003c/em\u003e exhibited cells with enlarged nuclei, producing FITC fluorescence signals within 24 h of incubation (\u003cstrong\u003eFig. 3\u003c/strong\u003e). These findings indicate that the in vitro microenvironment supports active DNA replication in \u003cem\u003eDaphnia\u003c/em\u003e cells. After 24 h, only 2-4% of cells showed a positive response, predominantly during the washing steps of the assay; it is common for some attached cells to be washed off. Due to the lower number of attached cells within the initial 24 h, most were washed away, resulting in minimal positive signals.\u0026nbsp;\u003c/p\u003e \u003cp\u003eUtilizing a dual staining method facilitated the discrimination between live and dead cells. Live cells emitted green fluorescence under UV light, while dead cells exhibited an orange-red coloration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Approximately 90% of cells displayed green fluorescence, signifying a predominance of viable cells after 24 h of growth. It is noteworthy that all experiments were conducted using 24 h old cells, and the assay confirmed a sufficient number of viable cells at this time point.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess cell viability and division in the \u003cem\u003eDaphnia\u003c/em\u003e culture, an XTT assay was conducted. The results revealed a gradual increase in cell number (cell density) when cultured using the modified Schneider's insect medium supplemented with glucose, vitamins, and selenium. Both analyses over 24 h and five-day intervals demonstrated in vitro cell division (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A 17.74% increase in growth was observed in a 48 h old plate compared to cells at 24 h. Additionally, 24.47% and 31.80% growth were noted in the subsequent 72- and 96-h plates compared to 24 h cells. After five days, a 22.06% increment was observed. Moreover, a 49.9% increase occurred after ten days, with 54.3% and 65.5% growth observed after 15 and 20 days, respectively, compared to the first day of seeding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Transgenic expression\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Transfection\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e. \u003cem\u003eTransfection experiment in D. magna primary embryonic cell cultures using Daphnia specific expression vector (pCS-EF1α1-DSRed2). Red fluorescent protein expression in D. magna primary embryonic cells shows that the DSRed2 proteins are expressed under the control of Daphnia specific promoter (EF1α1) in the expression vector pCS-EF 1α1-DSRed2. Scale bar: 50 \u0026micro;m\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the initial screening protocol, a dose-dependent increase in fluorescence activity, highest in terms of the number of expressed cells, was observed with 3 \u0026micro;g of DNA during a four-hour exposure. Extending the transfection time by an additional hour revealed a dose-dependent increase in expression, which decreased upon further incubation. While an increase in plasmid concentration had a slightly positive effect on the number of expressed cells, concentrations of Cellfectin exceeding 7 \u0026micro;L showed no additional benefits. Notably, cells did not exhibit signs of stress even with an increased Cellfectin concentration of up to 10 \u0026micro;L. Fluorescence in cells was started within 3 h of incubation but initially, a few cells showed positive results. The number of expressed cells gradually increased, reaching a maximum after 6 h of incubation.\u003c/p\u003e \u003cp\u003eIn summary, the experiments concluded that transfection of cells using 7 \u0026micro;L of Cellfectin reagent and 3 \u0026micro;g of plasmid for 6 h incubation resulted in robust fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This outcome indicates the successful transfection and expression of a \u003cem\u003eDaphnia\u003c/em\u003e-specific expression vector in the developed primary cell culture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Baculovirus transduction\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor this study, Baculovirus modified with Ie1 and P2 was employed. Notably, only PH-Ie1 demonstrated successful transfection into the \u003cem\u003eDaphnia\u003c/em\u003e primary culture. This outcome successfully demonstrates the efficiency of the hybrid promoter baculovirus for transduction in \u003cem\u003eDaphnia\u003c/em\u003e primary culture. GFP expression was detected on the third day in sf9 cells and the fourth day in \u003cem\u003eDaphnia cells\u003c/em\u003e. Approximately 20% of cells expressed GFP, with a slight increase up to 30% after one week (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Importantly, no signs of expression were observed in the negative control, confirming that the recombinant virus can effectively infect \u003cem\u003eDaphnia\u003c/em\u003e cells using a modified promoter system.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 DISCUSSION","content":"\u003cp\u003e \u003cem\u003eDaphnia\u003c/em\u003e spp. serves as a highly relevant model organism extensively studied in various fields such as ecology, predator-induced polyphenisms, evolution, ecotoxicology, and genomics (Hebert and Ward \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Koivisto \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Decaestecker et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). To further expand research on \u003cem\u003eDaphnia\u003c/em\u003e spp. at the cellular and molecular levels, the establishment of a cell line from \u003cem\u003eDaphnia\u003c/em\u003e species is imperative. Notably, there has been only one previous report on the development of primary cell culture from \u003cem\u003eD. magna\u003c/em\u003e, which was utilized for propagating viral particles (VSV) in Schneider's insect medium supplemented with 10% heat-inactivated FBS (Robinson et al., 2006). The cells in that study could be maintained for up to one week.