Estimation of Bisphenol A in mosquito breeding sites
Water samples (one litre) from four breeding sites with huge deposits of disposable plastics were collected in amber coloured bottles. BPA was quantitatively estimated by Gas chromatography – Mass spectroscopy with instrument Varian CP 3800 connected to MS model Saturn 2200.The capillary column with phenyl siloxane as packing material (Varian, USA) and the carrier gas used was chlorated helium. Ionisation mass of the mass spectrometer 70 eV. The detector was operated in positive ion mode using selected ion monitoring and peak obtained at retention time was analysed by Mass spectrum detector. The peaks detected were m/z 228(M+) and 214 ( M_CH3 ) (Barbalas and Garland 1991 ). The mass spectrum was recorded for mass range 40 – 600 m/z for 35 minutes.
Water samples were filtered through cellulose acetate filter with pore size of 0.45µm (Millipore HAWPO 4700) and 500ml was treated with 150 g NaCl and three drops of 1:1 aqueous HCl. The solution was mixed with 3.5 ml of dichloromethane in separating funnel. The organic phase was dropped into a small glass tube and treated with anhydrous sodium sulphate. The organic phase was evaporated to dryness under nitrogen and dissolved in ethylacetate, which is injected for analysis. Bisphenol A dissolved in ethylacetae was used as standard . The work was done in GC-MS facility available in Confederation of Ayurvedic Reniassance Keralam Ltd., Trichur, Kerala, India.
Collection of mosquito egg rafts
Egg rafts of Culex.sp. of mosquitoes were collected by keeping 3 litres of water mixed with contents of two raw eggs of domestic fowl in buckets placed at damp corners of the College campus. Mosquitoes started laying eggs after 2-3 days, once foul smell emanated from the bucket of water sample and egg rafts were collected everyday in the morning.
Identification of Culex at species level
Identification of adult mosquitoes were done as Culex quinquefasciatus Say. by using the taxonomic key (Barruad 1934) and instars of larvae were identified (Tripathy and Dash 1988) after killing by heat killed method. Larvae were killed in hot water just below boiling point and put them in 70 % ethanol, before mounting on a clean glass slide and observed under (10x) microscope (Leica,Germany).
Rearing of C.quinquefasciatus larvae
Collected egg rafts were transferred into enamelised metal pans (20 cm diameter and 10 cm depth) containing of egg- water medium(500 ml) and diluted to one litre with dechlorinated water. The rearing medium was changed every three days. The metal pan with developing larvae were kept in the insectarium (cage made of mosquito net and metal frame) for emergence of adults. Temperature of the insectarium ranged between 26 and 310C and humidity was kept above 75% by keeping the floor wet.
Mixing of BPA in water
BPA was dissolved in ethanol in such a way that 50µl of ethanol possessed different doses of BPA, starting from 1 to 50 mg. Fixed volume is added to one litre of rearing medium which resulted working dose of BPA starting from 1 to 50 ppm. Rearing medium applied with BPA was changed every three days.
LC50 study of BPA
As the length of each larval instar is approximately two days, 48 hours is considered as limit of activity/ toxicity. LC50 study was done in fourth instar larvae and it was calculated by Probit analysis.
Study on span of lifecycle from egg to adult
Newly laid egg rafts ( two, white coloured) were put on one litre larval rearing medium possessing 0, 1, 2 and 4 ppm BPA. Duration in hours, required for hatching of eggs, larval- larval instars, pupation and eclosion to imago were recorded. The experiment was repeated six times and during the whole study period temperature ranged from 26 - 310C .The experiment was also done during the month of May (at the peak of summer in Kerala) in which the temperature ranged between 30 and 370C.
SDS - PAGE Analysis
SDS – PAGE of whole body homogenate was done by standard method (Laemmli 1970).All protein samples contained 100µg protein and electrophoresis was carried out using 12% Polyacrylamide gel, pH 8.8. Acrylamide : Bisacrylamide ratio was 30 : 1.
