J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 39 http://jad.tums.ac.ir Published Online: April 27, 2019 Original Article Effect of Chlorine and Temperature on Larvicidal Activity of Cuban Bacillus thuringiensis Isolates *Aileen González-Rizo1; Camilo E Castañet2; Ariamys Companioni1; Zulema Menéndez1; Hilda Hernández3; M Magdalena-Rodríguez1; Rene Gato1 1Departamento Control de Vectores, Centro de Investigación Diagnóstico y Referencia, Instituto de Medicina Tropical ¨Pedro Kourí¨, La Habana, Cuba 2Facultad de Biología, Universidad de La Habana, La Habana, Cuba 3Departamento de Parasitología, Centro de Investigación Diagnóstico y Referencia, Instituto de Medicina Tropical ¨Pedro Kourí¨, La Habana, Cuba (Received 23 Jan 2018; accepted 8 Jan 2019) Abstract Background: The efficacy of biolarvicides may be influenced by species of mosquito, larval age and density, tem- perature, water quality, bacterial formulation, and others. The aim of this study was to evaluate the influence of tem- perature and chlorine on larvicidal activity of Bacillus thuringiensis Cuban isolates against Aedes aegypti. Methods: The influence of temperature (25, 30, 35 °C) and chlorine (2.25mg/L) on the larvicidal activity of eleven B. thuringiensis Cuban isolates (collected between 2007 and 2009) were tested under laboratory conditions following WHO protocols. Bioassay data were analyzed by Probit program. The effect of chlorine and temperature (25, 30, 35 and 40 °C) on the Cry and Cyt proteins of these isolates was determined by SDS-PAGE polyacrylamide gel electro- phoresis. Results: The pathogenicity of the isolates U81, X48 was affected at 35 °C. However, A21, A51, L910, and R89 isolates increase their entomopathogen activity at 35 °C. No differences were observed in toxicity of M29, R84, R85 and R87 isolates at different temperatures. The Cry 4, Cry 10 and Cry 11 proteins were reduced in A21, X48, R85 isolates at 35 and 40 °C. The Cyt proteins were reduced at 35 and 40 °C in A21, X48, R85, and A51 isolates. In L910 and R84 isolates, the Cyt toxin was degraded only at 40 °C. In chlorinated water, the lethal concentrations 50 and 90 in A21, A51, M29, R84, U81, and X48 isolates were increase. Conclusion: A21, A51, L910, R85, and X48 isolates have a strong larvicidal activity for the treatment of Ae. aegypti breeding’s sites exposed to high temperature and chlorine. Keywords: Mosquitoes; Biological control; Bacillus thuringiensis; Bioassays; Chlorine Introduction Mosquitos from the genus Aedes, specif- ically Ae. aegypti (Linnaeus) and Ae. albopictus (Skuse) are responsible to the arbovirus trans- mission and are closely related to urbanized en- vironments. For this reason, this genus has be- come the main vector of arboviruses in the world (1). Vector control is the best way to re- duce the arbovirus’s incidence. The Ae. aegypti control should be directed to the elimination of immature and adults’ stages near to the urban- izing sites (2). For many years, Bacillus thuringiensis for- mulations have been the most important bi- opesticide used for agriculture and health (3). They have the advantages of specificity, high efficiency and environmentally safe (4). Bacillus thuringiensis is a gram-positive sporulated bacterium. This bacterium in limited nutrient conditions sporulates and produces parasporal crystals with natural insecticidal