\u003c/p\u003e \u003cp\u003eIn our current investigation, we aimed to enhance the \u003cem\u003eDaphnia\u003c/em\u003e spp. cell culture system, focusing on improving the culture medium and conducting expression studies. Remarkably, our efforts led to the establishment of a cell culture system that could be maintained for over two months in vitro with medium modifications. The ongoing debate regarding the necessity of heat inactivation of FBS remains a question, but our findings suggest that heat inactivation is unnecessary and has neutral effects on \u003cem\u003eDaphnia\u003c/em\u003e spp. cell culture. These advancements contribute to the potential for further exploration of cellular and molecular aspects in \u003cem\u003eDaphnia\u003c/em\u003e research.\u003c/p\u003e \u003cp\u003eThe choice of tissue for cell culture is typically influenced by the nature of the studies being conducted. While results may vary depending on the body part and medium used for analysis, a general trend suggests that using younger animals as a tissue source tends to yield more successful cell culture initiation.\u003c/p\u003e \u003cp\u003eIn our studies, we utilized both the entire \u003cem\u003eDaphnia\u003c/em\u003e body and eggs for cell culture. The cell yield was higher when the whole body was employed, accompanied by an increased rate of attachment and predominantly healthy cells. However, a significant challenge encountered was regular contamination. The filter-feeding nature of \u003cem\u003eDaphnia\u003c/em\u003e, with associated microbes from the gut region, posed difficulties in overcoming such contaminations. Studies, such as one involving \u003cem\u003eArtemia sinica\u003c/em\u003e embryos, have demonstrated an extended cell lifespan (200 days) compared to studies using other tissues (Jiang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This suggests that embryonic cells might hold promise for cell culture, particularly in terms of self-renewal capacity. Ovary cells are often preferred due to their totipotency, self-renewal capacity and the potential for continuously dividing cells.\u003c/p\u003e \u003cp\u003eIn primary cell culture, the isolated tissue from the animal is typically not sterile, necessitating surface sterilization before initiating cell culture. In our studies, the entire body of the \u003cem\u003eDaphnia\u003c/em\u003e was sterilized before commencing tissue culture. The classical antibiotic combination of penicillin-streptomycin (Kasornchandra et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) was employed for this purpose along with ethanol and chlorine (Fraser and Hall, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) wash. Determining the optimal concentration of these agents to eliminate microbial contamination without causing harm to the desired cells is crucial and often requires empirical testing. Using a high concentration of antibiotics has been observed to adversely affect the health of \u003cem\u003eDaphnia\u003c/em\u003e cells, leading to senescence. In contrast, eggs are generally free from microbes and can be ensured to be contamination-free through additional washing. The careful removal of eggs from the brood was the only step that required extra attention.\u003c/p\u003e \u003cp\u003eThe concentration of FBS (Fetal Bovine Serum) is a critical factor in cell culture, and concentrations exceeding 15% have been reported to inhibit cell attachment in various studies. For example, in the lymphoid organ of \u003cem\u003eP. monodon\u003c/em\u003e, it was found that FBS concentrations greater than 15% hinder cell attachment (Hsu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Jayesh et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) clumping of cells in \u003cem\u003eP. semisulcatus\u003c/em\u003e (Rosenthal and Diamant, 1990), \u003cem\u003eLiocarcinus depurator\u003c/em\u003e and \u003cem\u003eCarcinus maenas\u003c/em\u003e (Walton and Smith, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Additionally, shorter cell survival was noted in Nephrops hematopoietic cultures (Mulford and Austin, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In alignment with these findings, our study similarly indicates that increasing the FBS concentration to 15% has no further effects compared to 10%, suggesting that 10% FBS is sufficient for promoting cell growth. This underscores the importance of optimizing FBS concentrations in cell culture to achieve the desired outcomes.\u003c/p\u003e \u003cp\u003eSeveral studies support the advantageous use of glucose supplements in cell culture medium, demonstrating a slight increase in cell attachment (Hsu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Goswami et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and enhanced cell migration (Hsu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Mulford and Austin, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Itami et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). In the context of our study, the division of \u003cem\u003eDaphnia\u003c/em\u003e primary culture initially exhibited increased cell division when 0.1% glucose was added.