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Lack of Resistance in Aedes vexans Field Populations After 36 Years of Bacillus thuringiensis subsp. Israelensis Applications in the Upper Rhine Valley, Germany

Lack of Resistance in Aedes vexans Field Populations After 36 Years of Bacillus thuringiensis... Downloaded from http://meridian.allenpress.com/jamca/article-pdf/34/2/154/1816233/17-6694_1.pdf by guest on 23 October 2021 Journal of the American Mosquito Control Association, 34(2):154–157, 2018 Copyright  2018 by The American Mosquito Control Association, Inc. SCIENTIFIC NOTE LACK OF RESISTANCE IN AEDES VEXANS FIELD POPULATIONS AFTER 36 YEARS OF BACILLUS THURINGIENSIS SUBSP. ISRAELENSIS APPLICATIONS IN THE UPPER RHINE VALLEY, GERMANY 1,2 1 3 NORBERT BECKER, MARIO LUDWIG AND TIANYUN SU ABSTRACT. Bacillus thuringiensis subsp. israelensis (Bti) has been widely and solely used against floodwater mosquitoes, mostly Aedes vexans, for 36 years in the Upper Rhine Valley by the German Mosquito Control Association. During this period, almost 5,000 tons of Bti formulations were applied to an area of approximately 400,000 ha. To investigate a possible resistance development after such a long-term and widespread application of Bti, the susceptibility of Ae. vexans larvae to Bti in 3 untreated (Lake Constance) and 6 treated areas on both sides of the Rhine within the Upper Rhine Valley was assessed by bioassays following World Health Organization guidelines. Comparing log-probit analyses, it was shown that neither the median lethal concentration (LC values) nor slopes of the probit lines of bioassays of the larvae deriving from treated and untreated areas showed significant differences. These results have been confirmed by resistance ratios, which varied from 0.80 to 1.12 in all tests. The results provided the evidence that no restistance in the target species Ae. vexans has developed in the areas of the Upper Rhine Valley, despite the large-scale use of Bti for 36 years. KEY WORDS Aedes vexans, bioassay, Bti, resistance, Upper Rhine valley In the Upper Rhine Valley, floodwater mosquitoes Usually two thirds of the total area were treated by play an important role as nuisance species and can helicopters. From 1982 to 1997, aerial application was significantly reduce the quality of life of the implemented with tailor-made Bti sand granules: 50 kg residents. The most abundant species is Aedes vexans of fire-dried quartz sand (1–2 mm diam) were mixed (Meigen), which frequently makes up more than 90% for 5 min with 1.4 liters of vegetable oil and 1.8 kg of of the mosquito population during the summer Bti powder (VectoBac TP, 5,000 ITU/mg), covering an (Becker and Ludwig 1983, Becker 1997). In response area of about 2 ha. Overall, 1,100 tons of sand granules to this nuisance, 100 communities on both sides of were applied over 44,111 ha. In 1998, Bti sand granules the Rhine River merged their common interest into a were replaced by Bti ice granules (Becker 2003). The united mosquito control program, the German production of ice granules took place by means of fluid Mosquito Control Association (Kommunale Aktions- nitrogen: 1,000 liters of water were mixed with 40 kg of gemelnschaft zur Bekampfung ¨ der Stechmuckenp- ¨ VectoBac WDG (3,000 ITU/mg) and transformed into lage-KABS) in 1976. In an integrated control icy pearls. From 1998 to 2016, 221,476 ha were treated program, at the beginning a lecithin surface layer with 3,811 tons of Bti ice granules. (Liparol) was used along with water management. Ground application within the Upper Rhine Valley Since 1981 formulations of Bacillus thuringiensis took place by means of knapsack sprayers. For the subsp. israelensis (Bti) were solely used against treatment of 1 ha, 500 g of VectoBac WDG, or 1 liter floodwater mosquitoes. All Bti formulations were of VectoBac 12AS, were mixed with 10 or 9 liters of sterilized by gamma radiation of 25 kGy and water (resulting in 10 liters of suspension per contained no viable spores or cells of Bti (Becker knapsack sprayer) and applied by about 300 field 2002). From 1981 to 2016, 4,988 tons of Bti workers. Between 1981 and 2016, about 145,687 ha formulations (4,911 tons of granules and 77 tons of were treated by ground application, using 77.4 tons powder and fluid concentrates) were successfully of powder/fluid formulations. The overall number of applied in multiple treatments (average 5) to a total rounds of treatments during the 36-year period was of more than 400,000 ha, resulting in at least 90% 189. Thus 189 populations (generations) of Ae. reduction of the mosquito populations in a treated vexans have been subjected to selection pressure of area of approximately 60,000 ha year after year. Bti. Treatments were usually necessary when egg horizons of the floodwater mosquitoes were flooded German Mosquito Control Association (KABS), (Becker 1989). The horizons corresponded to the Georg-Peter-Su¨ß-Strasse 3, 67346 Speyer, Germany. 