Resistance to Lysinibacillus sphaericus and Other Commonly Used Pesticides in Culex pipiens (Diptera: Culicidae) from Chico, California

Resistance to Lysinibacillus sphaericus and Other Commonly Used Pesticides in Culex pipiens... Abstract Bacillus sphaericus Neide, recently renamed as Lysinibacillus sphaericus Meyer and Neide, is a spore-forming bacterium that possesses various levels of larvicidal activity, depending on the strains, against some mosquito species. Products based on most active strains such as 2362, 2297, 1593, C3-41 that bear binary toxins, as well as mosquitocidal toxins at various levels, have been developed to combat mosquito larvae worldwide. Resistance in wild Culex mosquito populations has been reported since 1994 from France, Brazil, India, China, Thailand, and Tunisia. Laboratory studies to evaluate resistance development risk have been conducted by many groups of scientists worldwide. Products based on L. sphaericus strain 2362 were registered in the United States in 1990s, and their use for mosquito control has been increased considerably since invasion of West Nile virus. This report documents the first occurrence of high-level resistance to L. sphaericus in a natural population of Culex pipiens L. in Chico, CA, where resistance ratio was 537.0 at LC50 and 9,048.5 at LC90 when compared with susceptible laboratory colony of the same species. Susceptibility profile to other groups of pesticides with different modes of action was also determined. Various levels of resistance or tolerance were noticed to abamectin, pyriproxyfen, permethrin, and indoxacarb. Resistance management and susceptibility monitoring strategies are discussed and recommended. Lysinibacillus sphaericus, Culex pipiens, Diptera, Culicidae, resistance, susceptibility The mosquitocidal activity of some strains of Bacillus sphaericus, recently renamed as Lysinibacillus sphaericus (Ahmed et al. 2007), was recognized as early as 1960s. To date, 49 serotypes over 300 strains of L. sphaericus have been identified, among which 9 serotypes of 16 strains showed various levels of activity against mosquito larvae. Strains that possess high mosquitocidal activity are 2362, 1597, 2297, C3-41, and IAB-59. The mostly studied and developed strain, 2362, was isolated in 1984 from adult blackfly Simulium damnosum Theobald (Diptera: Simuliidae) in Nigeria (Su 2016a). Active strains produce parasporal inclusions during sporulation, containing crystal binary toxins A and B (BinA 42 kDa and 51 kDa BinB) that play the predominant role in larvicidal toxicity. Both BinA and BinB are required for larvicidal toxicity and an equimolar ratio of both proteins yields the greatest toxicity. Some strains also produce noncrystal mosquitocidal toxins (Mtx) such as Mtx 1, 2, and 3 during vegetative stage. Mtx possess lower larvicidal toxicity than crystal binary toxins. The target site of binary toxins is located at the brush border of the gut epithelium membrane. The receptor of the BinB is a 60-kDa alpha-glucosidase, which is anchored to the mosquito midgut membrane via a glycosyl-phosphatidylinositol anchor. It is generally believed that BinB opens the pathway on the membrane while BinA causes the actual pathological consequences leading the larval mortality. Together with Bacillus thuringiensis subsp. israelensis (B.t.i.), L. sphaericus also belongs to Group 11 (microbial disruptors of insect midgut membranes and derived toxins) by Insect Resistance Action Committee (IRAC). During the past decades, numerous products have been developed using various strains and applied to control mainly Culex spp. worldwide. Strain 2362 belonging to serotype H5a5b has been well studied, developed, and commercialized in the United States. Products that are solely based on this strain such as VectoLex CG, WDG, WSP and Spheratax SPH, WSP, and ones that contain this strain such as VectoMax CG, FG, WSP and FourStar CRG, MBG, WSP, Briquets have been registered since 1990s, and widely used to combat vectors of West Nile virus (WNV) and other nuisance mosquito species in the United States. Because of the simplicity of Bin toxins in L. sphaericus, the risk of resistance development in laboratory mosquito populations seemed quite imminent, which has been well documented in Culex pipiens L. complex (Wirth 2010, Su 2016b). Beside the toxin simplicity, long-term exposure of wild mosquito populations to naturally occurring strains associated with mosquito habitats may also have contributed to the resistance development after various periods of applications in different countries such as France, Brazil, India, Thailand, China, and Tunisia since 1994 (Sinègre et al. 1994, Wirth 2010, Su 2016b). In response to a control failure of mosquito populations in Chico, CA, after application of VectoLex WDG for 1.5 yr, investigation on potential resistance was initiated. Here in this paper we reported the first occurrence of L. sphaericus resistance in wild population of Cx. pipiens in the United States where the L. sphaericus-based products have been used most extensively, particularly since the invasion of the WNV. The resistance to other pesticides of public health, urban and agriculture uses was also evaluated in this population. Materials and Methods Mosquitoes A couple hundreds of mosquito larvae, mostly second and third instar larvae were collected from a storm drain located at Laburnum Avenue and E. 6th Street, Chico, CA, on 8 April 2015, by the Butte County Mosquito and Vector Control District (5117 Larkin Road, Oroville, CA). The specimens were provided to the West Valley Mosquito and Vector Control District (1295 E. Locust Street, Ontario, CA), from which a colony of Cx. pipiens was established. The third instars from generation F4–6 were used in bioassays to decide the potential resistance to L. sphaericus and other common pesticides of public health, urban and agriculture uses. A long-term laboratory colony of the same species was introduced from the San Mateo County Mosquito and Vector Control District (1351 Rollins Road, Burlingame, CA), which was assayed concurrently with the field population derived from collection in Chico, CA. Pesticides To generate immediately relevant susceptibility data for field application, the most commonly used commercial formulations with active ingredients of interest were chosen in bioassays. The pesticides tested included one pyrethroid (permethrin); six biological pesticides based on B.t.i., L. sphaericus, spinosad, spinetoram, and avermectins; four insect growth regulators based on methoprene, pyriproxyfen, diflubenzuron, and novaluron; and one from each of organophosphate (temephos), neonicotinoid (imidacloprid), phenylpyrazoles (fipronil), and oxadiazine (indoxacarb). Detailed information about the pesticides tested including trade names, active ingredients, lot numbers, and manufacturers was provided in Table 1. Table 1. Profile of the pesticides tested against a wild population of Culex pipiens L. from Chico, CA, along with laboratory colony Category  Products  Active ingredients  Concentration (%)  Lot no.  Manufacturers  Biological pesticides  VectoLex WDG  Lysinibacillus sphaericus  51.2  188-119-PG  Valent BioSciences Corp., Libertyville, IL    VectoBac WDG  Bacillus thuringiensis israelensis (B.t.i.)  37.4  201-391-PG  Valent BioSciences Corp., Libertyville, IL    VectoMax CG  B.t.i. + L. sphaericus  4.5 + 2.7  187-575-N8  Valent BioSciences Corp., Libertyville, IL    Natular G30  Spinosad  2.5    Clarke, St. Charles, IL.    Radiant SC  Spinetoram  11.7  XG14164911  Dow AgroSciences LLC, Indianapolis, IN    Advance 375A  Abamectin B1  0.011  81040334  BASF Corp., St. Louis, MO  Insect growth regulators (IGRs)  Altosid liquid larvicide  Methoprene  5.0  60111357  Wellmark International, Schaumburg, IL    NyGuard IGR  Pyriproxyfen  10.0  BAB6111  MGK, Minneapolis, MN    Dimilin 25W  Diflubenzuron  25.0  BA9D30P001  Chemtura Corp., Middlebury, CT    Mosquiron 0.12CRD  Novaluron  0.12  1012818  Makhteshim Agan North America, Inc., Raleigh, NC  Organophosphate  Skeeter Abate  Temephos  5.0  1009280003  Clarke Mosquito Control Products, Inc., Roselle, IL  Neonicotinoid  ImidaPro 4SC  Imidacloprid  40.7  12254PO42  Agrisel USA, Inc., Suwanee, GA  Phenylpyrazoles  Taurus SC  Fipronil  9.1  23204  CSI Control Solutions, Inc., Pasadena, TX    Advion RIFA bait  Indoxacarb  0.045    DuPont  Pyrethroid  Permethrin (Technical)  Permethrin (mixture of isomers)  99.9  2601000  Chem Service, West Chester, PA  Category  Products  Active ingredients  Concentration (%)  Lot no.  Manufacturers  Biological pesticides  VectoLex WDG  Lysinibacillus sphaericus  51.2  188-119-PG  Valent BioSciences Corp., Libertyville, IL    VectoBac WDG  Bacillus thuringiensis israelensis (B.t.i.)  37.4  201-391-PG  Valent BioSciences Corp., Libertyville, IL    VectoMax CG  B.t.i. + L. sphaericus  4.5 + 2.7  187-575-N8  Valent BioSciences Corp., Libertyville, IL    Natular G30  Spinosad  2.5    Clarke, St. Charles, IL.    