\u003c/p\u003e \u003cp\u003eThe role of vitamins in the medium is critical, as invertebrates, including \u003cem\u003eDaphnia\u003c/em\u003e, cannot synthesize carotenes, which are essential for their growth (Claydon and Owens, 2009). Schneider's insect medium also lacks biotin and riboflavin, considered crucial vitamins (Luedeman and Lightner, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Additionally, crustaceans lack the ability for de novo synthesis of sterols and require a dietary source of cholesterol for growth, development, and survival (Fungwe et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Surprisingly, our results indicate that the addition of cholesterol had no significant impact on cell culture, while survival was increased with vitamin supplementation.\u003c/p\u003e \u003cp\u003eSelenium, essential for \u003cem\u003eDaphnia\u003c/em\u003e reproduction and development (Keating and Dagbusan, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), demonstrated an improvement in cell attachment in our study at a concentration of 0.1 ppb, although gradual cell death was observed above this concentration. Initially, the attachment rate of \u003cem\u003eDaphnia\u003c/em\u003e cells in primary cell culture was prolonged, but after the addition of selenium, cells exhibited attachment within 12\u0026ndash;14 h. These findings contribute to understanding the specific requirements and responses of \u003cem\u003eDaphnia\u003c/em\u003e cells in culture conditions.\u003c/p\u003e \u003cp\u003eThe efficiency of gene transcription significantly influences gene expression levels, and the interaction between the RNA polymerase complex and promoter sequences is crucial for initiating this process. The resulting RNA transcript separates from the gene at the transcription signal, ready for eventual translation (Khan, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Successful gene expression in cells requires a suitable cell line and appropriate vectors. However, the lack of permanent cell lines and transgenic methods in crustaceans poses a challenge for such work.\u003c/p\u003e \u003cp\u003eTransfection studies are commonly conducted in primary cultures with oncogenes to induce cell immortality (Ratner et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Claydon and Owens, 2009; Puthumana et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Various transgenic methods, including lipofection-mediated (Claydon and Owens, 2009; Xu et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), electroporation-mediated (Li et al., 2011; Shi et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and microinjection-mediated (Preston et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Hiruta et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), are steadily advancing. Nevertheless, transfecting suspension cell lines, a common challenge, is often hindered by decreased attachment of the transfection complex to the cell surface, leading to reduced uptake of the target DNA (Landauer et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Montier et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Praveen et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, an effective transfection of \u003cem\u003eDaphnia\u003c/em\u003e cells was achieved using 7 \u0026micro;L of Cellfectin reagent and 3 \u0026micro;g plasmid DNA, incubated for 6 h before the medium change. This approach showed \u003cem\u003eDsRed2\u003c/em\u003e expression after 72 h of incubation, with nearly 60% of cells expressing \u003cem\u003eDsRed2\u003c/em\u003e. Our methodology provides a reliable, straightforward, and economically directed manipulation of suspension cells using Cellfectin. This advancement may contribute to the progress of genetic studies in \u003cem\u003eDaphnia\u003c/em\u003e and other suspension cell systems.\u003c/p\u003e \u003cp\u003eBaculovirus expression vector systems (BEVS) have been extensively employed since 1983 to produce recombinant proteins in insect cells, with the \u003cem\u003eAutographa californica\u003c/em\u003e multicapsid nucleopolyhedrovirus (AcMNPV) serving as the foundation for most BEVS (Smith et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Typically, strong insect-virus gene promoters such as polyhedrin or p10 are used to enhance the expression of recombinant proteins (Vaughn et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1977\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEnsuring the synthesis of proteins in optimal quantity and quality is crucial. Various modifications to baculovirus vectors have been explored to enhance the quantity and quality of recombinant proteins produced by BEVS, as reviewed by Hitchman et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In this context, we introduced a modified BEVS having the WSSV promoter gene Ie1 (Jayesh et al., 2015) to effectively transduce \u003cem\u003eDaphnia\u003c/em\u003e cells. Almost 30% of cells were successfully transfected after one week of incubation indicating the efficiency of baculovirus in \u003cem\u003eDaphnia\u003c/em\u003e cells.