Bisphenol A Acts As Developmental Agonist in Culex Quinquefasciatus Say

Plastic – derived Bisphenol A (BPA) contaminated sewages in Trivandrum, Kerala, India are mosquito breeding sites. After summer rain, BPA in the stagnant water samples ranged between 0.86 and 1.14ppm. 1.0 ppm BPA is considered as environmentally relevant concentration. Present study revealed that BPA is a developmental agonist of Culex quinquefasciatus. Embryonic and larval development are shortened by BPA but pupal development is unaffected. Under the atmospheric temperature of 26 to 31 0 C lifecycle was completed within 312 hours but during summer it was completed within 278 hours, meanwhile atmospheric temperature ranged between 30 and 37 0 C. Reduction in the duration of development due to BPA was 50 to 70 hours during summer and 60 to 80 hours in other seasons. Larval – pupal stadium of the mosquito has three surges of 20-hydroxy ecdysone(20-HE), at 24,32 and 48th hour of the 4th instar. BPA resulted dose-dependent advancement of 20-HE peaks, Phospholipase A 2 induction and expression of Ecdysone receptor EcRA and Ecdysone inducible gene E75A, which culminated in early pupation. Adults emerging from 1.0 ppm BPA treatment did not show signicant difference in sanguivory and fecundity compared to control but pupae developed in 2 and 4 ppm BPA were signicantly small.


Introduction
Accumulation of plastic wastes in sewage and formation of mosquito breeding sites are a major cause of vector borne diseases in urban areas. Studies on mosquito larval index of Thiruvananthapuram city, Kerala, India show swarms of mosquitoes after summer rain, especially Aedes albopictus in accumulated water in plastic wastes and Culex quinquefasciatus in such water with abundance of organic material (Sunilkumar et al. 2018). Coconut shells traditionally used in rubber plantations of Kerala to collect the tapped latex form breeding sites of A.albopictus during rainy season (Sumodan 2003). These are now completely replaced by plastic cups. Bisphenol A (BPA) is a bioactive compound released from plastic wastes (Yamamoto and Yasuhara 1999) and hence mosquito larvae developed in these sites have ample chances of being exposed to this compound.
BPA is a well known xenoestrogen which causes endocrine disruption in shes leading to expression of vitellogenin gene and oocyte development in testis (Yokota et al. 2000 ;Virk et al. 2014 ) and it interferes in metamorphosis of amphibian tadpoles (Fx Yang et al. 2005). Studies have shown that BPA also causes developmental abnormalities in invertebrates such as crustaceans (Biggers and Lanfer 2004) molluscs (Fabbri et al. 2014) and in insects (Watts et al. 2003). In Drosophila melanogaster, BPA caused delay on larval development and pupation time (Alti and Ulnu 2012). In a transgeneration study, D. ananassea reared in culture medium having high doses of BPA for ve generations do not cause any developmental arrest but there was protraction of life cycle by 7 to 9 hours. However, at higher doses of 10, 15 and 20mg/L, adult ies have shown abnormal phenotypic characters on the antennae (Anuji et al. 2018). Eventhough vector control measures coupled with scienti c research are adopted against mosquitoes, many urban and rural areas fail to effectively manage the twin problems such as vector borne diseases and plastic waste disposal. Rather than providing physical amenities to mosquito development residues of plastics may unravel a chemical environment through releasing a suite of bioactive molecules including BPA in the breeding sites. So the present study was undertaken to understand the effect of BPA on the developmental stages and metamorphosis in Culex quinquefasciatus.

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 _ CH 3 ) (Barbalas and Garland 1991 ). The mass spectrum was recorded for mass range 40 -600 m/z for 35 minutes.
Water samples were ltered through cellulose acetate lter 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.

Identi cation of Culex at species level
Identi cation of adult mosquitoes were done as Culex quinquefasciatus Say. by using the taxonomic key (Barruad 1934) and instars of larvae were identi ed (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 31 0 C and humidity was kept above 75% by keeping the oor 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. LC 50 study of BPA As the length of each larval instar is approximately two days, 48 hours is considered as limit of activity/ toxicity. LC 50 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 -31 0 C .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 37 0 C.

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 nal 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 Ultra ex 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 xed modi cation and oxidation of methionine as variable modi cation, 1.2 Da for peptide mass tolerance and 1.2 Da for fragment mass tolerance. Proteins identi ed were then analysed using the Uniprot data base ( http: //www.uniprot.org./). Estimation of 20 -hydroxy ecdysone Estimation of 20HE was done in nal instar larvae at every four hours, starting from 4 th 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 speci c 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 nal instar larvae of control group and larvae developed in 1 ppm BPA were done at the 30 th 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 Thermoscienti c, Verso cDNA kit. Quantitative real time PCR was carried out using SYBR Green Master Mix (Applied Biosystem,Life Technologies). Normalisation was performed using primer speci c 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 speci c 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 veri ed 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 lter 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 oor 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.