Radiant SC  Spinetoram  11.7  XG14164911  Dow AgroSciences LLC, Indianapolis, IN    Advance 375A  Abamectin B1  0.011  81040334  BASF Corp., St. Louis, MO  Insect growth regulators (IGRs)  Altosid liquid larvicide  Methoprene  5.0  60111357  Wellmark International, Schaumburg, IL    NyGuard IGR  Pyriproxyfen  10.0  BAB6111  MGK, Minneapolis, MN    Dimilin 25W  Diflubenzuron  25.0  BA9D30P001  Chemtura Corp., Middlebury, CT    Mosquiron 0.12CRD  Novaluron  0.12  1012818  Makhteshim Agan North America, Inc., Raleigh, NC  Organophosphate  Skeeter Abate  Temephos  5.0  1009280003  Clarke Mosquito Control Products, Inc., Roselle, IL  Neonicotinoid  ImidaPro 4SC  Imidacloprid  40.7  12254PO42  Agrisel USA, Inc., Suwanee, GA  Phenylpyrazoles  Taurus SC  Fipronil  9.1  23204  CSI Control Solutions, Inc., Pasadena, TX    Advion RIFA bait  Indoxacarb  0.045    DuPont  Pyrethroid  Permethrin (Technical)  Permethrin (mixture of isomers)  99.9  2601000  Chem Service, West Chester, PA  View Large Bioassays Cup Bioassay on Larvicides Best efforts were made to process commercial products for bioassay (Su and Cheng 2014b). Emulsifiable concentrates such as Radiant SC, Altosid liquid larvicide, NyGuard IGR, ImidaPro 4SC, and Taurus SC were suspended in tap water by gentle mixing. Small granules of Advance 375A ant bait and Advion RIFA bait or water dispersible granules of VectoBac WDG and VectoLex WDG, or wettable powders of Dimilin 25W were suspended in tap water by vigorous shaking. Large granular materials of VectoMax CG, or pellets of Skeeter Abate , were powdered in a coffee grinder (Hamilton Beach Custom Grind, Southern Pines, NC) at the maximum speed, then suspended in tap water by vortexing for 3 min. For briquet formulation such as Mosquiron 0.12CRD, fine pieces were shaved off using a razor blade and suspended in tap water by vortexing for 5 min (Vortex Mixer VX100, Labnet International, Inc., Edison, NJ). Bioassay was conducted as previously described (Su and Mulla 2004). Briefly, four to five concentrations of each test material within the concentration range resulting ~5–95% mortality were used in bioassay, with three replicates at each concentration. For larvicides based on B.t.i. and L. sphaericus, concentrations were determined by weight for the whole formulation in volume. Other bioassays were based on the concentration of weight of active ingredient of the insecticide in volume. For each replicate, 25 larvae were placed in 100 ml of tap water in a 120-ml disposable Styrofoam cup. Bioassays were conducted at 25 ± 1°C. In bioassays on B.t.i., the combination of B.t.i. and L. sphaericus, spinetoram, abamectin, temephos, imidacloprid, fipronil, and indoxacarb, late third instar larvae were used and larval mortality was recorded at 24 h post treatment. In bioassays using L. sphaericus, diflubenzuron and novaluron, early third instar larvae were preferred, and results were recorded at 48 h post treatment for L. sphaericus, and 72 h post treatment for diflubenzuron and novaluron. Moribund larvae were also considered dead. In bioassays of methoprene and pyriproxyfen, late fourth instar larvae were used, and mortality was read when all treated individuals emerged as adults or died prior to emergence. Three drops of 10% rabbit chow pellet suspension were added to each cup as larval food in all bioassays, except those using methoprene and pyriproxyfen where a small piece (~100 mg) of rabbit pellets was added to each bioassay cup to support them until pupation. Concentration-response data were analyzed using POLO-PC (LeOra Software 1987) to calculate lethal concentrations (LC) and their 95% confidence limits (CLs). For each pesticide tested, bioassays were also concurrently conducted on a reference laboratory colony of Cx. pipiens. Bottle Bioassay on Adulticide In bottle bioassay on susceptibility to permethrin, the interior of 250-ml glass bottle (Uline, Pleasant Prairie, WI) was evenly coated by 30 μg of permethrin in 1 ml of HPLC grade acetone (EMD Millipore, Temecula, CA) on an automatic roller (Fisher Scientific, Fisher Scientific, Hampton, NH) in a chemical fume hood (Hemco, Independence, MO). Bottles for untreated control were coated by acetone only. After the coated surface was completely dry in the hood, 25 of 3- to 5-d-old female mosquitoes were aspirated into each bottle. Mortality was read at 5, 10, 15, 30, 45, 60, 90, and 120 min, three replicates were made for each mosquito population. Mortality referred to individuals that did not show any movements of entire body or legs, wings, proboscis, antennae, or palpi. Bioassays were conducted at 25 ± 1°C and relative humidity 50–60%. Time mortality data were subject to probit analysis for calculations of LT50 and LT90 and their 95% CL (Throne et al. 1995). Bottle bioassays were also concurrently conducted on a reference laboratory colony of Cx. pipiens. Resistance Ratio Calculation The resistance ratios (RRs) were calculated by LC (LT)Field/LC (LT)Lab. Resistance was categorized as no resistance (RR ≤ 1), tolerance (RR = 1.1–5), low level (RR = 5.1–20), moderate level (RR = 20.1–100), and high level (RR > 100) (Su and Cheng 2014a). The significance at P < 0.05 in tolerance or resistance levels were validated by separated 95% CLs of LC (LT) levels between field population and laboratory colony. Results Moderate-to-High Resistance The collection from the field site in Chico, CA, showed significantly lower susceptibility (P < 0.05) to L. sphaericus (VectoLex WDG) when compared with the laboratory colony of the same species that was bioassayed concurrently (Table 2). The LC50 and LC90 in this field population were widely separated with a ratio of LC90/LC50 = 93.2, when compared with 2.8 in laboratory colony. This field population showed high RR of 687.4-fold at LC50 level and 22,878.6-fold at LC90 level (Table 3). The ratio of RR at LC90 and RR at LC50 (Table 3) was 33.3. Table 2. Susceptibility to Lysinibacillus sphaericus Meyer and Neide (VectoLex WDG) and other commonly used pesticides in a wild population of Culex pipiens L. from Chico, CA in comparison with laboratory colony Pesticides tested  Field collection  Laboratory colony  LC50 (ppm) or LT50 (min)* (95% CL)  LC90 (ppm) or LT90 (min)* (95% CL)  Slope  χ2/df  LC50 (ppm) or LT50 (min)* (95% CL)  LC90 (ppm) or LT90 (min)* (95% CL)  Slope  χ2/df  L. sphaericus (VectoLex WDG)  3.437 (1.925–5.889)  320.3 (158.3–773.9)  0.65 ± 0.05  0.65  0.005 (0.004–0.006)  0.014 (0.012–0.019)  3.04 ± 0.43  0.68  B.t.i. (VectoBac WDG)  0.033 (0.027–0.040)  0.095 (0.065–0.155)  2.91 ± 0.23  0.13  0.023 (0.020–0.027)  0.059 (0.050–0.074)  3.18 ± 0.30  0.82  B.t.i. + L. sphaericus (VectoMax FG)  0.200 (0.124–0.267)  0.499 (0.354–1.246)  3.23 ± 0.40  1.05  0.340 (0.231–0.516)  0.876 (0.561–3.013)  3.12 ± 0.32  1.51  Spinosad (Natular G30)  4.38 × 10−3 (3.75–4.90 × 10−3)  9.25x10−3 (8.00 × 10-3–1.16 × 10−2)  3.93 ± 0.52  0.82  4.88 × 10−3 (1.93–7.20 × 10−3)  1.28 × 10−2 (8.38 × 10−3–7.23 × 10−2)  3.05 ± 0.37  1.76  Spinetoram (Radiant SC)  8.10 × 10−4 (4.08 × 10−4–1.17 × 10−3)  2.38 × 10−3 (1.61–5.50 × 10−3)  2.73 ± 0.31  1.09  8.19 × 10−4 (4.68 × 10−4–1.05 × 10−3)  1.64 × 10−3 (1.17–3.51 × 10−3)  4.00 ± 0.44  1.22  Abamectin (Advance 375A)  1.42 × 10−2 (8.45 × 10−3–3.52 × 10−2)  1.02 (0.21–42.61)  0.69 ± 0.15  0.34  6.30 × 10−3 (5.73–6.90 × 10−3)  1.28 × 10−2 (1.11–1.56 × 10−2)  4.16 ± 0.44  0.12  S-methoprene (Altosid liquid larvicide)  1.61 × 10−3 (9.58 × 10−4–2.52 × 10−3)  7.02 × 10−2 (3.07 × 10−2–2.81 × 10−1)  0.78 ± 0.11  0.33  2.61 × 10−3 (1.62–4.14 × 10−3)  1.19 × 10−1 (4.92 × 10−2–5.18 × 10−1)  0.77 ± 0.11  0.90  Pyriproxyfen (NyGuard IGR)  8.90 × 10−5 (5.81 × 10−5–1.25 × 10−4)  1.79 × 10−3 (8.84 × 10−4–6.57 × 10−3)  0.98 ± 0.16  0.61  4.02 × 10−5 (2.46–5.64 × 10−5)  4.30 × 10−4 (2.86–8.12 × 10−4)  1.25 ± 0.17  0.10  Diflubenzuron (Dimilin 25 WP)  9.52 × 10−4 (3.85 × 10−4–1.60 × 10−3)  2.85 × 10−2 (1.51–9.32 × 10−2)  0.87 ± 0.16  0.17  3.03 × 10−4 (4.80 × 10−5– 6.92 × 10−4)  1.15 × 10−2 (6.55 × 10−3–3.34 × 10−2)  0.81 ± 0.17  0.72  Novaluron (Mosquiron 0.12CRD)  6.05 × 10−4 (4.86–7.63 × 10−4)  3.33 × 10−3 (2.29–5.65 × 10−−3)  1.73 ± 0.18  0.09  1.12 × 10−3 (9.51 × 10−4–1.33 × 10−3)  3.00 × 10−3 (2.36–4.19 × 10−3)  3.00 ± 0.31  0.62  Temephos (Skeeter Abate)  5.41 × 10−3 (5.00–5.85 × 10−3)  7.72 × 10−3 (6.93–9.33 × 10−3)  8.32 ± 1.38  0  3.25 × 10−3 (2.37–4.34 × 10−3)  7.11 × 10−3 (5.15 × 10−3–1.40 × 10−2)  3.78 ± 0.36  1.14  Imidacloprid (ImadPro 4SC)  0.035 (0.030–0.040)  0.079 (0.063–0.123)  3.60 ± 0.63  0.05  0.043 ( 0.040–0.048)  0.069 (0.060–0.086)  6.36 ± 0.89  0.00  Fipronil (Taurus SC)  1.76 × 10−3 (1.44–2.15 × 10−3)  8.00 × 10−3 (5.77 × 10−3–1.27 × 10−2)  1.95 ± 0.20  0.68  1.06 × 10−3 (8.90 × 10−4–1.29 × 10−3)  4.08 × 10−3 (2.96–6.57 × 10−3)  2.19 ± 0.24  0.54  Indoxacarb (Advion RIFA bait)  0.301 (0.234–0.387)  2.233 (1.440–4.349)  1.47 ± 0.17  0.91  0.141 (0.073–0.289)  0.534 (0.267–1.286)  2.22 ± 0.20  2.00  Permethrin  38.0 (30.7–45.2)  67.9 (55.8–96.0)  5.08 ± 0.47  2.08  7.1 (6.2–7.9)  13.7 (12.0–16.6)  4.46 ± 0.53  0.04  Pesticides tested  Field collection  Laboratory colony  LC50 (ppm) or LT50 (min)* (95% CL)  LC90 (ppm) or LT90 (min)* (95% CL)  Slope  χ2/df  LC50 (ppm) or LT50 (min)* (95% CL)  LC90 (ppm) or LT90 (min)* (95% CL)  Slope  χ2/df  L. sphaericus (VectoLex WDG)  3.437 (1.925–5.889)  320.3 (158.3–773.9)  0.65 ± 0.05  0.65  0.005 (0.004–0.006)  0.014 (0.012–0.019)  3.04 ± 0.43  0.68  B.t.i. (VectoBac WDG)  0.033 (0.027–0.040)  0.095 (0.065–0.155)  2.91 ± 0.23  0.13  0.023 (0.020–0.027)  0.059 (0.050–0.074)  3.18 ± 0.30  0.82  B.t.i. + L. sphaericus (VectoMax FG)  0.200 (0.124–0.267)  0.499 (0.354–1.246)  3.23 ± 0.40  1.05  0.340 (0.231–0.516)  0.876 (0.561–3.013)  3.12 ± 0.32  1.51  Spinosad (Natular G30)  4.38 × 10−3 (3.75–4.90 × 10−3)  9.25x10−3 (8.00 × 10-3–1.16 × 10−2)  3.93 ± 0.52  0.82  4.88 × 10−3 (1.93–7.20 × 10−3)  1.28 × 10−2 (8.38 × 10−3–7.23 × 10−2)  3.05 ± 0.37  1.76  Spinetoram (Radiant SC)  8.10 × 10−4 (4.08 × 10−4–1.17 × 10−3)  2.38 × 10−3 (1.61–5.50 × 10−3)  2.73 ± 0.31  1.09  8.19 × 10−4 (4.68 × 10−4–1.05 × 10−3)  1.64 × 10−3 (1.17–3.51 × 10−3)  4.00 ± 0.44  1.22  Abamectin (Advance 375A)  1.42 × 10−2 (8.45 × 10−3–3.52 × 10−2)  1.02 (0.21–42.61)  0.69 ± 0.15  0.34  6.30 × 10−3 (5.73–6.90 × 10−3)  1.28 × 10−2 (1.11–1.56 × 10−2)  4.16 ± 0.44  0.12  S-methoprene (Altosid liquid larvicide)  1.61 × 10−3 (9.58 × 10−4–2.52 × 10−3)  7.02 × 10−2 (3.07 × 10−2–2.81 × 10−1)  0.78 ± 0.11  0.33  2.61 × 10−3 (1.62–4.14 × 10−3)  1.19 × 10−1 (4.92 × 10−2–5.18 × 10−1)  0.77 ± 0.11  0.90  Pyriproxyfen (NyGuard IGR)  8.90 × 10−5 (5.81 × 10−5–1.25 × 10−4)  1.79 × 10−3 (8.84 × 10−4–6.57 × 10−3)  0.98 ± 0.16  0.61  4.02 × 10−5 (2.46–5.64 × 10−5)  4.30 × 10−4 (2.86–8.12 × 10−4)  1.25 ± 0.17  0.10  Diflubenzuron (Dimilin 25 WP)  9.52 × 10−4 (3.85 × 10−4–1.60 × 10−3)  2.85 × 10−2 (1.51–9.32 × 10−2)  0.87 ± 0.16  0.17  3.03 × 10−4 (4.80 × 10−5– 6.92 × 10−4)  1.15 × 10−2 (6.55 × 10−3–3.34 × 10−2)  0.81 ± 0.17  0.72  Novaluron (Mosquiron 0.12CRD)  6.05 × 10−4 (4.86–7.63 × 10−4)  3.33 × 10−3 (2.29–5.65 × 10−−3)  1.73 ± 0.18  0.09  1.12 × 10−3 (9.51 × 10−4–1.33 × 10−3)  3.00 × 10−3 (2.36–4.19 × 10−3)  3.00 ± 0.31  0.62  Temephos (Skeeter Abate)  5.41 × 10−3 (5.00–5.85 × 10−3)  7.72 × 10−3 (6.93–9.33 × 10−3)  8.32 ± 1.38  0  3.25 × 10−3 (2.37–4.34 × 10−3)  7.11 × 10−3 (5.15 × 10−3–1.40 × 10−2)  3.78 ± 0.36  1.14  Imidacloprid (ImadPro 4SC)  0.035 (0.030–0.040)  0.079 (0.063–0.123)  3.60 ± 0.63  0.05  0.043 ( 0.040–0.048)  0.069 (0.060–0.086)  6.36 ± 0.89  0.00  Fipronil (Taurus SC)  1.76 × 10−3 (1.44–2.15 × 10−3)  8.00 × 10−3 (5.77 × 10−3–1.27 × 10−2)  1.95 ± 0.20  0.68  1.06 × 10−3 (8.90 × 10−4–1.29 × 10−3)  4.08 × 10−3 (2.96–6.57 × 10−3)  2.19 ± 0.24  0.54  Indoxacarb (Advion RIFA bait)  0.301 (0.234–0.387)  2.233 (1.440–4.349)  1.47 ± 0.17  0.91  0.141 (0.073–0.289)  0.534 (0.267–1.286)  2.22 ± 0.20  2.00  Permethrin  38.0 (30.7–45.2)  67.9 (55.8–96.0)  5.08 ± 0.47  2.08  7.1 (6.2–7.9)  13.7 (12.0–16.6)  4.46 ± 0.53  0.04  *Bottle bioassay. View Large Table 3. RR to Lysinibacillus sphaericus Meyer and Neide (VectoLex WDG) and other commonly used pesticides in a wild population of Culex pipiens L. from Chico, CA Pesticides tested  At LC50 or LT50*  At LC90 or LT90*  L. sphaericus (VectoLex WDG)  687.4†  22,878.6†  B.t.i. (VectoBac WDG)  1.43  1.61  B.t.i. + L. sphaericus (VectoMax CG)  0.59  0.57  Spinosad (Natular G30)  0.90  0.72  Spinetoram (Radiant SC)  0.99  1.45  Abamectin (Advance 375A)  2.25†  79.69†  S-methoprene (Altosid liquid larvicide)  0.62  0.59  Pyriproxyfen (NyGuard IGR)  2.21†  4.16†  Diflubenzuron (Dimilin 25 WP)  2.37  2.48  Novaluron (Mosquiron 0.12CRD)  0.54  1.11  Temephos (Skeeter Abate)  1.80  1.10  Imidacloprid (ImidaPro 4SC)  0.81  1.14  Fipronil (Taurus SC)  1.66  1.96  Indoxacarb (Advion RIFA bait)  2.13  4.18†  Permethrin*  5.37†  4.95†  Pesticides tested  At LC50 or LT50*  At LC90 or LT90*  L. sphaericus (VectoLex WDG)  687.4†  22,878.6†  B.t.i. (VectoBac WDG)  1.43  1.61  B.t.i. + L. sphaericus (VectoMax CG)  0.59  0.57  Spinosad (Natular G30)  0.90  0.72  Spinetoram (Radiant SC)  0.99  1.45  Abamectin (Advance 375A)  2.25†  79.69†  S-methoprene (Altosid liquid larvicide)  0.62  0.59  Pyriproxyfen (NyGuard IGR)  2.21†  4.16†  Diflubenzuron (Dimilin 25 WP)  2.37  2.48  Novaluron (Mosquiron 0.12CRD)  0.54  1.11  Temephos (Skeeter Abate)  1.80  1.10  Imidacloprid (ImidaPro 4SC)  0.81  1.14  Fipronil (Taurus SC)  1.66  1.96  Indoxacarb (Advion RIFA bait)  2.13  4.18†  Permethrin*  5.37†  4.95†  *Bottle bioassay. †RR significant as indicated by separated. 95% CL of the LC(LT) levels. View Large In addition, this population also had significantly lower susceptibility (P < 0.05) to abamectin when compared with laboratory colony. The ratio of LC90/LC50 was 71.8 in this field population when compared with laboratory colony of 2.03. This field population bore a tolerance (RR 2.25-fold, see below) at LC50 and moderate level of resistance at LC90 (79.69-fold) to abamectin (Table 3). The ratio of RR at LC90 and RR at LC50 (Table 3) was 35.4. Tolerance Using the susceptibility of the laboratory colony as a baseline (Table 2), tolerance in this field population was noticed to pyriproxyfen and permethrin at both LC50 (LT50) and LC90 (LT90) levels. Tolerance was also encountered to Advance 375A ant bait at LC50 level, and to indoxacarb at LC90 level (Table 3). No Tolerance or Resistance Compared to laboratory colony of the same species bioassayed concurrently (Table 2), there was no tolerance or resistance in this field population to the following pesticides tested at both LC50 and LC90 levels: B.t.i., combination of B.t.i. and L. sphaericus, spinosad, spinetoram, methoprene, diflubenzuron, novaluron, temephos, imidacloprid, and fipronil. At the LC50 levels, this field population was as susceptible as laboratory colony to indoxacarb (Table 3). Discussion Global human population migration, freight exchange, demographic growth, and economic development have created new challenges for mosquito control and mosquito-borne disease management. In response to resurgence of traditional mosquito-borne illness and emergence of new mosquito-borne diseases worldwide, mosquito control by environmentally friendly interventions plays a crucial role in mitigation of disease burdens. Among the limited operational tools for mosquito control, products based on biorational active ingredients possess numerous advantages over conventional pesticides (Su 2016a,b). The efficacy of integrated mosquito control is often compromised by evolution of resistance to the pesticides used (Su 2016a). The resistance status mostly caught attention by reduced efficacy or control failure in mosquito control operations, if there is no routine pesticide susceptibility monitoring program in place along with product applications. A field population of Cx. pipiens that showed poor response to VectoLex WDG treatment was collected from downtown Chico, CA, the susceptibility of which to 15 common pesticides of public health, urban and agricultural uses was determined and compared with reference laboratory colony of the same species. High level of resistance to L. sphaericus was confirmed in this field collection of Cx. pipiens from Chico, CA. This is considered the first report of resistance in North America since the introduction of this biopesticide in 1990s (Su 2016a,b). The ratio of LC90/LC50 to VectoLex was 93.2, while this ratio was 2.8 in laboratory colony (Table 2), indicating the field population is highly heterogeneous when compared with laboratory colony in terms of response to L. sphaericus treatment. The ratio of RR at LC90 (22,878.6-fold) and RR at LC50 (687.4-fold) (Table 3) was high, indicating that there were extremely resistant individuals in this highly resistant population. The observed resistance development was assumedly attributable to continuous use of VectoLex WDG for 1.5 yr to the time when control failed. The first resistance to L. sphaericus in field populations occurred in Cx. pipiens in southern France with 70-fold at LC50 because of extended field applications (Sinègre et al. 1994). This first report was followed by more cases of resistance in Cx. pipiens complex in Brazil, India, China, Thailand, and Tunisia. The magnitude of resistance development in response to the field application varied greatly among countries with positive reports, depending on unknown historic exposure to naturally existing strains, population genetics, and gene exchange with refugee populations, as well as product applications. Furthermore, once mosquitoes have developed resistance to a given strain of L. sphaericus, they are also often resistant to other strains because of the similarity of the binary toxins in most strains. The cross-resistance among different strains is mild between the strains that also produce Mtx toxins. Fortunately, mosquitoes that have developed resistance to various strains of L. sphaericus remain susceptible to B.t.i. (Su 2016b). No case of resistance to L. sphaericus in North America has ever been reported in wild mosquito populations thus far, although a substantial amount of L. sphaericus products has been applied, particularly since the invasion of the WNV. During 1990–1993, the susceptibility of Cx. pipiens complex to L. sphaericus was determined in 31 collections across California, before the registration of this agent in the state in 1996. Variation was about five-fold at the LC50 and LC95 (Wirth et al. 2001). Reduced susceptibility was noticed in Culex spp. breeding in dairy lagoons in southern California soon after L. sphaericus was registered and applied for three consecutive yr during 1996–1998 (Su et