properties. These crystals showed specific tox- icity against invertebrates of the orders: Lep- idoptera, Diptera, Coleoptera, some nematodes, mites, and protozoa (5). *Corresponding author: Dr Aileen González-Rizo, E-mail: aileen@ipk.sld.cu, ailen@infomed.sld.cu mailto:ailen@infomed.sld.cu J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 40 http://jad.tums.ac.ir Published Online: April 27, 2019 Thousands of B. thuringiensis strains have shown considerable variability in their insec- ticidal toxicity. However, only highly toxic strains are used for biopesticide production (3). Different factors like temperature and wa- ter quality affect the larvicidal effect of B. thu- ringiensis suspension (6, 7). This lack of resid- ual activity is due to the low stability of its Cry and Cyt toxins and the poor recycling of spores under field conditions (6, 7). Therefore the pre- sent study assessed the influence of tempera- ture and water chlorination on the larvicidal ac- tivity of eleven B. thuringiensis Cuban native isolates (8, 9) in order to select the better iso- lates to control Ae. aegypti larvae in breed- ing sites. Materials and Methods Reference Strains Bacillus thuringiensis serotype H-14: IPS 82 from the International Entomopathogenic Bacillus Centre, Institute Pasteur; Paris, France. Mosquitoes: Ae. aegypti (Rockefeller strain). Mosquitoes were reared in 18x 18x 18 inch col- lapsible cages (Bio Quip, USA) maintained at 26 ºC±0.5 ºC in 80–85% relative humidity (RH) with a photoperiod of 12: 12h (light/ dark). A continuous supply of sucrose solution was provided. Female mosquitoes were giv- en access to an anesthetized mouse and al- lowed to blood feed for 30min weekly. The larvae were fed finely powdered fish food. Bacillus thuringiensis isolates A21, A51, L95, L910, M29, R84, R85, R87, R89, U81, X48 Cuban native isolates from soil samples of Cuba collected between 2007 and 2009 (8, 9). These isolates belongs to the entomopathogenic bacteria collection from the biological control laboratory of the Trop- ical Medicine Institute, ¨Pedro Kourí¨, IPK, Cuba. Bacterial formulations The bacterial isolates and reference strain were inoculated in a fermentation medium con- sisted of sucrose (2g/L), bacteriological peptone (2g/L), yeast extract (1g/L), and inorganic salts (12.5mmol/L MgSO4, 0.05mmol/L MnSO4, 1.2mmol/L FeSO4, 1.2mM ZnSO4, 25mmol/L CaCl2), incubated at 30 °C 48–72h at 150rpm (Retomed, Cuba). The bacterial sporulation was monitored through microscope. When more than 90% of cells lysed, the sporulated broth culture was transferred to 4 °C considered the final product (FP). Concentrations were ex- pressed in mg/L (dry weight). Larvicidal efficacy Quantitative bioassays were conducted fol- lowing WHO protocol (10). Twenty-five larvae (III-IV instar) were introduced into 120mL cups with 100mL of dechlorinating water. Four rep- licates per dose were included and the exper- iments were repeated at least three times. Five concentrations of FP that cause mortalities be- tween 10% and 90% were accepted for vali- dating the bioassay. Mortality data were rec- orded after 24h of exposure and were used to calculate the lethal concentrations for 50% and 90% of exposed individuals (LC50 and LC90, respectively) through log-probit analysis (11). Abbott formula was used and the survival per- centages were corrected if necessary (12). These values were compared to those obtained for the reference strain in order to estimate the efficacy of each isolate. The means of larval mortality caused by each isolate against Ae. aegypti were calculated. A value of P< 0.05 was considered statistically significant. Once the lethal doses were calculated, the CL95/CL50 ratio was performed to determine how many times it is necessary to increase the LC50 in order to obtain higher mortality. A lower ratio is indicative of better formulation efficiency (13). Effect of temperature on larvicida activity: taking into account the temperatures average in Cuba (14). Larvicidal efficacy bioassays were performed at temperatures of 25, 30 and 35 ºC. The effect of chlorine on larvicide efficacy: J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 41 http://jad.tums.ac.ir Published Online: April 27, 2019 the water for larvicidal efficacy bioassays was treated with sodium dichloroisocyanurate re- sulting in chlorine concentration of 2.25 mg/L, pH 6.8. The WHO guidelines on drink- ing water quality recommended not exceed the value of 5mg/L for free chlorine as sodium dichloroisocyanurate (15). The bioassays per- formed in dechlorinate water at 25 °C were used as control. The effect of abiotic factors on Bacillus thu- ringiensis Cry and Cyt proteins Temperature treatment: In order to deter- mine how the temperature affected the main virulence factors of B. thuringiensis native isolates. The FP of isolates was exposed to a range of temperatures 25, 30, 35 and 40 °C for 72h. Chlorine treatment: The FP was treated with sodium dichloroisocyanurate resulting in chlorine concentration of 2.25mg/L, pH 6.0 and incubated at 25 °C for 24h. Protein profiles: After each treatment (tem- perature and chlorine), the FPs were centrifuged (10,000 rpm for 20min) and the pellets were washed twice with 1mol/L NaCl and then with distilled water. The pellet was re-suspended in 100μL of distilled water and 100 of sample buffer (500mmol/L Tris-HCl pH 6.8, 10% SDS, 4% 2-mercaptoethanol, 8% glycerol, 0.1 % bromophenol blue), and boiled at wa- ter bath for 5min (16). The protein profiles of the crystal compo- nents were determined by SDS-polyacrylamide gel electrophoresis (PAGE) (17) with 10% acrylamide separating gels. Tenμl of each sam- ple was loaded onto a gel immediately before electrophoresis. FiveμL of a molecular weight marker (Broad Range Protein Molecular Weight Markers, Promega, USA) was added to each gel. The molecular weight of each protein was calculated with GelQuant software version 2.7.0 (Bio-Imaging Systems, Israel). Statistical analysis Data from the evaluation of temperature were subjected to analysis of variance (ANO- VA) and means were separated at the 5% sig- nificance level by using the Tukey HSD post- test. A log-transformation was used to calcu- late the slope values. The analysis of chlorine data was performed by t-Student test. Ethical approval This study was carried out according to the principles expressed in the Declaration of Hel- sinki. The protocols were approved by the In- stitutional Research Ethical Committee at the Institute of Tropical Medicine ¨Pedro Kourí¨. Results Influence of temperature on the toxicity of native isolates of Bacillus thuringiensis on Ae. aegypti larvae The pathogenicity of the isolates U81 and IPS-82 were affected at 35 °C. However, the isolates