2 middle water level and above of the Rhine River. The University of Heidelberg, Center for Organismal number of treatments varied from year to year owing Studies (COS), Im Neuenheimer Feld 230, 69120 to fluctuations in the Rhine water level. Mass Heidelberg, Germany. hatching of floodwater mosquitoes peaked between West Valley Mosquito and Vector Control District, 1295 E Locust Street, Ontario, CA 91761. April and September. On average, there were 1 to 11 154 Downloaded from http://meridian.allenpress.com/jamca/article-pdf/34/2/154/1816233/17-6694_1.pdf by guest on 23 October 2021 JUNE 2018 SCIENTIFIC NOTE 155 floods per year, with an average of 5 floods per summer during the 36 years of Bti applications. One of the main threats for an effective mosquito control is the ability of the target organisms to develop resistance to most control agents used. To address this concern, after 10 years of treatment with Bti within the Upper Rhine Valley, the KABS conducted its first investigation in 1991 to detemine if resistance had developed under constant selection pressure of Bti treatments (Becker and Ludwig 1993). This study was based on a comparison of the susceptibiliy of Ae. vexans populations obtained from selected untreated areas (Lake Constance) and treated areas (Upper Rhine Valley), which are about 300 km apart (Becker and Ludwig 1993). A similar study was repeated every third year (Ludwig and Becker 1997, Ludwig and Becker 2000, Ludwig and Becker 2005) to monitor mosquito resistance to Bti. To compare the susceptibility of Ae. vexans larvae from different field populations, soil samples con- taining eggs of Ae. vexans were collected in the following sites: 1) untreated sites, namely, 3 separate sites on the shore of Lake Constance, and 2) 6 treated areas under certain selection pressure for 36 years of Bti applications within the Upper Rhine Valley. Hereby, 3 areas were chosen on the left bank of the Rhine River and 3 sites on the right bank (Fig. 1). Within the larval habitats, 1 m soil samples from the upper layer (approximately 1 cm deep) were removed Fig. 1. Sampling sites of soil containing eggs of Aedes with a trowel and brought to the laboratory. In total, 9 vexans in Bti untreated (1–3) and treated (4–9) areas. samples were collected from each of the 3 untreated and 6 treated sites. The samples were kept for 14 Abbott’s formula (Abbott 1925), if needed. The days at 258C to ensure conditioning of the eggs results were subjected to log-probit analysis (Finney (Becker and Ludwig 1981, Becker 1989). After this 1971, Raymond 1985), and data means were period the soil samples were flooded in plastic compared using the Duncan’s multiple range test vessels (403 403 20 cm) with a water layer up to 20 and Student’s t-test (Kohler ¨ et al. 1984). cm above the soil. The hatched larvae were The median lethal concentration (LC values) and determined under a stereomicroscope, reared at slopes of concentration-response lines conducted 258C, and fed with fish food (Tetramin, Tetra GmbH, with larvae from from 3 indvidual untreated control Melle, Germany). sites and 6 treated sites showed no significant All bioassays were conducted with late third instars differences (Table 1). Furthermore, there were no according to World Health Organization guidelines significant differences between average LC and (WHO 1981), with slight modifications to meet the slope values of untreated sites and treated sites specific needs of this study. To prepare a stock (Table 1), and the average LC followed the same solution, 50 mg of gamma-irradiated Bti (VectoBac trend. The resistance ratios (RRs) of the 6 samples WDG, Valent BioSciences, Libertyville, IL) were from the treated sites as compared with the average added to 10 ml of destilled water and homogenized in susceptibility of the samples from the untreated sites a mixing machine (IKA Combimag REO) at 700 rpm ranged from 0.80 to 1.12 with an average of 0.97, for 10 min, then homogenized in an ultrasonic bath below the cutoff RR of 3, and showed that no (Branson Instruments, St. Louis, MO) for 15 min. One resistance developed in the treated sites (Table 1). milliliter of the homogenized solution was added to 99 The development of resistance is a major problem ml of destilled water. Depending on the concentration, in controlling insects with chemical and biological a range of 15 to 1,500 ll of homogenized, diluted insecticides (Mallet 1989, Su 2016). In contrast, the suspension was added to 200 ml plastic cups, which possibility of rapid development of resistance against had been previously filled with 148 ml of distilled Bti seems to be unlikely because of the complex mode water. To each cup, 25 larvae of Ae. vexans were of action of numerous synergistic endotoxins. Never- added in 2 ml of water. Tests were run at 6 different theless, a very low level of resistance against Bti can concentrations with controls and replicated 9 times per be induced in the laboratory when mosquito popula- sampling site. Larval mortility was evaluated at 24 and 48 h tions were subjected over a long period of selection postexposure. Mortality data were corrected, using pressure (Davidson 1992). For example, Vasquez- Downloaded from http://meridian.allenpress.com/jamca/article-pdf/34/2/154/1816233/17-6694_1.pdf by guest on 23 October 2021 156 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 34, NO.2 Table 1. The 48-h LC , slope, and resistance ratios for all 50 onset of resistance at various places depending on groups after 36 years of Bti applications (N ¼ 9). complexity of the combinations (Georghiou and Wirth 1 1 2 1997, Wirth et al. 2005). Similarly, the selection of Ae. Group LC (6 SD, mg/liters) Slope RR aegypti with field-persistent Bti collected from breed- Untreated areas ing sites resulted in a moderate level of resistance, as Site 1 0.04467 6 0.00426 2.78 n/a opposed to relatively high levels of resistance to Site 2 0.03850 6 0.00857 2.95 n/a individual Cry toxins. A 3.5-fold tolerance to Cry4Aa Site 3 0.04328 6 0.01593 2.42 n/a and 8-fold resistance to Cry11Aa was detected in one Average 0.04147 6 0.01090 2.71 n/a Ae. sticticus (Meigen) population (Tetreau et al. 2013). Treated areas There may be several reasons why resistance was Site 1 0.03331 6 0.00748 2.54 0.80 not found in Ae. vexans in spite of 36 years of Site 2 0.03558 6 0.00419 2.72 0.86 Site 3 0.04456 6 0.00335 3.11 1.07 extensive use to combat floodwater mosquitoes with Site 4 0.04177 6 0.00234 2.78 1.01 Bti. First is the complex mode of action of all intact Site 5 0.03952 6 0.00705 2.62 0.88 toxins. It is assumed that the lethal changes within the Site 6 0.04660 6 0.00194 3.11 1.12 cells are produced by the synergstic effects of the 4 Average 0.04022 6 0.00678 2.82 0.97 different proteins of the parasporal body, particularly Values under the LC levels and the slope values are not the spore-making toxin Cyt1A (Federici et al. 1990). significantly different among the replicates, averages, or areas (P With only protein crystals as in Btkurstaki (Tabashnik 0.05). 2 et al. 1990) or binary toxins in Lysinibacillus Resistance ratio at LC ¼ Value of individual treated sites/value sphaericus (Sinegre et al. 1994), the chance for a of the average untreated sites. faster development of resistance seems to be much higher (Su 2015). Second, the coevolution between Garcia (1983) treated laboratory populations of Culex the target insects and the mosquitocidal bacilli for quinquefasciatus Say with Bti at varying levels of more than 300 million years results in an effective selection over 32 generations and found a 5–7-fold toxin composition. It can be assumed that whenever decrease in susceptibility. The resistance phenomenon the insects started to develop resistance against the almost completely disappeared after a period of 3 toxin, the bacilli changed the composition of their generations without selection pressure. Goldman et al. toxins to keep the toxicity against the target organims. (1986) found a 2-fold increase in the resisance ratios The ecological advantage of the soil bacterium Bti, in only 1 out of 3 populations of Aedes aegypti (L.) namely, to kill the target, is that the insect cadaver can exposed to Bti pressure (LC values) for 14 serve ‘‘as a small fermenter’’ for the reproduction of generations. Similarly, Gharib and Szalay-Marzso the bacillus (Becker et al. 1995). Thus the insecticidal (1986) reported a 1.9-fold increase in LC values bacteria can better multiply in the soil, which is under Bti pressure for 25 generations. Georghiou et al. usually poor in nutrients. Third, variable gene pools (1983) found an 11-fold decrease of the Cx. exist within target populations. Aedes vexans is a quinquefasciatus susceptibility to Bti after 32 gener- strong flyer that facilitates the gene flow between ations, in response to a higher selection pressure untreated and treated areas, delaying the development (LC ). On the other hand, no or only very low of resistance. Maybe untreated areas serve as refugee resistance was detected in field population mosquitoes sites for the conservation of the gene