al. 2001). Furthermore, this population also showed a significantly lower susceptibility to abamectin B1 (IRAC Group 6—chloride channel activators) in a highly heterogeneous style, as indicated by high LC90/LC50 ratio (71.8). The tolerance level (RR 2.25) at LC50 and resistance level at LC90 (RR 79.69) were widely separated with a ratio of 35.4, indicating this population was blended with susceptible and significantly resistant individuals. Abamectin B1 is a mixture of 80% avermectin B1a and 20% avermectin B1b. Currently, there is no product based on abamectin B1 that is registered for mosquito control as larvicide or adulticide. Products containing abamectin B1 have been commonly used in bait and spray formulations to control ants, cockroaches, and mites in urban environment, gardens, lawns, and other horticulture. It is expected that mosquito populations can be exposed to these active ingredients unintentionally, leading to the development of tolerance even resistance. Tolerance of this field population to pyriproxyfen and permethrin at both LC50 (LT50) and LC90 (LT90) levels was indicated. Pyriproxyfen is a juvenile hormone analog belonging to IRAC Group 7 (juvenile hormone mimics), which interrupts the metamorphosis of mosquitoes from late fourth instars through pupae to adults. Pyriproxyfen was introduced to the United States in 1996 for controlling whiteflies. Various products containing this insect growth regulators are used to control a wide variety of urban and household pestiferous insects. It is not a surprise that mosquito populations have been exposed to pyriproxyfen in the products that were applied to control other target species. In a more obvious case, products based on pyrethrins and pyrethroids (IRAC Group 3A—sodium channel modulators) have been extensively used to combat pestiferous arthropods in agriculture, urban and households. Sublethal exposures to these ingredients are almost unavoidable. Tolerance or resistance to pyrethroids are quite common in mosquitoes, houseflies, and other urban pestiferous species (Zhu et al. 2016). Tolerance was also noticed to abamectin at LC50, which was in connection with high-level resistance to this active ingredient as described previously at LC90 level, a reflection of high genetic heterogeneity in terms of susceptibility to abamectin. Finally, the tolerance to indoxacarb (IRAC Group 22—voltage-dependent sodium channel blockers) was observed at LC90 level. This relatively new active ingredient has been formulated to baits to control ants, cockroaches, and others. Tolerance at LC90 only indicated existence of some hardy individuals that were mixed with the majority of susceptible ones. There was no tolerance or resistance in this field population to B.t.i., an IRAC Group 11 pesticide, which has very low risk of resistance development, and plays an important role in resistance management toward other public health pesticides (Su 2016b). Combination of B.t.i. and L. sphaericus as in commercial products VectoMax CG or FG, takes advantages of each microbial larvicide’s strengths, and reduces the limitations that each possesses. Mosquito populations that show resistance to other pesticides remained susceptible to this combination (Su and Cheng 2014a,b; Su 2016a,b). The studied field population was highly susceptible to spinosad and spinetoram, the IRAC Group 5 pesticides—nicotinic acetylcholine receptor allosteric modulators, although spinosad is prone to induce resistance development in insects (Su and Cheng 2014a; Sparks et al. 2012). The products based on methoprene (IRAC Group 7A), have been used to combat various mosquito species for decades, and low to high levels of resistance in Culex spp. and Aedes spp. have been documented in both laboratory colonies and field populations (Su 2016b). High susceptibility to methoprene was indicated in this field population, which might be attributable to the fact that products based on methoprene for urban pest control are not common. No tolerance was indicated to diflubenzuron and novaluron (IRAC Group 15—the chitin synthesis inhibitors), where diflubenzuron was not commonly used in urban areas, and novaluron is relatively new to pest and vector control. High susceptibility was observed to one of the organophosphate temephos (IRAC Group 1B—acetylcholinesterase inhibitors). The restrictive use of organophosphate products in urban areas may have reduced the exposure of mosquito populations to this group of pesticides. As to Imidacloprid (IRAC Group 4—nicotinic acetylcholine receptor agonists) and fipronil (IRAC Group 2—GABA-gated chloride channel antagonists), currently there are no products based on these two active ingredients labeled for mosquito control. Some products, mostly baits, are labeled to control garden and urban pests. These findings may shed some light to consider future use of these ingredients for mosquito control. There was no tolerance to indoxacarb at LC50, indicating ample numbers of susceptible individuals existed in the population studied even though tolerance was noticed at LC90 level as discussed earlier. The products based on above active ingredients can be considered to control larval population studied if product labels are in place. In summary, since mid-1990s, numerous cases of resistance to L. sphaericus in field mosquito populations have been reported in France, Brazil, India, China, Thailand, and Tunisia. The products based on L. sphaericus have been extensively applied to combat WNV vectors and other nuisance species since 1990s in the United States and elsewhere. It is important to monitor the susceptibility to L. sphaericus products periodically to ensure the field efficacy. At the same time, susceptibility monitoring to other pesticides of public health, urban and agriculture uses is also strongly recommended because mosquitoes are quite often exposed to other pesticides accidently and their susceptibility can be compromised such as in the current paper about resistance or tolerance to abamectin, pyriproxyfen, permethrin, and to a lesser extent to indoxacarb. Acknowledgments The authors are grateful to Dr. Alec Gerry, Department of Entomology, University of California at Riverside (Riverside, CA) for provision of Radiant SC, and to Dr. Barry Tyler, Pestalto Environmental Health Services Inc. (Hamilton, ON, Canada) for the product sample of Mosquiron 0.12CRD. They also specially thank Michelle Brown, Ph.D., Manager at the West Valley Mosquito and Vector Control District, Ontario, CA, for her constructive review of this manuscript. References Cited Ahmed, I., A. Yokota, A. Yamazoe, and Fujiwara T.. 2007. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Int. J. Syst. Evol. Microbiol . 57: 1117– 1125. Google Scholar CrossRef Search ADS PubMed  LeOra Software. 1987. POLO-PC: A user’s guide to probit or logit analysis . LeOra Software, Berkeley, CA Sinègre, G., Babinot M., Quermel J. M., and Gavon B.. 1994. First field occurrence of Culex pipiens resistance to Bacillus sphaericus in southern France. In Proceedings of 8thh European Meet. Soc. Vector Ecol, September 5–8, 1994, Barcelona, Spain, Society for Vector Ecology, Santa Ana, CA, 1997. P17. Sparks, Dripps T. C., J. E., Watson G. B., and Paroonagian D.. 2012. Resistance and cross-resistance to the spinosyn - a review and analysis. Pestic. Biochem. Physiol . 102: 1– 10. Su, T. 2016a. Microbial control of pest and vector mosquitoes in North America north of Mexico. In Lacey, L. (ed.), Microbial control of insect and mite pests . Academic Press, San Diego, CA. pp. 393– 407. Google Scholar CrossRef Search ADS   Su, T. 2016b. Resistance and its management to microbial and insect growth regulator larvicides in mosquitoes. In Trdan, S. (ed.), Insecticides resistance , InTech Europe, Rijeka, Croatia. pp. 135– 154. Google Scholar CrossRef Search ADS   Su, T., and Cheng M. L.. 2014a. Laboratory selection of resistance to spinosad in Culex quinquefasciatus (Diptera: Culicidae). J. Med. Entomol . 51: 421– 427. Google Scholar CrossRef Search ADS   Su, T., and Cheng M. L.. 2014b. Cross resistances in spinosad-resistant Culex quinquefasciatus (Diptera: Culicidae). J. Med. Entomol . 51: 428– 435. Google Scholar CrossRef Search ADS   Su, T., and Mulla M. S.. 2004. Documentation of high level Bacillus sphaericus-resistance in tropical Culex quinquefasciatus populations from Thailand. J. Am. Mosq. Control Assoc . 20: 405– 411. Google Scholar PubMed  Su, T., Soliman B. A., Chaney J. D., Mulla M. S., and Beehler J. W.. 2001. Susceptibility of Culex mosquitoes breeding in dairy ponds before and after treatment with Bacillus sphaericus formulation. Proc. Pap. Mosq. Vector Control Assoc. Calif . 69: 110– 116. Throne, J., Weaver D. K., Chew V., and Baker J. E.. 1995. Probit analysis of correlated data: Multiple observations over time at one concentration. J. Econ. Entomol . 88: 1510– 1512. Google Scholar CrossRef Search ADS   Zhu, F., Lavine L., O’Neal S., Lavine M., Foss C., and Walsh D.. 2016. Insecticide resistance and management strategies in urban ecosystems. Insects  7: 2. Google Scholar CrossRef Search ADS   Wirth, M. C. 2010. Mosquito resistance to bacterial larvicidal toxins. OpenToxinol. J . 3: 126– 140. Google Scholar CrossRef Search ADS   Wirth, M. C., Ferrari J. A., and Georghiou G. P.. 2001. Baseline susceptibility to bacterial insecticides in populations of Culex pipiens complex (Diptera: Culicidae) from California and from the Mediterranean Island of Cyprus. J. Med. Entomol . 94: 920– 928. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Medical Entomology Oxford University Press