A21, A51, L910, R84, R85, and R89 showed a significant improvement of their con- centration lethal 90 (CL90) at 35 °C compared to the CL90 obtained at 25 °C (P< 0.05). How- ever, only the R89 and X48 isolates improve their efficiency at high temperature. No dif- ferences were observed in toxicity of M29 and R87 isolates when bioassays were performed at different temperatures (Table 1). Effect of temperature on protein profile The stability of these proteins at different temperatures (25, 30, 35 and 40 °C) was test- ed by SDS-PAGE (Fig. 1). All four bands cor- responding with Cry and Cyt proteins were ob- served after treatment at 25 °C. After treatment at 35 and 40 °C the bands corresponding to Cry 4, Cry 10 and Cry11 proteins were ob- served reduced in A21, X48, R85 isolates. By another hand, Cyt protein reduction by temperature treatment was observed in A21, X48, R85 and A51 isolates at 35 and 40 °C. In L910 and R84 isolates, the Cyt toxin was degraded only by 40 °C treatment (Fig. 1). J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 42 http://jad.tums.ac.ir Published Online: April 27, 2019 The effect of chlorine on the larvicidal ac- tivity of native isolates Chlorine had negative effects on the larvi- cidal activity of B. thuringiensis Cuban isolates. Lethal concentrations 50 and 90 were increased significantly in A21, A51, M29, R84, U81 and X48 isolates (Table 2). However, A21 efficien- cy was improved in chlorinate water. The A51 and M29 were in the same range at both con ditions (chlorinated and dechlorinated water). There were not differences in lethal concen- trations 50 and 90 of L95 and L910 isolates. The LC90 of R89 was reducing significantly in chlorinated water (Table 2). The protein analysis by SDS-PAGE does not reveal visible reduction of the major vir- ulence factors of these isolates (Fig. 2). Table 1. Entomopathogenic activity [lethal concentration (LC50 and LC90)] and efficiency of isolates of B. thuringiensis on Aedes aegypti at 25, 30 and 35 °C Isolates and strains Variable 25 °C 30 °C 35 °C A21 LC50 (mg/L) (CL) 0.00374 (0.00329–0.00422) 0.00133 (0.00121–0.00145) 0.00070 (0.00062–0.00079)* LC90 (mg/L) (CL) 0.01278 (0.0106–0.01609) 0.00411 (0.00352–0.00508)* 0.00464 (0.0037–0.00606)* LC95 (mg/L) (CL) 0.01810 (0.01455–0.02400) 0.005704 (0.00487–0.00790)* 0.00790 (0.00603–0.01105)* Efficiency 4.84 4.29 11.2 A51 LC50 (mg/L) (CL) 0.00153 (0.00135–0.00173) 0.00070 (0.00061–0.00080)* 0.00002 (0.000001–0.00007)* LC90 (mg/L) (CL) 0.00455 (0.00374–0.00593) 0.001602 (0.00132–0.00211)* 0.00012 (0.00007–0.00030)* LC95 (mg/L) (CL) 0.00621 (0.00491–0.00855) 0.00226 (0.00202–0.03230)* 0.00035 (0.00023–0.00093)* Efficiency 4.05 3.74 13.78 L95 LC50 (mg/L) (CL) 0.09819 (0.08325–0.11700) 0.07864 (0.06237–0.10706)* 0.06140 (0.05490–0.069680)* LC90 (mg/L) (CL) 0.21750 (0.17275–0.30899) 0.38221 (0.23742–0.82760) 0.20240 (0.15830–0.28520) LC95 (mg/L) (CL) 0.2724 (0.23088–0.3247) 0.5983 (0.34174–1.5000) 0.9345 (0.83600–1.05940) Efficiency 2.77 7.6 15.22 L910 LC50 (mg/L) (CL) 0.01019 (0.00941–0.01093) 0.01010 (0.00808–0.01300) 0.00199 (0.00179–0.00219)* LC90 (mg/L) (CL) 0.01920 (0.01757–0.02185) 0.02119 (0.01577–0.03689) 0.00593 (0.00520–0.00695)* LC95 (mg/L) (CL) 0.02316 (0.0206–0.02706) 0.02614 (0.01864–0.0508) 0.00808 (0.00689–0.00989)* Efficiency 2.55 2.59 4 M29 LC50 (mg/L) (CL) 0.06569 (0.05833–0.07440) 0.05458 (0.04310–0.07440) 0.02447 (0.02111–0.03178) LC90 (mg/L) (CL) 0.13652 (0.11449–0.17510) 0.32005 (0.18700–0.83119) 0.06622 (0.05640–0.15280) LC95 (mg/L) (CL) 0.1678 (0.1368–0.2250) 0.5284 (0.2779–0.71686) 0.08746 (0.07544–0.11357) Efficiency 2.56 9.62 3.57 J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 43 http://jad.tums.ac.ir Published Online: April 27, 2019 R84 LC50 (mg/L) (CL) 0.00954 (0.00862–0.10358) 0.00293 (0.00254–0.00337) 0.00284 (0.00244–0.00339) LC90 (mg/L) (CL) 0.01913 (0.01741–0.02162) 0.00717 (0.00588–0.00958)* 0.00803 (0.00610–0.01230)* LC95 (mg/L) (CL) 0.02573 (0.0235–0.0298) 0.00924 (0.00800–0.01219) 0.01080 (0.00779–0.01794) Efficiency 2.72 3.15 3.8 R85 LC50 (mg/L) (CL) 0.00793 (0.00697–0.00901) 0.00189 (0.00153–0.00220) 0.00107 (0.00091–0.00126) LC90 (mg/L) (CL) 0.01700 (0.01478–0.02176) 0.00496 (0.00410–0.00665)* 0.00507 (0.00359–0.00855)* LC95 (mg/L) (CL) 0.02170 (0.01792–0.02847) 0.00652 (0.00516–0.00959)* 0.00788 (0.00518–0.01498)* Efficiency 2.77 3.45 7.37 R87 LC50 (mg/L) (CL) 0.02897 (0.02616–0.03232) 0.00565 (0.00500–0.00643) 0.00253 (0.00199–0.00325) LC90 (mg/L) (CL) 0.07831 (0.06713–0.09530) 0.0105 (0.00886–0.01351) 0.01242 (0.00821–0.02462) LC95 (mg/L) (CL) 0.10400 (0.0866–0.13102) 0.0125 (0.0109–0.0151) 0.01950 (0.01182–0.0.04533) Efficiency 3.58 2.21 7.7 R89 LC50 (mg/L) (CL) 0.07146 (0.06444–0.07903) 0.01252 (0.00527–0.01637) 0.00529 (0.00302–0.00649) LC90 (mg/L) (CL) 0.17308 (0.14962–0.20820) 0.05094 (0.03350–0.27200)* 0.01191 (0.01020–0.04760)* LC95 (mg/L) (CL) 0.2223 (0.1873–0.27823) 0.07584 (0.043416– 0.78746)* 0.01499 (0.00799–0.09271)* Efficiency 3.14 6.06 2.83 U81 LC50 (mg/L) (CL) 0.00461 (0.00424–0.00491) 0.00163 (0.00145–0.00181)* 0.00466 (0.00346–0.00683) LC90 (mg/L) (CL) 0.00905 (0.00810–0.01043) 0.00332 (0.00285–0.00409)* 0.04605 (0.02259–0.15460)* LC95 (mg/L) (CL) 0.01095 (0.0096v0.01304) 0.00406 (0.00339–0.00523)* 0.08895 (0.03814–0.3774)* Efficiency 2.37 2.48 19.08 X48 LC50 (mg/L) (CL) 0.00213 (0.00192–0.00236) 0.00079 (0.00060–0.00090)* 0.00222 (0.00200–0.00248) LC90 (mg/L) (CL) 0.00527 (0.00450–0.00643) 0.00094 (0.00082–0.00125)* 0.00585 (0.00486–0.00746) LC95 (mg/L) (CL) 0.00945 (0.00825–0.01127) 0.00108 (0.009212– 0.01576)* 0.007677 (0.00619–0.01028) Efficiency 4.44 1.36 3.46 IPS-82 LC50 (mg/L) (CL) 0.00224 (0.00170–0.00273) 0.00067 (0.00061–0.00073)* 0.00567 (0.00490–0.00680)* LC90 (mg/L) (CL) 0.00567 (0.00457–0.00749) 0.00177 (0.00157–0.00207)* 0.02417 (0.01740–0.03865)* LC95 (mg/L) (CL) 0.00892 (0.00731–0.01174) 0.002128 (0.001805–0.00262) 0.0364 (0.0246–0.06371)* Efficiency 3.3 3.17 6.41 *P≤ 0.05 CL: 95% confidence limits Efficiency: LC95/LC50 Table 1. Continued … J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 44 http://jad.tums.ac.ir Published Online: April 27, 2019 Table 2. Entomopathogenic activity (Lethal Concentration (LC) 50 and 90) and efficiency of isolates of Bacillus thuringiensis on Aedes aegypti in dechlorinated water and chlorinated water Isolates and strains Variable Dechlorinate water 25 °C Chlorinate water 25 °C A21 LC50 (mg/L) (CL) 0.00374 (0.00329–0.00422) 0.02342 (0.02052–0.02707)* LC90 (mg/L) (CL) 0.01278 (0.0106–0.01609) 0.05545 (0.04470–0.07637)* LC95 (mg/L) (CL) 0.01810 (0.01455–0.02400) 0.0689 (0.05568–0.10375)* Efficiency 4.84 2.79 A51 LC50 (mg/L) (CL) 0.00153 (0.00135–0.00173) 0.01132 (0.00901–0.01404)* LC90 (mg/L) (CL) 0.00455 (0.00374–0.00593) 0.04702 (0.03150–0.10350)* LC95 (mg/L) (CL) 0.00621 (0.00491–0.00855) 0.04700 (0.03730–0.05800)* Efficiency 4.05 4.14 L95 LC50 (mg/L) (CL) 0.09819 (0.08325–0.11700) 0.12627 (0.11321–0.14025) LC90 (mg/L) (CL) 0.21750 (0.17275–0.30899) 0.23140 (0.19980–0.28970) LC95 (mg/L) (CL) 0.2724 (0.23088–0.3247) 0.27534 (0.23099–0.3615) Efficiency 2.77 2.14 L910 LC50 (mg/L) (CL) 0.01019 (0.00941–0.01093) 0.01730 (0.01460–0.02122) LC90 (mg/L) (CL) 0.01920 (0.01757–0.02185) 0.03460 (0.02680–0.05490) LC95 (mg/L) (CL) 0.02316 (0.0206–0.02706) 0.04157 (0.01849–0.24528) Efficiency 2.55 2.42 M29 LC50 (mg/L) (CL) 0.06569 (0.05833–0.07440) 0.10393 (0.09322–0.11521)* LC90 (mg/L) (CL) 0.13652 (0.11449–0.17510) 0.21480 (0.18660–0.25910)* LC95 (mg/L) (CL) 0.1678 (0.1368–0.2250) 0.2639 (0.2237–0.33110)* Efficiency 2.56 2.53 R84 LC50 (mg/L) (CL) 0.00954 (0.00862–0.10358) 0.03412 (0.02797–0.04334)* LC90 (mg/L) (CL) 0.01913 (0.01741–0.02162) 0.14650 (0.09805–0.28373)* LC95 (mg/L) (CL) 0.02573 (0.0235–0.0298) 0.16262 (0.14396–0.22360)* Efficiency 2.72 5.16 R85 LC50 (mg/L) (CL) 0.00793 (0.00697–0.00901) 0.01244 (0.01097–0.01425) LC90 (mg/L) (CL) 0.01700 (0.01478–0.02176) 0.02232 (0.01925–0.03167)* LC95 (mg/L) (CL) 0.02170 (0.01792–0.02847) 0.02772 (0.02223–0.04034)* Efficiency 2.77 2.15 R87 LC50 (mg/L) (CL) 0.02897 (0.02616–0.03232) 0.03800 (0.03280–0.04600) LC90 (mg/L) (CL) 0.07831 (0.06713–0.09530) 0.10720 (0.08040–0.16730) LC95 (mg/L) (CL) 0.10400 (0.0866–0.13102) 0.14250 (0.12460–0.18640) Efficiency 3.58 3.75 R89 LC50 (mg/L) (CL) 0.07146 (0.06444–0.07903) 0.04899 (0.04208–0.05907) LC90 (mg/L) (CL) 0.17308 (0.14962–0.20820) 0.13740 (0.10310–0.21440)* LC95 (mg/L) (CL) 0.2223 (0.1873–0.27823) 0.1841 (0.1313–0.31279) Efficiency 3.14 3.76 U81 LC50 (mg/L) (CL) 0.00461 (0.00424–0.00491) 0.01682 (0.01250–0.03050)* LC90 (mg/L) (CL) 0.00905 (0.00810–0.01043) 0.06720 (0.03501–0.03630)* LC95 (mg/L) (CL) 0.01095 (0.0096–0.01304) 0.0996 (0.04610–0.74310)* Efficiency 2.37 5.9 X48 LC50 (mg/L) (CL) 0.00213 (0.00192–0.00236) 0.01140 (0.01140–0.01860)* LC90 (mg/L) (CL) 0.00527 (0.00450–0.00643) 0.05950 (0.03850–0.12500)* LC95 (mg/L) (CL) 0.00945 (0.00825–0.01127) 0.07182 (0.03585–0.19502)* Efficiency 4.44 6.3 IPS-82 LC50 (mg/L) (CL) 0.00224 (0.00170–0.00273) 0.01803 (0.01440–0.02332)* LC90 (mg/L) (CL) 0.00567 (0.00457–0.00749) 0.07023 (0.04757–0.13180)* LC95 (mg/L) (CL) 0.00892 (0.00731–0.01174) 0.12100 (0.08250–0.23779)* Efficiency 3.3 6.73 *P≤ 0.05 CL: 95% confidence limits Efficiency: LC95/LC50 J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 45 http://jad.tums.ac.ir Published Online: April 27, 2019 Fig. 1. Analysis of Cry and Cyt protein of A51, A21, R85 and R84 isolates by SDS-PAGE (Coomassie brilliant blue-stained gel) Lane MM: Molecular weight Marker, Lanes-1: Proteins profiles of FP treated at 25 °C, Lanes-2: Proteins profiles of FP treated at 30 °C, Lanes-3: Proteins profiles of FP treated at 35 °C, Lanes-4: Proteins profiles of FP treated at 40 °C Fig. 2. Analysis of Cry and Cyt protein by SDS- PAGE (Coomassie brilliant blue-stained gel) Lane- MM: Molecular weight Marker, and Lanes-1: Pro- teins profiles of FP without chlorine, Lanes-2: FP exposed to chlorine (2.25mg/L, pH 6.0) Discussion Bacillus thuringiensis is a viable alternative for insect control due to its specific toxicity against insect larvae. Nevertheless, parasporal crystals