Results And Discussion
The present study reveals that the developmental stages of Culex quinquefasciatus can tolerate BPA up to a limited extent without causing lethal effects at a concentration of 5ppm , LD 50 of BPA in fourth instar larvae being 22.334 ppm (Table S.2). Transgeneration study in D. ananassea also showed high tolerance towards BPA (Anuji, et al. 2018 ). The concentration of BPA in mosquito breeding sites of Thiruvananthapuram showed variation in relation to monsoon. During mosquito swarms after summer rain, these sites detected BPA at a range between 0.86 and 1.14 ppm but during continuous rain it declined sharply to 9 ppb (Table 1) The present study shows that BPA in uences the development of C.quinquefasciatus, from egg to pupation (Table 2). Eggs deposited in water possessing 1 to 4 ppm BPA showed 14 to 18 hours of reduction in the duration for hatching of rst instar larvae (Table 2) The results of the present study revealed that BPA had no effect on the duration required for the transformation of pupae to adults ( Table 2). Under the experimental condition with atmosphere temperature varying between 26 and 31 0 C, adults of the control group emerged on the 13 th day of egg laying, while treatment with 1 ppm BPA shortened life cycle from egg to adult emergence to 11 days i.e. , early compared to controls. The decrease in duration for adult emergence was found to be 72 h and 81h when the concentration of BPA was increased to 2 and 4 ppm respectively. The decrease in duration of larval period would affect feeding and cause a corresponding decrease in body weight of pupae (Table  3). However, at the environmentally relevant concentration of BPA at 1 ppm, such effects are not prominent..
Since temperature in uences mosquito development (Ciota et al. 2014), the same experiment was conducted during the peak of summer at which the temperature ranged between 30 and 37 0 C. Under this temperature, adult mosquitoes of the control group emerged 24 hours earlier, on 12 th day (Table 4), when compared to the controls emerging during rainy season with atmosphere temperature ranging between 26 and 31 0 C.In larvae exposed to increasing concentration of BPA viz. 1, 2 and 4 ppm, the duration required for adult emergence was further reduced to 10, 9 and 8.5 days respectively ( Table 4). Elevation of temperature also resulted elevated activity of phenol oxidase (PO), its co-stimulator trypsin like serine protease (TLSP) and increased expression of phospholipase A 2 activating protein, as an adaptive response of to overcome temperature-induced stress (Gayathri and Evans 2018) and simultaneously, temperature and BPA act together with further reduction of life cycle span (Table 4) SDS-PAGE analysis of proteins extracted from BPA treated nal instar C.quinquefasciatus larvae and their controls can be resolved into 15 bands with molecular weight ranging from 6.5 to 205 kDa. Larvae exposed to BPA showed increase in band volume and band width between 66 -97.4 kDa. (Fig. 1). New bands or disappearance of existing bands were not observed. Exposure of C.quinquefasciatus to ambient temperatures of 35 0 C to 10 or 15 0 C below or 5 0 C above the temperature also resulted identical changes in the protein pro le in electropherogram (Gayathri and Evans 2018). MALDI -TOF /TOF analysis of the excised ten protein bands between 66 -97.4 kDa from both control and larvae developed in BPA revealed that rst eight bands are importin sub unit alpha and set domain proteins in control and test. Bands of 9 and 10 were identi ed as putative uncharacterized protein and Phospholipase A2 activating protein respectively( Table 5). Exposure of C.quinquefasciatus larvae to hypothermia or hyperthermia also resulted expression of Phospholipase A2 activating protein and putative uncharacterized protein (Gayathri and Evans 2018 ).Identical physiological response exhibited by C.quinquefasciatus larvae reared in the presence of a xenoestrogen, which is actually a growth enhancer to this species and under exposure to extremes of temperature are interesting because both these agents have profound in uence on the development of this poikilotherm. In the present study, it was found that BPA treated C. quinquefasciatus, maintained at temperature ranging between 26-31°C completed life cycle within 11 days compared to 13 days required for controls. This was further reduced for 10 days, while there was an elevation of temperature by 6°C (30-37°C). In Spodoptera exigua , Phospholipase A2 plays crucial role in insect immunity and larval growth (Youngjin et al. 2015 ). BPA induced expression of Phospholipase A2 in C.quinquefasciatus larvae might have played a signi cant role in inducing rapid development.
Studies in various species of arthropods have shown that BPA has adversely affects or delays larval development, while larvae were allowed to develop in presence of this compound (Watts et  In house y larvae, anti ecdysteroid activity of BPA appears to be mediated through excess synthesis of juvenile hormone II and III (Izumi et al. 2008) and in water ea it is found to be the result of enhanced activity of crustacean juvenile hormone methyl farnesoate ( Mu Xueyan, et al.2005). The present observation in C. quinquefasciatus larvae was distinct from other reports in that BPA had signi cantly shortened the stadium in all four larval instars and the compound did not cause any reduced availability or synthesis of endogenous ecdysone or any endocrine disruptor. Eventhough BPA caused endocrine disruption in many insect larvae, it did not exhibit any ecdysteroid receptor antagonism ( Mu Xueyan, et al.2005).