Resistance to Lysinibacillus sphaericus and Other Commonly Used Pesticides in Culex pipiens (Diptera: Culicidae) from Chico, California

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Abstract

Abstract Bacillus sphaericus Neide, recently renamed as Lysinibacillus sphaericus Meyer and Neide, is a spore-forming bacterium that possesses various levels of larvicidal activity, depending on the strains, against some mosquito species. Products based on most active strains such as 2362, 2297, 1593, C3-41 that bear binary toxins, as well as mosquitocidal toxins at various levels, have been developed to combat mosquito larvae worldwide. Resistance in wild Culex mosquito populations has been reported since 1994 from France, Brazil, India, China, Thailand, and Tunisia. Laboratory studies to evaluate resistance development risk have been conducted by many groups of scientists worldwide. Products based on L. sphaericus strain 2362 were registered in the United States in 1990s, and their use for mosquito control has been increased considerably since invasion of West Nile virus. This report documents the first occurrence of high-level resistance to L. sphaericus in a natural population of Culex pipiens L. in Chico, CA, where resistance ratio was 537.0 at LC50 and 9,048.5 at LC90 when compared with susceptible laboratory colony of the same species. Susceptibility profile to other groups of pesticides with different modes of action was also determined. Various levels of resistance or tolerance were noticed to abamectin, pyriproxyfen, permethrin, and indoxacarb. Resistance management and susceptibility monitoring strategies are discussed and recommended. Lysinibacillus sphaericus, Culex pipiens, Diptera, Culicidae, resistance, susceptibility The mosquitocidal activity of some strains of Bacillus sphaericus, recently renamed as Lysinibacillus sphaericus (Ahmed et al. 2007), was recognized as early as 1960s. To date, 49 serotypes over 300 strains of L. sphaericus have been identified, among which 9 serotypes of 16 strains showed various levels of activity against mosquito larvae. Strains that possess high mosquitocidal activity are 2362, 1597, 2297, C3-41, and IAB-59. The mostly studied and developed strain, 2362, was isolated in 1984 from adult blackfly Simulium damnosum Theobald (Diptera: Simuliidae) in Nigeria (Su 2016a). Active strains produce parasporal inclusions during sporulation, containing crystal binary toxins A and B (BinA 42 kDa and 51 kDa BinB) that play the predominant role in larvicidal toxicity. Both BinA and BinB are required for larvicidal toxicity and an equimolar ratio of both proteins yields the greatest toxicity. Some strains also produce noncrystal mosquitocidal toxins (Mtx) such as Mtx 1, 2, and 3 during vegetative stage. Mtx possess lower larvicidal toxicity than crystal binary toxins. The target site of binary toxins is located at the brush border of the gut epithelium membrane. The receptor of the BinB is a 60-kDa alpha-glucosidase, which is anchored to the mosquito midgut membrane via a glycosyl-phosphatidylinositol anchor. It is generally believed that BinB opens the pathway on the membrane while BinA causes the actual pathological consequences leading the larval mortality. Together with Bacillus thuringiensis subsp. israelensis (B.t.i.), L. sphaericus also belongs to Group 11 (microbial disruptors of insect midgut membranes and derived toxins) by Insect Resistance Action Committee (IRAC). During the past decades, numerous products have been developed using various strains and applied to control mainly Culex spp. worldwide. Strain 2362 belonging to serotype H5a5b has been well studied, developed, and commercialized in the United States. Products that are solely based on this strain such as VectoLex CG, WDG, WSP and Spheratax SPH, WSP, and ones that contain this strain such as VectoMax CG, FG, WSP and FourStar CRG, MBG, WSP, Briquets have been registered since 1990s, and widely used to combat vectors of West Nile virus (WNV) and other nuisance mosquito species in the United States. Because of the simplicity of Bin toxins in L. sphaericus, the risk of resistance development in laboratory mosquito populations seemed quite imminent, which has been well documented in Culex pipiens L. complex (Wirth 2010, Su 2016b). Beside the toxin simplicity, long-term exposure of wild mosquito populations to naturally occurring strains associated with mosquito habitats may also have contributed to the resistance development after various periods of applications in different countries such as France, Brazil, India, Thailand, China, and Tunisia since 1994 (Sinègre et al. 1994, Wirth 2010, Su 2016b). In response to a control failure of mosquito populations in Chico, CA, after application of VectoLex WDG for 1.5 yr, investigation on potential resistance was initiated. Here in this paper we reported the first occurrence of L. sphaericus resistance in wild population of Cx. pipiens in the United States where the L. sphaericus-based products have been used most extensively, particularly since the invasion of the WNV. The resistance to other pesticides of public health, urban and agriculture uses was also evaluated in this population. Materials and Methods Mosquitoes A couple hundreds of mosquito larvae, mostly second and third instar larvae were collected from a storm drain located at Laburnum Avenue and E. 6th Street, Chico, CA, on 8 April 2015, by the Butte County Mosquito and Vector Control District (5117 Larkin Road, Oroville, CA). The specimens were provided to the West Valley Mosquito and Vector Control District (1295 E. Locust Street, Ontario, CA), from which a colony of Cx. pipiens was established. The third instars from generation F4–6 were used in bioassays to decide the potential resistance to L. sphaericus and other common pesticides of public health, urban and agriculture uses. A long-term laboratory colony of the same species was introduced from the San Mateo County Mosquito and Vector Control District (1351 Rollins Road, Burlingame, CA), which was assayed concurrently with the field population derived from collection in Chico, CA. Pesticides To generate immediately relevant susceptibility data for field application, the most commonly used commercial formulations with active ingredients of interest were chosen in bioassays. The pesticides tested included one pyrethroid (permethrin); six biological pesticides based on B.t.i., L. sphaericus, spinosad, spinetoram, and avermectins; four insect growth regulators based on methoprene, pyriproxyfen, diflubenzuron, and novaluron; and one from each of organophosphate (temephos), neonicotinoid (imidacloprid), phenylpyrazoles (fipronil), and oxadiazine (indoxacarb). Detailed information about the pesticides tested including trade names, active ingredients, lot numbers, and manufacturers was provided in Table 1. Table 1. Profile of the pesticides tested against a wild population of Culex pipiens L. from Chico, CA, along with laboratory colony Category  Products  Active ingredients  Concentration (%)  Lot no.  Manufacturers  Biological pesticides  VectoLex WDG  Lysinibacillus sphaericus  51.2  188-119-PG  Valent BioSciences Corp., Libertyville, IL    VectoBac WDG  Bacillus thuringiensis israelensis (B.t.i.)  37.4  201-391-PG  Valent BioSciences Corp., Libertyville, IL    VectoMax CG  B.t.i. + L. sphaericus  4.5 + 2.7  187-575-N8  Valent BioSciences Corp., Libertyville, IL    Natular G30  Spinosad  2.5    Clarke, St. Charles, IL.    Radiant SC  Spinetoram  11.7  XG14164911  Dow AgroSciences LLC, Indianapolis, IN    Advance 375A  Abamectin B1  0.011  81040334  BASF Corp., St. Louis, MO  Insect growth regulators (IGRs)  Altosid liquid larvicide  Methoprene  5.0  60111357  Wellmark International, Schaumburg, IL    NyGuard IGR  Pyriproxyfen  10.0  BAB6111  MGK, Minneapolis, MN    Dimilin 25W  Diflubenzuron  25.0  BA9D30P001  Chemtura Corp., Middlebury, CT    Mosquiron 0.12CRD  Novaluron  0.12  1012818  Makhteshim Agan North America, Inc., Raleigh, NC  Organophosphate  Skeeter Abate  Temephos  5.0  1009280003  Clarke Mosquito Control Products, Inc., Roselle, IL  Neonicotinoid  ImidaPro 4SC  Imidacloprid  40.7  12254PO42  Agrisel USA, Inc., Suwanee, GA  Phenylpyrazoles  Taurus SC  Fipronil  9.1  23204  CSI Control Solutions, Inc., Pasadena, TX    Advion RIFA bait  Indoxacarb  0.045    DuPont  Pyrethroid  Permethrin (Technical)  Permethrin (mixture of isomers)  99.9  2601000  Chem Service, West Chester, PA  Category  Products  Active ingredients  Concentration (%)  Lot no.  Manufacturers  Biological pesticides  VectoLex WDG  Lysinibacillus sphaericus  51.2  188-119-PG  Valent BioSciences Corp., Libertyville, IL    VectoBac WDG  Bacillus thuringiensis israelensis (B.t.i.)  37.4  201-391-PG  Valent BioSciences Corp., Libertyville, IL    VectoMax CG  B.t.i. + L. sphaericus  4.5 + 2.7  187-575-N8  Valent BioSciences Corp., Libertyville, IL    Natular G30  Spinosad  2.5    Clarke, St. Charles, IL.    