pool. The treated with Bti (Liu et al. 2004, Akiner et al. 2009, phased eclosion of Ae. vexans produces generations Vasquez et al. 2009, Loke et al. 2010). In contrast to that are not homogenous, which leads to an increase the foregoing reports, Paul et al. (2005) found in diversity of the gene pool within the populations significant levels of resistance in a field population (Becker 1989). Last, the rather short exposure period of Cx. pipiens (L.) in an urban area in Syracuse, NY, of the toxins and quick kill may also be attributable to treated for some years with Bti formulations. lack of resistance development. The confrontation The insecticidal effect of Bti strains against some between the toxins and the target organisms takes members of nematoceran insects, especially mosqui- place only for a short time after field application. toes and blackfly larvae, emanates from the parasporal In summary, Bti has the unique story of success in body (crystal protein), which contains 4 major peptides combating mosquitoes in Germany and many other of different molecular weights, referred to as Cry4Aa, countries worldwide. It offers quick kill, high efficacy, Cry4Ba, Cry11Aa, and CRY11Aa (Federici et al. and safety to the nontargets, the environment, and the 1990, Wirth et al. 2005). A fifth toxin, called the applicators. Most strikingly, no resistance has devel- Cyt1Aa protein, is the principal factor for delaying the oped after 36 years of massive application. It is evolution and expression of resistance to mosquitocidal anticipated that Bti will continue to play an important Cry proteins (Wirth et al. 2005). It is the synergism of role in mosquito control for decades to come. the Cry proteins and the CytA proteins that results in We thank the employees of the KABS, who have the high toxicity against mosquito and black fly larvae. checked and treated the mosquito breeding habitats Each single Cry toxin is mosquitocidal, but none is as and conducted hard physical work, regardless of toxic as the intact combination of all toxins. Selections weekends or holidays. We are grateful to the with single purified toxins or combinations of less Gesellschaft zur Fo¨rderung der Stechmuc ¨ ken- toxin in the absence of Cyt1A toxin resulted in the bekampfung ¨ for financing the study. Downloaded from http://meridian.allenpress.com/jamca/article-pdf/34/2/154/1816233/17-6694_1.pdf by guest on 23 October 2021 JUNE 2018 SCIENTIFIC NOTE 157 REFERENCES CITED and Florida strains of Culex quinquefasciatus. J Med Entomol 41:408–413. Abbott WS. 1925. A method of computing the effectiveness Loke SR, Andy-Tan WA, Benjamin S, Lee HL, Sofian- of an insecticide. J Econ Entomol 18:265–267. Azirun M. 2010. Susceptibility of field-collected Aedes Akiner MM, Simsek FM, Caglar SS. 2009. Insecticide aegypti (L.) (Diptera: Culicidae) to Bacillus thuringien- resistance of Culex pipiens (Diptera: Culicidae) in sis israelensis and temephos. Trop Biomed 27:493–503. Turkey. J Pestic Sci. 34:259–264. Ludwig M, Becker N. 1997. Untersuchungen zum Auf- Becker N. 1989. Life strategies of mosquitoes as an treten von moglichen ¨ Resistenzerscheinungen bei Aedes adaptation to their habitats. Bull Soc Vector Ecol 14:6– vexans (Diptera, Culicinae) nach 15 Jahren Applikation von Bacillus thuringiensis israelensis. Zool Beitrage N F Becker N. 1997. Microbial control of mosquitoes: man- 38:167–174. agement of the Upper Rhine mosquito population as a Ludwig M, Becker N. 2000. Stechmuckenbek ¨ ampfung im model programme. Parasitol Today 13:485–487. Oberrheingebiet. Der prakt Schadlingsbek ¨ 12:20–22. Becker N. 2002. Sterilization of Bacillus thuringiensis Ludwig M, Becker N. 2005. Stechmuckenbek ¨ ampfung ¨ am israelensis products by gamma radiation. J Am Mosq Oberrhein. Gibt es Resistenzen nach 20 Jahren B.t.i.? Control Assoc 18:57–62. Der prakt Schadlingsbek ¨ 1:15–17. Becker N. 2003. Ice granules containing endotoxins of Mallet J. 1989.The evolution of insecticide resistance: have microbial control agents for the control of mosquito the insects won? 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In: Abstract resistance in Aedes vexans field populations after a 10- volume of the VIII European Meeting of Society for year application of Bacillus thuringiensis israelensis. J Vector Ecology. 1994 September 5–8; Barcelona, Spain. Am Mosq Control Assoc 9:221–224. p17. Becker N, Zgomba M, Petric´ D, Beck M, Ludwig M. 1995. Su T. 2016. Resistance and its management to microbial Role of larval cadavers in recycling processes of Bacillus and insect growth regulator larvicides in mosquitoes. In: sphaericus. J Am Mosq Control Assoc 11:329–334. Trdan S, ed. Insecticides resistance. Rijeka, Croatia: Davidson EW. 1992. Development of insect resistance to InTech Europe. p 135–154. biopesticides. Pesq Agropec Brasil 27:47–57. Tabashnik BE, Cushing NL, Finson N, Johnson MW. 1990. Federici BA, Luthy ¨ P, Ibarra J. 1990. Parasporal Body of Development of resistance to Bacillus thuringiensis in Bacillus thuringiensis israelensis. 