Induction of moulting in insects coincides with a surge in the release of 20-hydroxy ecdysone (20HE) prior to each larval moult and pupation. Periodic pulses of 20HE and juvenile hormone orchestrates the timing of moulting (Zhu and Norigea 2016). In C.quinquefasciatus, larval-pupal stadium having a total duration of 50-52 hours is characterized by three surges of 20-HE secretion (Fig. 2). The rst surge of 20-HE occurred as the larvae passed one third of the stadium i.e. after 20h, producing a prominent peak. Increase in the titre of 20-HE continued and reached a second peak around two third of the stadium, which is followed by a third spike after 8 or 10 h. Under the environmental temperature of 26 to 31 0 C, the three peaks occurred approximately at 24,32 and 44 h leading to pupation after a lapse of 8h (Fig.2). Larval-pupal stadium of Aedes aegypti also includes three peaks of 20-HE (Margam et al. 2006). It was found in the study that in C. quinquefasciatus, nal instar larvae treated with BPA, the 20-HE peaks were advanced by 8-10h when compared with controls which culminated into pupation earlier by 10 hours (Table 2 and Fig. 2). This is shown to be a dose-dependent effect. The duration of larval instars too decreases with increasing doses of BPA. It is well known that moulting hormone functions as a key temporal signal in directing moulting and development (Zhu and Norigea 2016;Denlinger 2002) and hence early pupation in the presence of BPA could be the effect of early appearance of 20-HE peaks.
Quantitative RT-PCR study on Ecdysone receptor A (EcRA) and Ecdy sone inducible gene E75A of nal instar larvae of C. quinquefasciatus in presence of 1ppm BPA showed contrasting difference on their expression pro les (Fig.3). The relative EcRA and E75A mRNA showed double-fold elevation in BPA treated larvae, compared to controls during the two third of larval-pupal stadium (Fig. 4). Ecdysteroids in concert with EcR complex play important role in regulating reproduction and development in insects (Kumar et al.2002). In Chironomus tentans, increased EcRA expression occurs in response to 20-HE (Rouch et al. 1988). Larvae of C.riparius exposed to BPA also results in increased level of m-RNA of EcRA (Planello et al. 2008 ). During larval-pupal transition in Drosophila, activation of EcR/USP and E75A are under the direct control of 20-HE (Johnston 2011) and ecdysteroid induced E75A expression de nes a feed forward pathway that ampli es or maintains the ecdysteroid titre during larval-pupal development ensuring proper temporal progression through lifecycle (Bialecki et al.2002). The present study indicates that BPA induced early peaks of 20-HE may have modulatory effects on EcRA and E75A through feed forward mechanism which inturn leads to shortened larval-larval and larval-pupal stadia, and thus the span of the life cycle as a whole.
The span of lifecycle and number of instars in holometabolous insects are genetically programmed. Being a poikilotherm, in mosquitoes lifecycle shortens as temperature increases (Ciota et al. 2014). This is further reduced by 60 or 80h subsequent to treatment with BPA. The present study reveals that BPA acts as a developmental agonist in C.quinquefasciatus. Exogenous compounds such as fennoxycarb (Dedos et al. 2001 ), phytoecdysones such as 22-hydroxy cholesterol and 25-hydroxy cholesterol which are converted into 20-HE by the prothoracic gland of larvae (Warren et al. 2001 ) and anti JH compound uoromevalonate (Farag and Varjas, 1983) are also developmental agonists in various holometabolous insects. These compounds cause precocious metamorphosis and adults thus formed are often nonviable and malformed (Warren et al. 2001;Farag and Varjas 1983 ). It was found in this study that, in C.quinquefasciatus, exposure of larvae to environmentally relevant concentration of BPA(1 ppm) caused signi cant reduction in the duration of larval period, while preserving all the larval instars. Though a signi cant reduction in pupal weight was observed, the duration of pupal period was same as in controls. The adults emerged appeared normal, fully viable and did not exhibit any signi cant difference with respect to sanguivory and fecundity (Table 6) Accumulation of plastic wastes in sewages not only created stagnation and subsequent mosquito breeding but also facilitated a favourable environment which aggravated mosquito menace through shortening the lifecycle span. Plastic waste management itself can act as a vector control strategy through which release of mosquito developmental agonist in the ecosystem can be minimized.

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Corresponding author
Ethics of Approval (Not applicable) a. The results incorporated in this paper did not include any information made on humans or on higher animals.

b. Individual person's data is not incorporated in this
All data incorporated in the paper is based on observations made on Culex quinquefasciatus.

Competing Interests
Both authors have no competing interests with any person, Institution or with any Organization.    Values are mean + SE, p <0.05 , n = 6.
(All values except pupal life duration are signi cantly different from control) Mr-relative molecular mass.
Ion score is -10* Log(P), where P is the probability that the observed match is a random event. Individual ion scores >31 indicate identity or extensive homology (P<0.05). * 120 pupae developed in control and different doses of BPA were allowed to develop as adult mosquitoes and were provided blood meal and allowed to lay eggs. Figure 1 Electropherogram of fourth instar C.quinquefasciatus larvae reared in selected concentrations of BPA.