Radiant SC  Spinetoram  11.7  XG14164911  Dow AgroSciences LLC, Indianapolis, IN    Advance 375A  Abamectin B1  0.011  81040334  BASF Corp., St. Louis, MO  Insect growth regulators (IGRs)  Altosid liquid larvicide  Methoprene  5.0  60111357  Wellmark International, Schaumburg, IL    NyGuard IGR  Pyriproxyfen  10.0  BAB6111  MGK, Minneapolis, MN    Dimilin 25W  Diflubenzuron  25.0  BA9D30P001  Chemtura Corp., Middlebury, CT    Mosquiron 0.12CRD  Novaluron  0.12  1012818  Makhteshim Agan North America, Inc., Raleigh, NC  Organophosphate  Skeeter Abate  Temephos  5.0  1009280003  Clarke Mosquito Control Products, Inc., Roselle, IL  Neonicotinoid  ImidaPro 4SC  Imidacloprid  40.7  12254PO42  Agrisel USA, Inc., Suwanee, GA  Phenylpyrazoles  Taurus SC  Fipronil  9.1  23204  CSI Control Solutions, Inc., Pasadena, TX    Advion RIFA bait  Indoxacarb  0.045    DuPont  Pyrethroid  Permethrin (Technical)  Permethrin (mixture of isomers)  99.9  2601000  Chem Service, West Chester, PA  View Large Bioassays Cup Bioassay on Larvicides Best efforts were made to process commercial products for bioassay (Su and Cheng 2014b). Emulsifiable concentrates such as Radiant SC, Altosid liquid larvicide, NyGuard IGR, ImidaPro 4SC, and Taurus SC were suspended in tap water by gentle mixing. Small granules of Advance 375A ant bait and Advion RIFA bait or water dispersible granules of VectoBac WDG and VectoLex WDG, or wettable powders of Dimilin 25W were suspended in tap water by vigorous shaking. Large granular materials of VectoMax CG, or pellets of Skeeter Abate , were powdered in a coffee grinder (Hamilton Beach Custom Grind, Southern Pines, NC) at the maximum speed, then suspended in tap water by vortexing for 3 min. For briquet formulation such as Mosquiron 0.12CRD, fine pieces were shaved off using a razor blade and suspended in tap water by vortexing for 5 min (Vortex Mixer VX100, Labnet International, Inc., Edison, NJ). Bioassay was conducted as previously described (Su and Mulla 2004). Briefly, four to five concentrations of each test material within the concentration range resulting ~5–95% mortality were used in bioassay, with three replicates at each concentration. For larvicides based on B.t.i. and L. sphaericus, concentrations were determined by weight for the whole formulation in volume. Other bioassays were based on the concentration of weight of active ingredient of the insecticide in volume. For each replicate, 25 larvae were placed in 100 ml of tap water in a 120-ml disposable Styrofoam cup. Bioassays were conducted at 25 ± 1°C. In bioassays on B.t.i., the combination of B.t.i. and L. sphaericus, spinetoram, abamectin, temephos, imidacloprid, fipronil, and indoxacarb, late third instar larvae were used and larval mortality was recorded at 24 h post treatment. In bioassays using L. sphaericus, diflubenzuron and novaluron, early third instar larvae were preferred, and results were recorded at 48 h post treatment for L. sphaericus, and 72 h post treatment for diflubenzuron and novaluron. Moribund larvae were also considered dead. In bioassays of methoprene and pyriproxyfen, late fourth instar larvae were used, and mortality was read when all treated individuals emerged as adults or died prior to emergence. Three drops of 10% rabbit chow pellet suspension were added to each cup as larval food in all bioassays, except those using methoprene and pyriproxyfen where a small piece (~100 mg) of rabbit pellets was added to each bioassay cup to support them until pupation. Concentration-response data were analyzed using POLO-PC (LeOra Software 1987) to calculate lethal concentrations (LC) and their 95% confidence limits (CLs). For each pesticide tested, bioassays were also concurrently conducted on a reference laboratory colony of Cx. pipiens. Bottle Bioassay on Adulticide In bottle bioassay on susceptibility to permethrin, the interior of 250-ml glass bottle (Uline, Pleasant Prairie, WI) was evenly coated by 30 μg of permethrin in 1 ml of HPLC grade acetone (EMD Millipore, Temecula, CA) on an automatic roller (Fisher Scientific, Fisher Scientific, Hampton, NH) in a chemical fume hood (Hemco, Independence, MO). Bottles for untreated control were coated by acetone only. After the coated surface was completely dry in the hood, 25 of 3- to 5-d-old female mosquitoes were aspirated into each bottle. Mortality was read at 5, 10, 15, 30, 45, 60, 90, and 120 min, three replicates were made for each mosquito population. Mortality referred to individuals that did not show any movements of entire body or legs, wings, proboscis, antennae, or palpi. Bioassays were conducted at 25 ± 1°C and relative humidity 50–60%. Time mortality data were subject to probit analysis for calculations of LT50 and LT90 and their 95% CL (Throne et al. 1995). Bottle bioassays were also concurrently conducted on a reference laboratory colony of Cx. pipiens. Resistance Ratio Calculation The resistance ratios (RRs) were calculated by LC (LT)Field/LC (LT)Lab. Resistance was categorized as no resistance (RR ≤ 1), tolerance (RR = 1.1–5), low level (RR = 5.1–20), moderate level (RR = 20.1–100), and high level (RR > 100) (Su and Cheng 2014a). The significance at P < 0.05 in tolerance or resistance levels were validated by separated 95% CLs of LC (LT) levels between field population and laboratory colony. Results Moderate-to-High Resistance The collection from the field site in Chico, CA, showed significantly lower susceptibility (P < 0.05) to L. sphaericus (VectoLex WDG) when compared with the laboratory colony of the same species that was bioassayed concurrently (Table 2). The LC50 and LC90 in this field population were widely separated with a ratio of LC90/LC50 = 93.2, when compared with 2.8 in laboratory colony. This field population showed high RR of 687.4-fold at LC50 level and 22,878.6-fold at LC90 level (Table 3). The ratio of RR at LC90 and RR at LC50 (Table 3) was 33.3. Table 2. Susceptibility to Lysinibacillus sphaericus Meyer and Neide (VectoLex WDG) and other commonly used pesticides in a wild population of Culex pipiens L. from Chico, CA in comparison with laboratory colony Pesticides tested  Field collection  Laboratory colony  LC50 (ppm) or LT50 (min)* (95% CL)  LC90 (ppm) or LT90 (min)* (95% CL)  Slope  χ2/df  LC50 (ppm) or LT50 (min)* (95% CL)  LC90 (ppm) or LT90 (min)* (95% CL)  Slope  χ2/df  L. sphaericus (VectoLex WDG)  3.437 (1.925–5.889)  320.3 (158.3–773.9)  0.65 ± 0.05  0.65  0.005 (0.004–0.006)  0.014 (0.012–0.019)  3.04 ± 0.43  0.68  B.t.i. (VectoBac WDG)  0.033 (0.027–0.040)  0.095 (0.065–0.155)  2.91 ± 0.23  0.13  0.023 (0.020–0.027)  0.059 (0.050–0.074)  3.18 ± 0.30  0.82  B.t.i. + L. sphaericus (VectoMax FG)  0.200 (0.124–0.267)  0.499 (0.354–1.246)  3.23 ± 0.40  1.05  0.340 (0.231–0.516)  0.876 (0.561–3.013)  3.12 ± 0.32  1.51  Spinosad (Natular G30)  4.38 × 10−3 (3.75–4.90 × 10−3)  9.25x10−3 (8.00 × 10-3–1.16 × 10−2)  3.93 ± 0.52  0.82  4.88 × 10−3 (1.93–7.20 × 10−3)  1.28 × 10−2 (8.38 × 10−3–7.23 × 10−2)  3.05 ± 0.37  1.76  Spinetoram (Radiant SC)  8.10 × 10−4 (4.08 × 10−4–1.17 × 10−3)  2.38 × 10−3 (1.61–5.50 × 10−3)  2.73 ± 0.31  1.09  8.19 × 10−4 (4.68 × 10−4–1.05 × 10−3)  1.64 × 10−3 (1.17–3.51 × 10−3)  4.00 ± 0.44  1.22  Abamectin (Advance 375A)  1.42 × 10−2 (8.45 × 10−3–3.52 × 10−2)  1.02 (0.21–42.61)  0.69 ± 0.15  0.34  6.30 × 10−3 (5.73–6.90 × 10−3)  1.28 × 10−2 (1.11–1.56 × 10−2)  4.16 ± 0.44  0.12  S-methoprene (Altosid liquid larvicide)  1.61 × 10−3 (9.58 × 10−4–2.52 × 10−3)  7.02 × 10−2 (3.07 × 10−2–2.81 × 10−1)  0.78 ± 0.11  0.33  2.61 × 10−3 (1.62–4.14 × 10−3)  1.19 × 10−1 (4.92 × 10−2–5.18 × 10−1)  0.77 ± 0.11  0.90  Pyriproxyfen (NyGuard IGR)  8.90 × 10−5 (5.81 × 10−5–1.25 × 10−4)  1.79 × 10−3 (8.84 × 10−4–6.57 × 10−3)  0.98 ± 0.16  0.61  4.02 × 10−5 (2.46–5.64 × 10−5)  4.30 × 10−4 (2.86–8.12 × 10−4)  1.25 ± 0.17  0.10  Diflubenzuron (Dimilin 25 WP)  9.52 × 10−4 (3.85 × 10−4–1.60 × 10−3)  2.85 × 10−2 (1.51–9.32 × 10−2)  0.87 ± 0.16  0.17  3.03 × 10−4 (4.80 × 10−5– 6.92 × 10−4)  1.15 × 10−2 (6.55 × 10−3–3.34 × 10−2)  0.81 ± 0.17  0.72  Novaluron (Mosquiron 0.12CRD)  6.05 × 10−4 (4.86–7.63 × 10−4)  3.33 × 10−3 (2.29–5.65 × 10−−3)  1.73 ± 0.18  0.09  1.12 × 10−3 (9.51 × 10−4–1.33 × 10−3)  3.00 × 10−3 (2.36–4.19 × 10−3)  3.00 ± 0.31  0.62  Temephos (Skeeter Abate)  5.41 × 10−3 (5.00–5.85 × 10−3)  7.72 × 10−3 (6.93–9.33 × 10−3)  8.32 ± 1.38  0  3.25 × 10−3 (2.37–4.34 × 10−3)  7.11 × 10−3 (5.15 × 10−3–1.40 × 10−2)  3.78 ± 0.36  1.14  Imidacloprid (ImadPro 4SC)  0.035 (0.030–0.040)  0.079 (0.063–0.123)  3.60 ± 0.63  0.05  0.043 ( 0.040–0.048)  0.069 (0.060–0.086)  6.36 ± 0.89  0.00  Fipronil (Taurus SC)  1.76 × 10−3 (1.44–2.15 × 10−3)  8.00 × 10−3 (5.77 × 10−3–1.27 × 10−2)  1.95 ± 0.20  0.68  1.06 × 10−3 (8.90 × 10−4–1.29 × 10−3)  4.08 × 10−3 (2.96–6.57 × 10−3)  2.19 ± 0.24  0.54  Indoxacarb (Advion RIFA bait)  0.301 (0.234–0.387)  2.233 (1.440–4.349)  1.47 ± 0.17  0.91  0.141 (0.073–0.289)  0.534 (0.267–1.286)  2.22 ± 0.20  2.00  Permethrin  38.0 (30.7–45.2)  67.9 (55.8–96.0)  5.08 ± 0.47  2.08  7.1 (6.2–7.9)  13.7 (12.0–16.6)  4.46 ± 0.53  0.04  Pesticides tested  Field collection  Laboratory colony  LC50 (ppm) or