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Ann Rpt populations in Cyprus to conventional organic insecti- Mosq Control Res, University of California: 86–90. cides, Bacillus thuringiensis subsp. israelensis,and Georghiou GP, Wirth MC. 1997. Influence of exposure to methoprene. J Med Entomol 46:881–887. single versus multiple toxins of Bacillus thuringiensis Vasquez-Garcia M. 1983. Investigations of the potentiality subsp. israelensis on development of resistance in the of resistance to Bacillus thuringiensis ser. H-14 in Culex mosquito Culex quinquefasciatus (Diptera: Culicidae). quinquefasciatus through accelerated selection pressure Appl Environ Microbiol 63:1095–1101. in the laboratory [Ph.D. dissertation]. University of Gharib AH, Szalay-Marzso L. 1986. Selection for resis- California, Riverside, CA. tance to Bacillus thuringiensis serotype H-14 in a Wirth MC, Park HW, Walton WE, Federici BA. 2005. laboratory strain of Aedes aegypti L. In: Samson RA, Cyt1A of Bacillus thuringiensis delays evolution of Vlak JM, Peters D, eds. Fundamental and applied resistance to Cry11A in the mosquito Culex quinque- aspects of invertebrate pathology. Fourth International fasciatus. Appl Environ Microbiol 71:185–189. Colloquium of Invertebrate Pathology, Wageningen, the Netherlands. p 37. Wirth MC, Walton WE, Federici BA. 2004. Cyt1A from Goldman IF, Arnold J, Carlton BC. 1986. Selection for Bacillus thuringiensis restores toxicity of Bacillus resistance to Bacillus thuringiensis subsp. israelensis in sphaericus against resistant Culex quinquefasciatus field and laboratory populations of the mosquito Aedes (Diptera: Culicidae). J Med Entomol 37:401–407. aegypti. J Invertebr Pathol 47:317–324. WHO [World Health Organization]. 1981. Report of Kohler ¨ W, Schachtel G, Voleske P. 1984. Biometrie. informal consultation on standardization of Bacillus Berlin, Germany: Springer. thuringiensis H-14. Mimeographed documents TDR/ Liu, H, Cupp, EW, Micher, KM, Guo, A, Liu N. 2004. BCV/BTH-14/811, WHO/VBC/81-828. Geneva, Swit- Insecticide resistance and cross-resistance in Alabama zerland: WHO. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of the American Mosquito Control Association Unpaywall

Lack of Resistance in Aedes vexans Field Populations After 36 Years of Bacillus thuringiensis subsp. Israelensis Applications in the Upper Rhine Valley, Germany

Becker, Norbert; Ludwig, Mario; Su, Tianyun
Journal of the American Mosquito Control Association – Jun 1, 2018
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Downloaded from http://meridian.allenpress.com/jamca/article-pdf/34/2/154/1816233/17-6694_1.pdf by guest on 23 October 2021 Journal of the American Mosquito Control Association, 34(2):154–157, 2018 Copyright  2018 by The American Mosquito Control Association, Inc. SCIENTIFIC NOTE LACK OF RESISTANCE IN AEDES VEXANS FIELD POPULATIONS AFTER 36 YEARS OF BACILLUS THURINGIENSIS SUBSP. ISRAELENSIS APPLICATIONS IN THE UPPER RHINE VALLEY, GERMANY 1,2 1 3 NORBERT BECKER, MARIO LUDWIG AND TIANYUN SU ABSTRACT. Bacillus thuringiensis subsp. israelensis (Bti) has been widely and solely used against floodwater mosquitoes, mostly Aedes vexans, for 36 years in the Upper Rhine Valley by the German Mosquito Control Association. During this period, almost 5,000 tons of Bti formulations were applied to an area of approximately 400,000 ha. To investigate a possible resistance development after such a long-term and widespread application of Bti, the susceptibility of Ae. vexans larvae to Bti in 3 untreated (Lake Constance) and 6 treated areas on both sides of the Rhine within the Upper Rhine Valley was assessed by bioassays following World Health Organization guidelines. Comparing log-probit analyses, it was shown that neither the median lethal concentration (LC values) nor slopes of the probit lines of bioassays of the larvae deriving from treated and untreated areas showed significant differences. These results have been confirmed by resistance ratios, which varied from 0.80 to 1.12 in all tests. The results provided the evidence that no restistance in the target species Ae. vexans has developed in the areas of the Upper Rhine Valley, despite the large-scale use of Bti for 36 years. KEY WORDS Aedes vexans, bioassay, Bti, resistance, Upper Rhine valley In the Upper Rhine Valley, floodwater mosquitoes Usually two thirds