\u003c/p\u003e \u003cp\u003eIn conclusion, this study successfully modified and validated Schneider's insect-based cell culture medium for the development of a primary cell culture from \u003cem\u003eD. magna\u003c/em\u003e, utilizing various tissues and embryos. The improvements observed in terms of extended longevity, enhanced DNA synthesis, metabolic activity, and increased transfection/transduction efficiency of the primary cell culture highlight the potential of \u003cem\u003eDaphnia\u003c/em\u003e cell culture as a valuable 'model' in vitro system for crustaceans. The findings of this research are expected to contribute to the advancement of crustacean cell line studies.\u003c/p\u003e \u003cp\u003eFurthermore, the methodologies and techniques outlined in this paper provide valuable insights that can assist researchers in developing and establishing cell lines from the crustacean model, particularly \u003cem\u003eD. magna\u003c/em\u003e, for diverse applications. This work contributes to the broader field of cell culture research and underscores the potential of \u003cem\u003eDaphnia\u003c/em\u003e as an effective model organism for further studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interests\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eContributions\u003c/h2\u003e \u003cp\u003eSCP carried out the formal analysis and investigation. SCP and JP conducted the writing original draft and SB provided experimental support and made critical revisions. JP conceptualized, designed the methodology, and supervised SCP for all the experiments.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003eAll investigations were conducted as per guidelines provided by the Institutional Biosafety Committee (IBSC) and Ethical Committee of NCAAH, CUSAT in Kerala, India.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by the Government of India: the Department of Biotechnology under the Innovative Young Biotechnologist Award (DBT-IYBA; BT/09/IYBA/2015/11).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSCP carried out the formal analysis and investigation. SCP and JP conducted the writing original draft and SB provided experimental support and made critical revisions. JP conceptualized, designed the methodology, and supervised SCP for all the experiments.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThe authors thank the Department of Biotechnology (DBT), Govt. of India, for the Innovative Young Biotechnologist Award (DBT-IYBA; BT/09/IYBA/2015/11) to Dr. Jayesh Puthumana and the funding for this research work. The first author thanks DBT for the fellowship.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbul-Hassan K, Walmsley R, Boulton M (2000) Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells. 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Int J Pharm 390(2):198\u0026ndash;207\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Daphnia magna, Primary cell culture, BrdU assay, XTT assay, Transformation, Transduction","lastPublishedDoi":"10.21203/rs.3.rs-3841832/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3841832/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCell culture represents an indispensable tool for investigating fundamental biological processes. Nevertheless, technical challenges such as low cell yield, sub-optimal cell differentiation, and inadequate attachment to the growth substrate have restricted the application of this tool in many studies. Here, we introduce an easy protocol for the preparation of primary cell cultures from \u003cem\u003eDaphnia magna\u003c/em\u003e embryos, offering a versatile approach to address cell biological questions in conjunction with the robust in vivo model of \u003cem\u003eD. magna\u003c/em\u003e. The development of transgenic cells is an emerging interdisciplinary field that can be used for the fundamental understanding of normal and pathological responses of cells and the improvement of tissue functionality. The application of this technology to primary cells is still in its infancy but promises to accelerate research. In this work, embryonic cell culture is developed from \u003cem\u003eD. magna\u003c/em\u003e; and is used to standardize viral (BacIe1-\u003cem\u003eGFP\u003c/em\u003e) and plasmid vector (pCS-\u003cem\u003eEF1α1-DSRed2\u003c/em\u003e)-mediated transgenic experiments. The standardized conditions methodology for developing embryonic cell culture, Cellfectin-mediated transfection and baculovirus-mediated transduction methods envisage strengthening the crustacean cell line research and bringing forth the \u003cem\u003eDaphnia\u003c/em\u003e cell culture system as a 'model' in vitro system for crustaceans. Additionally, the simplicity and flexibility of the methodology described are expected to lead to widespread use in many biological research areas, including their wide application to ecotoxicological and epigenetic studies which are currently limited to in vivo studies. This is the first report on the optimization of cell culture medium for freshwater crustaceans and the use of baculovirus for transduction studies in \u003cem\u003eD. magna\u003c/em\u003e embryonic cell culture.\u003c/p\u003e","manuscriptTitle":"Baculovirus and plasmid vector-mediated transgenic experiments in the embryonic cell cultures developed from the freshwater crustacean Daphnia magna","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 07:01:10","doi":"10.21203/rs.3.rs-3841832/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ed10ba4-8132-4401-81d1-5acb0efa6959","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-29T00:53:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-17 07:01:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3841832","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3841832","identity":"rs-3841832","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}