In - gel tryptic digestion and Mass Spectroscopy – MALDI TOF / TOF
Protein bands showing increase in band density and band volume (ten bands) between 66 and 97.4 kDa corresponding to control and final instar larvae of C.quinquefasciatus developed in 1 and 2 ppm BPA as obtained in 2D SDS PAGE analysis were excised, destained ,subjected to in – gel trypsin digestion and prepared for matrix - assisted laser desorption / ionization (MALDI) MS analysis. The MS analysis was done using Ultraflex III MALDI TOF/ TOF Mass spectrometer (Bruker Daltonik, Bremen, Germany). Spectra obtained were searched against Culex quinquefasciatus data base using the program MASCOT (Mascot Daemon; Matrix Science, Inc.,Boston, MA,USA). The search parameters were enzyme trypsin with one mixed cleavage, carbamidomethylation as fixed modification and oxidation of methionine as variable modification, 1.2 Da for peptide mass tolerance and 1.2 Da for fragment mass tolerance. Proteins identified were then analysed using the Uniprot data base ( http: //www.uniprot.org./).
Estimation of 20 -hydroxy ecdysone
Estimation of 20HE was done in final instar larvae at every four hours, starting from 4th hour of their emergence to pupation. Enzyme immunoassay of 20HE in the whole body homogenate was done by using the assay kit (Porcheron et al. 1989),Caymon Chemicals, (AO 5120# 96 wells), France. Assay is based on competition between unlabelled 20HE and acetylcholine labelled 20HE for a limited amount of specific antiserum raised against 20HE in rabbit.
Five larvae, each of treatment and control were homogenized in 250µl of ice cold methanol to extract 20HE. The homogenate was centrifuged at 8000g in refrigerated centrifuge and supernatant was evaporated to dryness and dissolved in 100µl buffer supplied along with the kit. Assay was done in 96 well microplate which was added with Ellman reagent, leading to chromogen formation. Absorbance of the chromogen was read by microplate reader at 405 nm.
Relative expression of genes by quantitative real time PCR
Activity state of Ecdysone receptor A (EcRA) and Ecdysone regulated gene (E75A) in final instar larvae of control group and larvae developed in 1 ppm BPA were done at the 30th hour after the formation of the instar ( usually 50 to 52 hours is the length of the stadium). RNA was isolated by RNA isolation kit. Larval tissue (100 mg) was homogenized in 1.0 ml of Trizol reagent and shaken well with 200µl of chloroform for 15 minutes, centrifuged after three minutes at 14000 rpm. Aqueous layer was mixed with 500 µl of isopropanol and centrifuged at 14000 rpm for 15 minutes. Supernatant was discarded and the pellet was washed with 200 µl of 75% ethanol. After centrifugation for 15 minutes at 14000 rpm, the pellet was dissolved in buffer. (Refrigerated centrifuge was used for all the above studies).
After assessing the purity and concentration of the extracted RNA, cDNA was prepared by Thermoscientific, Verso cDNA kit. Quantitative real time PCR was carried out using SYBR Green Master Mix (Applied Biosystem,Life Technologies). Normalisation was performed using primer specific for S7 ribosomal m-RNA. All the reactions were performed in triplicate and data were analysed. The primer sequences (Table S 1) used against each specific genes and after gene expression, values are normalized against S7 ribosomal protein gene (S7RP) as standard and relative expression level was calculated. The level of expression of two genes in control larvae is considered as background level or 1.0. Reproducibility of the experiments were verified by independent triplicate experiments
Viability testing of adult mosquitoes developed in BPA
Adult mosquitoes emerged from control and BPA treatments such as 1, 2 and 4 ppm were tested for their sanguivorous behavior and egg laying performance. Pupae (120 numbers) developed in control and BPA groups were separately kept in mosquito cages for adult emergence. They were provided with 2% glucose by placing a 250 ml beaker with the solution in which a filter paper roll was placed. They were provided with blood meal by allowing an albino rabbit which is partially immobilized by keeping in wire mesh and pinnae were shaved and kept overnight . After providing blood meal, fresh set up of glucose solution and a metal pan with one litre of larval rearing medium were kept in the cage. Humidity was maintained in the room by keeping the floor wet. Metal pan with larval rearing medium was observed for next six days for the presence of egg rafts.
Recording the body weight of Pupa
Pupae (50 number) were pooled together in clean water to free them off the dirts and gently blotted in tissue paper and weighed them together using an electronic balance (Shimadzu, Japan ). Weight of ten samples, each one with 50 pupae were recorded for each dose of BPA.