of the total area were treated by play an important role as nuisance species and can helicopters. From 1982 to 1997, aerial application was significantly reduce the quality of life of the implemented with tailor-made Bti sand granules: 50 kg residents. The most abundant species is Aedes vexans of fire-dried quartz sand (1–2 mm diam) were mixed (Meigen), which frequently makes up more than 90% for 5 min with 1.4 liters of vegetable oil and 1.8 kg of of the mosquito population during the summer Bti powder (VectoBac TP, 5,000 ITU/mg), covering an (Becker and Ludwig 1983, Becker 1997). In response area of about 2 ha. Overall, 1,100 tons of sand granules to this nuisance, 100 communities on both sides of were applied over 44,111 ha. In 1998, Bti sand granules the Rhine River merged their common interest into a were replaced by Bti ice granules (Becker 2003). The united mosquito control program, the German production of ice granules took place by means of fluid Mosquito Control Association (Kommunale Aktions- nitrogen: 1,000 liters of water were mixed with 40 kg of gemelnschaft zur Bekampfung ¨ der Stechmuckenp- ¨ VectoBac WDG (3,000 ITU/mg) and transformed into lage-KABS) in 1976. In an integrated control icy pearls. From 1998 to 2016, 221,476 ha were treated program, at the beginning a lecithin surface layer with 3,811 tons of Bti ice granules. (Liparol) was used along with water management. Ground application within the Upper Rhine Valley Since 1981 formulations of Bacillus thuringiensis took place by means of knapsack sprayers. For the subsp. israelensis (Bti) were solely used against treatment of 1 ha, 500 g of VectoBac WDG, or 1 liter floodwater mosquitoes. All Bti formulations were of VectoBac 12AS, were mixed with 10 or 9 liters of sterilized by gamma radiation of 25 kGy and water (resulting in 10 liters of suspension per contained no viable spores or cells of Bti (Becker knapsack sprayer) and applied by about 300 field 2002). From 1981 to 2016, 4,988 tons of Bti workers. Between 1981 and 2016, about 145,687 ha formulations (4,911 tons of granules and 77 tons of were treated by ground application, using 77.4 tons powder and fluid concentrates) were successfully of powder/fluid formulations. The overall number of applied in multiple treatments (average 5) to a total rounds of treatments during the 36-year period was of more than 400,000 ha, resulting in at least 90% 189. Thus 189 populations (generations) of Ae. reduction of the mosquito populations in a treated vexans have been subjected to selection pressure of area of approximately 60,000 ha year after year. Bti. Treatments were usually necessary when egg horizons of the floodwater mosquitoes were flooded German Mosquito Control Association (KABS), (Becker 1989). The horizons corresponded to the Georg-Peter-Su¨ß-Strasse 3, 67346 Speyer, Germany. 2 middle water level and above of the Rhine River. The University of Heidelberg, Center for Organismal number of treatments varied from year to year owing Studies (COS), Im Neuenheimer Feld 230, 69120 to fluctuations in the Rhine water level. Mass Heidelberg, Germany. hatching of floodwater mosquitoes peaked between West Valley Mosquito and Vector Control District, 1295 E Locust Street, Ontario, CA 91761. April and September. On average, there were 1 to 11 154 Downloaded from http://meridian.allenpress.com/jamca/article-pdf/34/2/154/1816233/17-6694_1.pdf by guest on 23 October 2021 JUNE 2018 SCIENTIFIC NOTE 155 floods per year, with an average of 5 floods per summer during the 36 years of Bti applications. One of the main threats for an effective mosquito control is the ability of the target organisms to develop resistance to most control agents used. To address this concern, after 10 years of treatment with Bti within the Upper Rhine Valley, the KABS conducted its first investigation in 1991 to detemine if resistance had developed under constant selection pressure of Bti treatments (Becker and Ludwig 1993). This study was based on a comparison of the susceptibiliy of Ae. vexans populations obtained from selected untreated areas (Lake Constance) and treated areas (Upper Rhine Valley), which are about 300 km apart (Becker and Ludwig 1993). A similar study was repeated every third year (Ludwig and Becker 1997, Ludwig and Becker 2000, Ludwig and Becker 2005) to monitor mosquito resistance to Bti. To compare the susceptibility of Ae. vexans larvae from different field populations, soil samples con- taining eggs of Ae. vexans were collected in the following sites: 1) untreated sites, namely, 3 separate sites on the shore of Lake Constance, and 2) 6 treated areas under certain selection pressure for 36 years of Bti applications within