activity can be affected by abiotic fac- tors, such as high temperature (18). In Cuba, the annual temperature average has increased since 1951, and in 1997 and 1998 reached the highest values throughout its his- tory. Overall, the temperature average of the years after 2000 was the warmest of all avail- able climate records (14, 19) increased in dry season, +2.0 °C, and in Jun–Aug: +0.8 °C with higher percentage of days with maximum tem- perature ≥ 30 °C (20). Climate variability influences vector popu- lation dynamics, distribution and disease trans- mission (21). Dengue transmission is associat- ed in space and time with local climate effects on survival of its vector Ae. aegypti. Thus, more rain and higher temperature generates more transmission (22, 23). Obviously, the biolar- J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 46 http://jad.tums.ac.ir Published Online: April 27, 2019 vicides should keep high entomopathogen ac- tivity at temperatures values over annual av- erage. Most of our isolates increased their toxicity when temperature raise from 25 to 30 °C and five isolates from 30 to 35 °C. Similar results were obtained with B. thuringiensis var. is- raelensis on Chironomus kiiensis larvae, re- sults showed changes of LC50 doses with a tem- peratures variation from 15 ºC to 30 ºC (24). However, no detected differences in larvicidal activity of B. thuringiensis (Vectobac AS12, ABG-6164, and AC-513695) against Culex quinquesfasciatus with water temperature var- iations from 15 to 30 °C. The reduction in LC90 in the A21, A51, L910, R84, R85, R89, U81, and X48 isolates, when the temperature increase from 25 to 30 °C may be due to the influence of this factor on the Ae. aegypti larvae behavior. The growth of larvae is accelerated with temperature raise, so they begin to feed faster and more toxins Cry and Cyt are ingested (24, 26). In previous studies, Cry4, Cry10, Cry11 and Cyt proteins were established as the prin- cipal virulence factors of Cuban native B. thu- ringiensis isolates (8, 9). Nevertheless we as- sociated the reduction of the Cry and Cyt tox- ins under then effect of temperature with lack of toxicity of U81 isolate (25–35 °C) and worse efficiency of A21 and A51 isolates. Taking in- to account that Cyt proteins potentiate the ac- tion of Cry (7), the loss of these proteins may be the cause of the significant reduction in the toxicity and efficiency. We also demonstrated reduction of Cry and Cyt proteins at 40 °C but no bioassays were performed. We speculate that in natural breeding sites the toxicity of biolarvicides fail by the loss of these proteins at temperatures over 40 °C. This result should be taken into account for the preservation of B. thuringiensis aqueous formulations. The influence of temperature over Cry pro- teins has been demonstrated by different au- thors. The Cry1Ac protein was degraded by ex- posure to temperature of 35 °C (27). A reduc- tion in Cry protein after 50 °C treatment was demonstrated by SDS-PAGE. They achieved temperature protection of the internal crystals by micro-capsulation (18). According to our results the isolates, A21, A51, L910, R84, R85 and X48 keep accepta- ble larvicidal activity at high temperatures, so they would be excellent candidates for the de- velopment of formulations better adapted to the climate change effects. The variability of temperature