LT50 (min)* (95% CL)  LC90 (ppm) or LT90 (min)* (95% CL)  Slope  χ2/df  LC50 (ppm) or LT50 (min)* (95% CL)  LC90 (ppm) or LT90 (min)* (95% CL)  Slope  χ2/df  L. sphaericus (VectoLex WDG)  3.437 (1.925–5.889)  320.3 (158.3–773.9)  0.65 ± 0.05  0.65  0.005 (0.004–0.006)  0.014 (0.012–0.019)  3.04 ± 0.43  0.68  B.t.i. (VectoBac WDG)  0.033 (0.027–0.040)  0.095 (0.065–0.155)  2.91 ± 0.23  0.13  0.023 (0.020–0.027)  0.059 (0.050–0.074)  3.18 ± 0.30  0.82  B.t.i. + L. sphaericus (VectoMax FG)  0.200 (0.124–0.267)  0.499 (0.354–1.246)  3.23 ± 0.40  1.05  0.340 (0.231–0.516)  0.876 (0.561–3.013)  3.12 ± 0.32  1.51  Spinosad (Natular G30)  4.38 × 10−3 (3.75–4.90 × 10−3)  9.25x10−3 (8.00 × 10-3–1.16 × 10−2)  3.93 ± 0.52  0.82  4.88 × 10−3 (1.93–7.20 × 10−3)  1.28 × 10−2 (8.38 × 10−3–7.23 × 10−2)  3.05 ± 0.37  1.76  Spinetoram (Radiant SC)  8.10 × 10−4 (4.08 × 10−4–1.17 × 10−3)  2.38 × 10−3 (1.61–5.50 × 10−3)  2.73 ± 0.31  1.09  8.19 × 10−4 (4.68 × 10−4–1.05 × 10−3)  1.64 × 10−3 (1.17–3.51 × 10−3)  4.00 ± 0.44  1.22  Abamectin (Advance 375A)  1.42 × 10−2 (8.45 × 10−3–3.52 × 10−2)  1.02 (0.21–42.61)  0.69 ± 0.15  0.34  6.30 × 10−3 (5.73–6.90 × 10−3)  1.28 × 10−2 (1.11–1.56 × 10−2)  4.16 ± 0.44  0.12  S-methoprene (Altosid liquid larvicide)  1.61 × 10−3 (9.58 × 10−4–2.52 × 10−3)  7.02 × 10−2 (3.07 × 10−2–2.81 × 10−1)  0.78 ± 0.11  0.33  2.61 × 10−3 (1.62–4.14 × 10−3)  1.19 × 10−1 (4.92 × 10−2–5.18 × 10−1)  0.77 ± 0.11  0.90  Pyriproxyfen (NyGuard IGR)  8.90 × 10−5 (5.81 × 10−5–1.25 × 10−4)  1.79 × 10−3 (8.84 × 10−4–6.57 × 10−3)  0.98 ± 0.16  0.61  4.02 × 10−5 (2.46–5.64 × 10−5)  4.30 × 10−4 (2.86–8.12 × 10−4)  1.25 ± 0.17  0.10  Diflubenzuron (Dimilin 25 WP)  9.52 × 10−4 (3.85 × 10−4–1.60 × 10−3)  2.85 × 10−2 (1.51–9.32 × 10−2)  0.87 ± 0.16  0.17  3.03 × 10−4 (4.80 × 10−5– 6.92 × 10−4)  1.15 × 10−2 (6.55 × 10−3–3.34 × 10−2)  0.81 ± 0.17  0.72  Novaluron (Mosquiron 0.12CRD)  6.05 × 10−4 (4.86–7.63 × 10−4)  3.33 × 10−3 (2.29–5.65 × 10−−3)  1.73 ± 0.18  0.09  1.12 × 10−3 (9.51 × 10−4–1.33 × 10−3)  3.00 × 10−3 (2.36–4.19 × 10−3)  3.00 ± 0.31  0.62  Temephos (Skeeter Abate)  5.41 × 10−3 (5.00–5.85 × 10−3)  7.72 × 10−3 (6.93–9.33 × 10−3)  8.32 ± 1.38  0  3.25 × 10−3 (2.37–4.34 × 10−3)  7.11 × 10−3 (5.15 × 10−3–1.40 × 10−2)  3.78 ± 0.36  1.14  Imidacloprid (ImadPro 4SC)  0.035 (0.030–0.040)  0.079 (0.063–0.123)  3.60 ± 0.63  0.05  0.043 ( 0.040–0.048)  0.069 (0.060–0.086)  6.36 ± 0.89  0.00  Fipronil (Taurus SC)  1.76 × 10−3 (1.44–2.15 × 10−3)  8.00 × 10−3 (5.77 × 10−3–1.27 × 10−2)  1.95 ± 0.20  0.68  1.06 × 10−3 (8.90 × 10−4–1.29 × 10−3)  4.08 × 10−3 (2.96–6.57 × 10−3)  2.19 ± 0.24  0.54  Indoxacarb (Advion RIFA bait)  0.301 (0.234–0.387)  2.233 (1.440–4.349)  1.47 ± 0.17  0.91  0.141 (0.073–0.289)  0.534 (0.267–1.286)  2.22 ± 0.20  2.00  Permethrin  38.0 (30.7–45.2)  67.9 (55.8–96.0)  5.08 ± 0.47  2.08  7.1 (6.2–7.9)  13.7 (12.0–16.6)  4.46 ± 0.53  0.04  *Bottle bioassay. View Large Table 3. RR to Lysinibacillus sphaericus Meyer and Neide (VectoLex WDG) and other commonly used pesticides in a wild population of Culex pipiens L. from Chico, CA Pesticides tested  At LC50 or LT50*  At LC90 or LT90*  L. sphaericus (VectoLex WDG)  687.4†  22,878.6†  B.t.i. (VectoBac WDG)  1.43  1.61  B.t.i. + L. sphaericus (VectoMax CG)  0.59  0.57  Spinosad (Natular G30)  0.90  0.72  Spinetoram (Radiant SC)  0.99  1.45  Abamectin (Advance 375A)  2.25†  79.69†  S-methoprene (Altosid liquid larvicide)  0.62  0.59  Pyriproxyfen (NyGuard IGR)  2.21†  4.16†  Diflubenzuron (Dimilin 25 WP)  2.37  2.48  Novaluron (Mosquiron 0.12CRD)  0.54  1.11  Temephos (Skeeter Abate)  1.80  1.10  Imidacloprid (ImidaPro 4SC)  0.81  1.14  Fipronil (Taurus SC)  1.66  1.96  Indoxacarb (Advion RIFA bait)  2.13  4.18†  Permethrin*  5.37†  4.95†  Pesticides tested  At LC50 or LT50*  At LC90 or LT90*  L. sphaericus (VectoLex WDG)  687.4†  22,878.6†  B.t.i. (VectoBac WDG)  1.43  1.61  B.t.i. + L. sphaericus (VectoMax CG)  0.59  0.57  Spinosad (Natular G30)  0.90  0.72  Spinetoram (Radiant SC)  0.99  1.45  Abamectin (Advance 375A)  2.25†  79.69†  S-methoprene (Altosid liquid larvicide)  0.62  0.59  Pyriproxyfen (NyGuard IGR)  2.21†  4.16†  Diflubenzuron (Dimilin 25 WP)  2.37  2.48  Novaluron (Mosquiron 0.12CRD)  0.54  1.11  Temephos (Skeeter Abate)  1.80  1.10  Imidacloprid (ImidaPro 4SC)  0.81  1.14  Fipronil (Taurus SC)  1.66  1.96  Indoxacarb (Advion RIFA bait)  2.13  4.18†  Permethrin*  5.37†  4.95†  *Bottle bioassay. †RR significant as indicated by separated. 95% CL of the LC(LT) levels. View Large In addition, this population also had significantly lower susceptibility (P < 0.05) to abamectin when compared with laboratory colony. The ratio of LC90/LC50 was 71.8 in this field population when compared with laboratory colony of 2.03. This field population bore a tolerance (RR 2.25-fold, see below) at LC50 and moderate level of resistance at LC90 (79.69-fold) to abamectin (Table 3). The ratio of RR at LC90 and RR at LC50 (Table 3) was 35.4. Tolerance Using the susceptibility of the laboratory colony as a baseline (Table 2), tolerance in this field population was noticed to pyriproxyfen and permethrin at both LC50 (LT50) and LC90 (LT90) levels. Tolerance was also encountered to Advance 375A ant bait at LC50 level, and to indoxacarb at LC90 level (Table 3). No Tolerance or Resistance Compared to laboratory colony of the same species bioassayed concurrently (Table 2), there was no tolerance or resistance in this field population to the following pesticides tested at both LC50 and LC90 levels: B.t.i., combination of B.t.i. and L. sphaericus, spinosad, spinetoram, methoprene, diflubenzuron, novaluron, temephos, imidacloprid, and fipronil. At the LC50 levels, this field population was as susceptible as laboratory colony to indoxacarb (Table 3). Discussion Global human population migration, freight exchange, demographic growth, and economic development have created new challenges for mosquito control and mosquito-borne disease management. In response to resurgence of traditional mosquito-borne illness and emergence of new mosquito-borne diseases worldwide, mosquito control by environmentally friendly interventions plays a crucial role in mitigation of disease burdens. Among the limited operational tools for mosquito control, products based on biorational active ingredients possess numerous advantages over conventional pesticides (Su 2016a,b). The efficacy of integrated mosquito control is often compromised by evolution of resistance to the pesticides used (Su 2016a). The resistance status mostly caught attention by reduced efficacy or control failure in mosquito control operations, if there is no routine pesticide susceptibility monitoring program in place along with product applications. A field population of Cx. pipiens that showed poor response to VectoLex WDG treatment was collected from downtown Chico, CA, the susceptibility of which to 15 common pesticides of public health, urban and agricultural uses was determined and compared with reference laboratory colony of the same species. High level of resistance to L. sphaericus was confirmed in this field collection of Cx. pipiens from Chico, CA. This is considered the first report of resistance in North America since the introduction of this biopesticide in 1990s (Su 2016a,b). The ratio of LC90/LC50 to VectoLex was 93.2, while this ratio was 2.8 in laboratory colony (Table 2), indicating the field population is highly heterogeneous when compared with laboratory colony in terms of response to L. sphaericus treatment. The ratio of RR at LC90 (22,878.6-fold) and RR at LC50 (687.4-fold) (Table 3) was high, indicating that there were extremely resistant individuals in this highly resistant population. The observed resistance development was assumedly attributable to continuous use of VectoLex WDG for 1.5 yr to the time when control failed. The first resistance to L. sphaericus in field populations occurred in Cx. pipiens in southern France with 70-fold at LC50 because of extended field applications (Sinègre et al. 1994). This first report was followed by more cases of resistance in Cx. pipiens complex in Brazil, India, China, Thailand, and Tunisia. The magnitude of resistance development in response to the field application varied greatly among countries with positive reports, depending on unknown historic exposure to naturally existing strains, population genetics, and gene exchange with refugee populations, as well as product applications. Furthermore, once mosquitoes have developed resistance to a given strain of L. sphaericus, they are also often resistant to other strains because of the similarity of the binary toxins in most strains. The cross-resistance among different strains is mild between the strains that also produce Mtx toxins. Fortunately, mosquitoes that have developed resistance to various strains of L. sphaericus remain susceptible to B.t.i. (Su 2016b). No case of resistance to L. sphaericus in North America has ever been reported in wild mosquito populations thus far, although a substantial amount of L. sphaericus products has been applied, particularly since the invasion of the WNV. During 1990–1993, the susceptibility of Cx. pipiens complex to L. sphaericus was determined in 31 collections across