In Vitro Cell.Dev.Biol.—Animal (2014) 50:296–303 DOI 10.1007/s11626-013-9694-1
Establishing midgut cell culture from Rhynchophorus ferrugineus (Olivier) and toxicity assessment against ten different insecticides Ahmed Mohammed Aljabr & Muhammad Rizwan-ul-Haq & Abid Hussain & Abdullah I. Al-Mubarak & Hassan Y. AL-Ayied
Received: 3 April 2013 / Accepted: 12 September 2013 / Published online: 7 November 2013 / Editor: T. Okamoto # The Society for In Vitro Biology 2013
Abstract Midgut epithelial cell culture was successfully developed from red palm weevil (Rhynchophorus ferrugineus) during this study and named as RPW-1. Optimum conditions for four different commercial media were also worked out to successfully maintain the culture. Grace’s medium was found to be the most effective for RPW-1 culturing which resulted in the highest cell density of 7.5×106 cells/ml after 72 h of cell seeding with 96% cell viability. It was followed by Schneider’s medium and TNMFH medium where cell densities reached up to 7.4×106 and 5.9× 106 cells/ml, respectively, after 72 h having 91 and 89% cell viability. Comparatively, Media-199 was least effective for RPW-1 cell culturing. As a whole, temperature at 27°C and pH 6.3 were the best for RPW-1 culturing where the highest cell Ahmed Mohammed Aljabr and Muhammad Rizwan-ul-Haq equally participated in the research work. A. M. Aljabr : M. Rizwan-ul-Haq (*) : A. Hussain Department of Arid Land Agriculture, College of Agriculture and Food Sciences, King Faisal University, Hofuf, Saudi Arabia e-mail:
[email protected] M. Rizwan-ul-Haq e-mail:
[email protected] A. M. Aljabr e-mail:
[email protected] A. Hussain e-mail:
[email protected] A. I. Al-Mubarak Department of Microbiology and Parasitology, College of Veterinary Medicine and Animal Resources, King Faisal University, Hofuf, Saudi Arabia e-mail:
[email protected] H. Y. AL-Ayied Natural Resources and Environment Research Institute (NRERI), King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia e-mail:
[email protected]
density and maximum cell viability were noted. Individually, Grace’s medium, Schneider’s medium, TNM-FH medium, and Media-199 produced better results at 27°C, 27°C, 24°C, and 21°C and pH 6.3, 6.4, 5.3, and 7.1, respectively. The toxicity assay and MTT cell proliferation assay revealed that, out of the ten insecticides used in this study, emamectin benzoate was the most toxic insecticide to RPW-1 cells resulting in 92% cell mortality and 74% cell growth inhibition. Dieldrin was the least potent, causing only 19% cell mortality and 18% cell growth inhibition. Keywords Midgutcellculture . Rhynchophorus ferrugineus . Insecticidal toxicity . MTT assay
Introduction Insects cause huge economic losses of crops and orchards worldwide. Different methodologies have been developed over time and are adopted to control pests, but synthetic insecticides are still the main tool of insect pest control. The problems related to insecticides, such as of resistance, environmental disturbances, and health concerns are increasing over time, and a number of insecticides have been reduced by law in many countries (http://www.irac-online.org; Alford 2000; Ware et al. 2003). Thus, efforts are devoted to the discovery of new bioinsecticidal molecules with lower impact on the environment and high specificity against target pests. Date palm Phoenix dactylifera is the most important fruit tree in the Kingdom of Saudi Arabia, which represents the historic symbol of the country, and approximately 23.5 million trees are planted in the kingdom. Red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier), a concealed tissue borer, is a lethal pest of palms and is reported to attack 17 palm species worldwide. Though the weevil was first reported on coconut Cocos nucifera from
ESTABLISHING CELL CULTURE FROM R. FERRUGINEUS
South Asia, during the last two decades, it has also been established as a pest of date palm in several Middle Eastern countries (Faleiro 2006). Although integrated control methods are employed to control this pest, the main method is the use of insecticides (Mahmoud et al. 2012). Among the body tissues of insects, the alimentary canal is one of the most important tissues playing a crucial role in the insect life. Study on the insect alimentary canal is important as it is the site of digestion, detoxification, transport, and semiochemical production, which are important processes in the insect life cycle (Hall et al. 2002a, 2002b; Nardi et al. 2002). The insect gut can be subdivided into three regions with different functional roles. The foregut and hindgut are derived from the embryonic