the Upper Rhine Valley. Hereby, 3 areas were chosen on the left bank of the Rhine River and 3 sites on the right bank (Fig. 1). Within the larval habitats, 1 m soil samples from the upper layer (approximately 1 cm deep) were removed Fig. 1. Sampling sites of soil containing eggs of Aedes with a trowel and brought to the laboratory. In total, 9 vexans in Bti untreated (1–3) and treated (4–9) areas. samples were collected from each of the 3 untreated and 6 treated sites. The samples were kept for 14 Abbott’s formula (Abbott 1925), if needed. The days at 258C to ensure conditioning of the eggs results were subjected to log-probit analysis (Finney (Becker and Ludwig 1981, Becker 1989). After this 1971, Raymond 1985), and data means were period the soil samples were flooded in plastic compared using the Duncan’s multiple range test vessels (403 403 20 cm) with a water layer up to 20 and Student’s t-test (Kohler ¨ et al. 1984). cm above the soil. The hatched larvae were The median lethal concentration (LC values) and determined under a stereomicroscope, reared at slopes of concentration-response lines conducted 258C, and fed with fish food (Tetramin, Tetra GmbH, with larvae from from 3 indvidual untreated control Melle, Germany). sites and 6 treated sites showed no significant All bioassays were conducted with late third instars differences (Table 1). Furthermore, there were no according to World Health Organization guidelines significant differences between average LC and (WHO 1981), with slight modifications to meet the slope values of untreated sites and treated sites specific needs of this study. To prepare a stock (Table 1), and the average LC followed the same solution, 50 mg of gamma-irradiated Bti (VectoBac trend. The resistance ratios (RRs) of the 6 samples WDG, Valent BioSciences, Libertyville, IL) were from the treated sites as compared with the average added to 10 ml of destilled water and homogenized in susceptibility of the samples from the untreated sites a mixing machine (IKA Combimag REO) at 700 rpm ranged from 0.80 to 1.12 with an average of 0.97, for 10 min, then homogenized in an ultrasonic bath below the cutoff RR of 3, and showed that no (Branson Instruments, St. Louis, MO) for 15 min. One resistance developed in the treated sites (Table 1). milliliter of the homogenized solution was added to 99 The development of resistance is a major problem ml of destilled water. Depending on the concentration, in controlling insects with chemical and biological a range of 15 to 1,500 ll of homogenized, diluted insecticides (Mallet 1989, Su 2016). In contrast, the suspension was added to 200 ml plastic cups, which possibility of rapid development of resistance against had been previously filled with 148 ml of distilled Bti seems to be unlikely because of the complex mode water. To each cup, 25 larvae of Ae. vexans were of action of numerous synergistic endotoxins. Never- added in 2 ml of water. Tests were run at 6 different theless, a very low level of resistance against Bti can concentrations with controls and replicated 9 times per be induced in the laboratory when mosquito popula- sampling site. Larval mortility was evaluated at 24 and 48 h tions were subjected over a long period of selection postexposure. Mortality data were corrected, using pressure (Davidson 1992). For example, Vasquez- Downloaded from http://meridian.allenpress.com/jamca/article-pdf/34/2/154/1816233/17-6694_1.pdf by guest on 23 October 2021 156 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 34, NO.2 Table 1. The 48-h LC , slope, and resistance ratios for all 50 onset of resistance at various places depending on groups after 36 years of Bti applications (N ¼ 9). complexity of the combinations (Georghiou and Wirth 1 1 2 1997, Wirth et al. 2005). Similarly, the selection of Ae. Group LC (6 SD, mg/liters) Slope RR aegypti with field-persistent Bti collected from breed- Untreated areas ing sites resulted in a moderate level of resistance, as Site 1 0.04467 6 0.00426 2.78 n/a opposed to relatively high levels of resistance to Site 2 0.03850 6 0.00857 2.95 n/a individual Cry toxins. A 3.5-fold tolerance to Cry4Aa Site 3 0.04328 6 0.01593 2.42 n/a and 8-fold resistance to Cry11Aa was detected in one Average 0.04147 6 0.01090 2.71 n/a Ae. sticticus (Meigen) population (Tetreau et al. 2013). Treated areas There may be several reasons why resistance was Site 1 0.03331 6 0.00748 2.54 0.80 not found in Ae. vexans in spite of 36 years of Site 2 0.03558 6 0.00419 2.72 0.86 Site 3 0.04456 6 0.00335 3.11 1.07 extensive use to combat floodwater mosquitoes with Site 4 0.04177 6 0.00234 2.78 1.01 Bti. First is the complex mode of action of all intact Site 5 0.03952 6 0.00705 2.62 0.88 toxins. It is assumed that the lethal changes within the Site 6 0.04660 6 0.00194 3.11 1.12 cells are produced by the synergstic effects of the 4 Average 0.04022 6 0.00678 2.82 0.97 different proteins of the parasporal body, particularly Values under the LC levels and the slope values are not the spore-making toxin Cyt1A (Federici et al. 1990). significantly different among the replicates, averages, or areas (P With only protein crystals as in Btkurstaki (Tabashnik 0.05). 