average, in- crease the incidence of water and foodborne diseases (20). Chlorine is the most used do- mestic water disinfectant in the world to pre- vent these diseases (28), and the mainly breed- ing sites of Ae. aegypti are domestic water con- tainers (29). Therefore, an ideal biolarvicide should keep its activity in chlorinated water. A biologist from the Cuban Vector Con- trol Programme in Matanzas and Santiago de Cuba provinces referred to the reduction of op- erational effectiveness of biolarvicides based in B. thuringiensis in chlorinated water (per- sonal communications). In our study, the bioassays showed a sig- nificantly increase in CL50 in chlorinated wa- ter but there are not visible reduction of Cry and Cyt toxins by treatment with chlorine in SDS-PAGE analysis. Chlorine is a non-selec- tive oxidant with a number of effects over the living systems: reacts with a variety of cellu- lar components (30), deactivates enzymatic ac- tive sites, decreases the biological functions of proteins and produces deleterious effects on DNA (31). In addition chlorinated waters af- fect the permeability of the cytoplasmic mem- brane (32), which could lead to cell death and inhibit the growth of B. thuringiensis (33). Different layers of proteinaceous present in the spore act as a protection from chemical attacks, including oxidizing agents like hydro- gen peroxide, sodium hypochlorite, chlorine dioxide, or ozone (34). In case of the chlorine- releasing products produce the lost of refrac- tivity, separation of the spore coats from the cortex, extensive discharge of Ca+, dipicolinic J Arthropod-Borne Dis, March 2019, 13(1): 39–49 A González-Rizo et al.: Effect of Chlorine … 47 http://jad.tums.ac.ir Published Online: April 27, 2019 acid, and DNA and finally lysis occurred (35). However, B. thuringiensis subs israelensis spores are more resistant to chlorine than other spores of either B. anthracis or B. cereus (33). It may be that B. thuringiensis spores resistance to the chlorine effect allows the larvicidal ef- fect in our native isolates when the doses were increases and explain why there were not a significant dose variation in a L95, L910 iso- lates and the increase of the toxicity 90 in R89 isolate. In spite of the significantly doses variations under chlorine treatment, A21, A51, L910, R85 and X48 isolates had a better larvicidal activ- ity than IPS 82 control strain. For this reason these isolates will be good candidate for Ae. aegypti breeding site treatment. Our results have shown the possibility to treat the chlo- rinated Ae. aegypti breeding sites with B. thu- ringiensis based products. Conclusion Our results showed that A21, A51, L910, R85, and X48 isolates have a strong larvicidal activity at 25, 30, 35 °C and chlorinated water. The use of these isolates as biolarvicides can reduce the operational problems in Ae. aegypti breeding´s sites. Acknowledgements We are thankful to our colleagues Jorge Anaya and Israel Garcia who provided exper- tise in the maintened and breeding of Ae. ae- gypti (Rockefeller strain) in the laboratory con- ditions. The authors would like to thank to the Pub- lic Cuban Ministry of Health (MINSAP) for its supports to this research as a task of the project 1701041. The authors declare that there is no conflict of interest. References 1. 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