ectodermal layer and are primarily responsible for ingestion of food (foregut), water absorption, and osmoregulation (hindgut), whereas the primary function of the midgut is the absorption of nutrients (Corley and Lavine 2006). Midgut tissue is also important for the production and secretion of luminal enzymes and final digestion by microvillar enzymes. Midgut epithelium consists of columnar absorptive cells with apical microvilli, pear-shaped goblet cells that transport ions in lepidopteran insects, and small round stem cells located at the base of the epithelium (Hakim et al. 2001; Smagghe et al. 2005). Round stem cells are undifferentiated cells that are mitotically active and proliferate to produce additional undifferentiated cells that differentiate through extrinsic or intrinsic signals (Young and Black 2004; Li and Xie 2005). In the last four decades, cell lines have been established from different insect species. Mostly, these cell lines originated from serious agricultural pests. Noctuid species are the origins of three most popular cell lines such as Spodoptera frugiperda (SF21 and SF9 cells) and the cabbage looper Trichoplusia ni (high five cells). Thus, the use of cell lines for research and commercial applications is currently dominated by three cell lines. Nevertheless, the continued development of new cell cultures from other species is important for the future growth of insect cell studies. Cell lines are perfect tools not only for the screening of insecticides but also for unraveling their mechanism of action and the mechanism of resistance (Smagghe et al. 2009). So, there is a need to develop and establish cell lines from notorious insect pest species like RPW. The current study was designed to establish the cell culture from the midgut tissues of RPW larvae. The techniques and optimum conditions to maintain the cell culture were also devised during the study. Impacts of synthetic insecticides belonging to pyrethroids (αcypermethrin, bifenthrin), carbamates (sevin, carbofuran), organophosphates (dursban), avermectins (emamectin benzoate, abamectin), organochlorines (dieldrin), phenylpyrazoles (fipronil), and pyrroles (chlorfenapyr) on the RPW cells were
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studied to provide a clear picture about the sensitivity to these insecticides.
Material and Methods Reagents. All the reagents required for the cell isolation and culturing were purchased from Sigma-Aldrich, St. Louis, MO. Four insect cell culture media (Grace’s insect cell medium containing L -glutamine and sodium bicarbonate; Schneider’s insect cell medium containing L -glutamine and sodium bicarbonate; TNM-FH insect cell medium containing L -glutamine and sodium bicarbonate; and Medium-199 containing Hanks’ salt and L -glutamine) were used in this study. All the insecticides (bifenthrin, dieldrin, emamectin benzoate, fipronil, αcypermethrin, abamectin, sevin, carbofuran, chlorfenapyr, and dursban) were of technical grade and purchased from SigmaAldrich. Cell isolation. Fifth-instar larvae were selected from the RPW colony maintained under the laboratory conditions as described by Al-Ayedh (2011). Midgut epithelial cells were isolated according to Lynn (2001) with some modifications as follows: 1. Insect larvae were submerged for 3–5 min in 0.05% sodium hypochlorite and disinfected by submersion in 70% ethanol. 2. Larvae were rinsed twice with sterile distilled water. 3. Larvae were then transferred to a sterile Petri dish containing 3 ml of culture media and antibiotic (gentamicin) at the concentration of 50 μg/ml. 4. Sterile instruments were used to remove the tissue of interest and transferred to a new dish with additional media. 5. Contaminating tissues were teased away, and the dish was left for 2 h to settle, thus diffusing away the contaminating cells and hemocytes. 6. Contents of the midgut lumen were removed, and the tissue of interest was transferred to a new dish containing a drop of media and cut into small pieces. 7. The edge of the dish was sealed with Parafilm and incubated at 25°C. 8. Additional 1 ml of fresh media was added after 48 h. 9. The culture was centrifuged at 400 g for 5 min, and the supernatant was discarded. Cells were resuspended in the media and evenly distributed into six-well plates. The plate was sealed with Parafilm and incubated at 25°C; 0.5 ml of media was added every7 d. 10. Once established, the cell culture was named RPW1 and subcultured every third day. RPW-1 cells