2 et al. 1990) or binary toxins in Lysinibacillus Resistance ratio at LC ¼ Value of individual treated sites/value sphaericus (Sinegre et al. 1994), the chance for a of the average untreated sites. faster development of resistance seems to be much higher (Su 2015). Second, the coevolution between Garcia (1983) treated laboratory populations of Culex the target insects and the mosquitocidal bacilli for quinquefasciatus Say with Bti at varying levels of more than 300 million years results in an effective selection over 32 generations and found a 5–7-fold toxin composition. It can be assumed that whenever decrease in susceptibility. The resistance phenomenon the insects started to develop resistance against the almost completely disappeared after a period of 3 toxin, the bacilli changed the composition of their generations without selection pressure. Goldman et al. toxins to keep the toxicity against the target organims. (1986) found a 2-fold increase in the resisance ratios The ecological advantage of the soil bacterium Bti, in only 1 out of 3 populations of Aedes aegypti (L.) namely, to kill the target, is that the insect cadaver can exposed to Bti pressure (LC values) for 14 serve ‘‘as a small fermenter’’ for the reproduction of generations. Similarly, Gharib and Szalay-Marzso the bacillus (Becker et al. 1995). Thus the insecticidal (1986) reported a 1.9-fold increase in LC values bacteria can better multiply in the soil, which is under Bti pressure for 25 generations. Georghiou et al. usually poor in nutrients. Third, variable gene pools (1983) found an 11-fold decrease of the Cx. exist within target populations. Aedes vexans is a quinquefasciatus susceptibility to Bti after 32 gener- strong flyer that facilitates the gene flow between ations, in response to a higher selection pressure untreated and treated areas, delaying the development (LC ). On the other hand, no or only very low of resistance. Maybe untreated areas serve as refugee resistance was detected in field population mosquitoes sites for the conservation of the gene pool. The treated with Bti (Liu et al. 2004, Akiner et al. 2009, phased eclosion of Ae. vexans produces generations Vasquez et al. 2009, Loke et al. 2010). In contrast to that are not homogenous, which leads to an increase the foregoing reports, Paul et al. (2005) found in diversity of the gene pool within the populations significant levels of resistance in a field population (Becker 1989). Last, the rather short exposure period of Cx. pipiens (L.) in an urban area in Syracuse, NY, of the toxins and quick kill may also be attributable to treated for some years with Bti formulations. lack of resistance development. The confrontation The insecticidal effect of Bti strains against some between the toxins and the target organisms takes members of nematoceran insects, especially mosqui- place only for a short time after field application. toes and blackfly larvae, emanates from the parasporal In summary, Bti has the unique story of success in body (crystal protein), which contains 4 major peptides combating mosquitoes in Germany and many other of different molecular weights, referred to as Cry4Aa, countries worldwide. It offers quick kill, high efficacy, Cry4Ba, Cry11Aa, and CRY11Aa (Federici et al. and safety to the nontargets, the environment, and the 1990, Wirth et al. 2005). A fifth toxin, called the applicators. Most strikingly, no resistance has devel- Cyt1Aa protein, is the principal factor for delaying the oped after 36 years of massive application. It is evolution and expression of resistance to mosquitocidal anticipated that Bti will continue to play an important Cry proteins (Wirth et al. 2005). It is the synergism of role in mosquito control for decades to come. the Cry proteins and the CytA proteins that results in We thank the employees of the KABS, who have the high toxicity against mosquito and black fly larvae. checked and treated the mosquito breeding habitats Each single Cry toxin is mosquitocidal, but none is as and conducted hard physical work, regardless of toxic as the intact combination of all toxins. Selections weekends or holidays. 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