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were used for further experiments after 40 passages in the laboratory. Optimization of conditions for cell cultures. Optimization of insect cell growth media. Four Insect growth media (Grace’s medium, Schneider’s medium, TNM-FH medium, and Medium-199) were used to conduct this study. Into six-well plates containing 3 ml of insect cell growth media, 1×106 cells/ml were seeded. Cells were counted in triplicate every 24 h for each medium with a Neubauer hemocytometer. Cell density and viable cell percentage were observed up to 72 h to find the optimum growth media for RPW-1 cells. Cell calculations were made as follows: % Cell viability ¼ Total viable cellsðunstainedÞ=Total cellsðviable þ deadÞ 100 Viable cells=ml ¼ Average viable cell count per square dilution factor 104
Optimization of temperature. RPW-1 cells were exposed to a range of five different temperatures: 18°C, 21°C, 24°C, 27°C, and 30°C. Into six-well plates containing 3 ml of insect cell growth medium, 1×106 cells/ml were seeded. Each of the four insect cell culture medium, mentioned above, was used separately to be exposed to five different temperatures (18°C, 21°C, 24°C, 27°C, and 30°C). Efficiency of the RPW-1 cells and insect cell culture media at these temperatures was independently assessed by calculating the cell density and percentage of viable cells, every 24 up to 72 h. Optimization of pH. Based on the preliminary studies (data not included), three different pH levels were set for each insect cell media separately using 1 M NaOH and 1 M HCl. pH 5.8, 6.3, and 6.8 were set for Grace’s medium whereas 5.9, 6.4, and 6.9 for Schneider’s medium. Similarly, pH 4.8, 5.3, and 5.8 were set for TNM-FH medium and 7.1, 7.6, and 8.1 for Medium-199. Into six-well plates containing 3 ml of each insect cell growth media with three different pH levels, 1× 106 cells/ml were seeded and exposed to a range of five different temperatures (18°C, 21°C, 24°C, 27°C, and 30°C) separately, and data was recorded after every 24 up to 72 h based on the cell density and viability percentage. Insecticide applications . Ten insecticides from different groups (bifenthrin, dieldrin, emamectin benzoate, fipronil, αcypermethrin, abamectin, sevin, carbofuran, chlorfenapyr, and dursban) were used during this study. Five concentrations of each insecticide were prepared in 0.01% DMSO as follows: 500, 100, 50, 10, and 1 ppm. In six-well plate containing 3 ml of Grace’s medium, 1×106 cells/ml were seeded and incubated for 4 h at 27°C. Cell cultures were then treated with 10 μg/ml of the above concentrations for each insecticide. Controls were treated with equal volumes of 0.01% DMSO. Cells were harvested after 24 h, and trypan blue assay was conducted according to Oh et al. (2004) to assess the percent mortality of the cells. After
treatment and incubation of cells, 10 μl of cell solution was mixed with 10 μl 0.4% trypan blue solution (Sigma-Aldrich) and incubated for 3 min. The number of blue (dead) cells was counted under the microscope by using a Neubauer hemocytometer, and the percentage of dead cells was calculated. Cell mortality percentages and LC 50 values. Cell mortality percentages were noted after 24 h of treatment and worked out separately for each concentration (500, 100, 50, 10, and 1 ppm) of all the ten insecticides. LC50 values were determined using probit regression analysis (Grimm et al. 2001) and represented in a graph. MTT cell proliferation assay. The Vybrant® MTT Cell Proliferation Assay Kit was used to perform MTT cell proliferation assay (Invitrogen, Life Technologies, www.probes.com). RPW-1 cell suspensions of 180 μl/well (1×106 cells/ml) were seeded in 96-well plates. After 24 h of incubation, 20 μl of each of the five insecticide concentrations from ten insecticides was added, and 0.01% DMSO was used as the control. After 24 h of treatment, 10 μL of the 12 mM MTT stock solution was added to each well and incubated for 4 h at 37°C. Then 100 μL of the SDS-HCl solution (10% SDS solution in 0.01 M HCl) was added to each well and mixed thoroughly using the pipette. The plate was again incubated at 37°C for 4 h to dissolve the formazan crystals. Later on, each sample was mixed properly with the pipette, and the absorbance was measured at 570 nm by a microplate reader. Cytotoxic effect was expressed as a relative percentage of inhibition calculated as follows: Cell growth inhibition rateð%Þ ¼ ððControl−Insecticide TreatmentÞ=ControlÞ 100Þ
Statistical analysis. Data analysis was carried out using analysis of variance (ANOVA), and statistical significance was established by using the SAS software (SAS 2000). Differences between the treatments were determined by Tukey’s multiple range tests at P