California, before the registration of this agent in the state in 1996. Variation was about five-fold at the LC50 and LC95 (Wirth et al. 2001). Reduced susceptibility was noticed in Culex spp. breeding in dairy lagoons in southern California soon after L. sphaericus was registered and applied for three consecutive yr during 1996–1998 (Su et al. 2001). Furthermore, this population also showed a significantly lower susceptibility to abamectin B1 (IRAC Group 6—chloride channel activators) in a highly heterogeneous style, as indicated by high LC90/LC50 ratio (71.8). The tolerance level (RR 2.25) at LC50 and resistance level at LC90 (RR 79.69) were widely separated with a ratio of 35.4, indicating this population was blended with susceptible and significantly resistant individuals. Abamectin B1 is a mixture of 80% avermectin B1a and 20% avermectin B1b. Currently, there is no product based on abamectin B1 that is registered for mosquito control as larvicide or adulticide. Products containing abamectin B1 have been commonly used in bait and spray formulations to control ants, cockroaches, and mites in urban environment, gardens, lawns, and other horticulture. It is expected that mosquito populations can be exposed to these active ingredients unintentionally, leading to the development of tolerance even resistance. Tolerance of this field population to pyriproxyfen and permethrin at both LC50 (LT50) and LC90 (LT90) levels was indicated. Pyriproxyfen is a juvenile hormone analog belonging to IRAC Group 7 (juvenile hormone mimics), which interrupts the metamorphosis of mosquitoes from late fourth instars through pupae to adults. Pyriproxyfen was introduced to the United States in 1996 for controlling whiteflies. Various products containing this insect growth regulators are used to control a wide variety of urban and household pestiferous insects. It is not a surprise that mosquito populations have been exposed to pyriproxyfen in the products that were applied to control other target species. In a more obvious case, products based on pyrethrins and pyrethroids (IRAC Group 3A—sodium channel modulators) have been extensively used to combat pestiferous arthropods in agriculture, urban and households. Sublethal exposures to these ingredients are almost unavoidable. Tolerance or resistance to pyrethroids are quite common in mosquitoes, houseflies, and other urban pestiferous species (Zhu et al. 2016). Tolerance was also noticed to abamectin at LC50, which was in connection with high-level resistance to this active ingredient as described previously at LC90 level, a reflection of high genetic heterogeneity in terms of susceptibility to abamectin. Finally, the tolerance to indoxacarb (IRAC Group 22—voltage-dependent sodium channel blockers) was observed at LC90 level. This relatively new active ingredient has been formulated to baits to control ants, cockroaches, and others. Tolerance at LC90 only indicated existence of some hardy individuals that were mixed with the majority of susceptible ones. There was no tolerance or resistance in this field population to B.t.i., an IRAC Group 11 pesticide, which has very low risk of resistance development, and plays an important role in resistance management toward other public health pesticides (Su 2016b). Combination of B.t.i. and L. sphaericus as in commercial products VectoMax CG or FG, takes advantages of each microbial larvicide’s strengths, and reduces the limitations that each possesses. Mosquito populations that show resistance to other pesticides remained susceptible to this combination (Su and Cheng 2014a,b; Su 2016a,b). The studied field population was highly susceptible to spinosad and spinetoram, the IRAC Group 5 pesticides—nicotinic acetylcholine receptor allosteric modulators, although spinosad is prone to induce resistance development in insects (Su and Cheng 2014a; Sparks et al. 2012). The products based on methoprene (IRAC Group 7A), have been used to combat various mosquito species for decades, and low to high levels of resistance in Culex spp. and Aedes spp. have been documented in both laboratory colonies and field populations (Su 2016b). High susceptibility to methoprene was indicated in this field population, which might be attributable to the fact that products based on methoprene for urban pest control are not common. No tolerance was indicated to diflubenzuron and novaluron (IRAC Group 15—the chitin synthesis inhibitors), where diflubenzuron was not commonly used in urban areas, and novaluron is relatively new to pest and vector control. High susceptibility was observed to one of the organophosphate temephos (IRAC Group 1B—acetylcholinesterase inhibitors). The restrictive use of organophosphate products in urban areas may have reduced the exposure of mosquito populations to this group of pesticides. As to Imidacloprid (IRAC Group 4—nicotinic acetylcholine receptor agonists) and fipronil (IRAC Group 2—GABA-gated chloride channel antagonists), currently there are no products based on these two active ingredients labeled for mosquito control. Some products, mostly baits, are labeled to control garden and urban pests. These findings may shed some light to consider future use of these ingredients for mosquito control. There was no tolerance to indoxacarb at LC50, indicating ample numbers of susceptible individuals existed in the population studied even though tolerance was noticed at LC90 level as discussed earlier. The products based on above active ingredients can be considered to control larval population studied if product labels are in place. In summary, since mid-1990s, numerous cases of resistance to L. sphaericus in field mosquito populations have been reported in France, Brazil, India, China, Thailand, and Tunisia. The products based on L. sphaericus have been extensively applied to combat WNV vectors and other nuisance species since 1990s in the United States and elsewhere. It is important to monitor the susceptibility to L. sphaericus products periodically to ensure the field efficacy. At the same time, susceptibility monitoring to other pesticides of public health, urban and agriculture uses is also strongly recommended because mosquitoes are quite often exposed to other pesticides accidently and their susceptibility can be compromised such as in the current paper about resistance or tolerance to abamectin, pyriproxyfen, permethrin, and to a lesser extent to indoxacarb. Acknowledgments The authors are grateful to Dr. Alec Gerry, Department of Entomology, University of California at Riverside (Riverside, CA) for provision of Radiant SC, and to Dr. Barry Tyler, Pestalto Environmental Health Services Inc. (Hamilton, ON, Canada) for the product sample of Mosquiron 0.12CRD. They also specially thank Michelle Brown, Ph.D., Manager at the West Valley Mosquito and Vector Control District, Ontario, CA, for her constructive review of this manuscript. References Cited Ahmed, I., A. Yokota, A. Yamazoe, and Fujiwara T.. 2007. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Int. J. Syst. Evol. Microbiol . 57: 1117– 1125. Google Scholar CrossRef Search ADS PubMed  LeOra Software. 1987. POLO-PC: A user’s guide to probit or logit analysis . LeOra Software, Berkeley, CA Sinègre, G., Babinot M., Quermel J. M., and Gavon B.. 1994. First field occurrence of Culex pipiens resistance to Bacillus sphaericus in southern France. In Proceedings of 8thh European Meet. Soc. Vector Ecol, September 5–8, 1994, Barcelona, Spain, Society for Vector Ecology, Santa Ana, CA, 1997. P17. Sparks, Dripps T. C., J. E., Watson G. B., and Paroonagian D.. 2012. Resistance and cross-resistance to the spinosyn - a review and analysis. Pestic. Biochem. Physiol . 102: 1– 10. Su, T. 2016a. Microbial control of pest and vector mosquitoes in North America north of Mexico. In Lacey, L. (ed.), Microbial control of insect and mite pests . Academic Press, San Diego, CA. pp. 393– 407. Google Scholar CrossRef Search ADS   Su, T. 2016b. Resistance and its management to microbial and insect growth regulator larvicides in mosquitoes. In Trdan, S. (ed.), Insecticides resistance , InTech Europe, Rijeka, Croatia. pp. 135– 154. Google Scholar CrossRef Search ADS   Su, T., and Cheng M. L.. 2014a. Laboratory selection of resistance to spinosad in Culex quinquefasciatus (Diptera: Culicidae). J. Med. Entomol . 51: 421– 427. Google Scholar CrossRef Search ADS   Su, T., and Cheng M. L.. 2014b. Cross resistances in spinosad-resistant Culex quinquefasciatus (Diptera: Culicidae). J. Med. Entomol . 51: 428– 435. Google Scholar CrossRef Search ADS   Su, T., and Mulla M. S.. 2004. Documentation of high level Bacillus sphaericus-resistance in tropical Culex quinquefasciatus populations from Thailand. J. Am. Mosq. Control Assoc . 20: 405– 411. Google Scholar PubMed  Su, T., Soliman B. A., Chaney J. D., Mulla M. S., and Beehler J. W.. 2001. Susceptibility of Culex mosquitoes breeding in dairy ponds before and after treatment with Bacillus sphaericus formulation. Proc. Pap. Mosq. Vector Control Assoc. Calif . 69: 110– 116. Throne, J., Weaver D. K., Chew V., and Baker J. E.. 1995. Probit analysis of correlated data: Multiple observations over time at one concentration. J. Econ. Entomol . 88: 1510– 1512. Google Scholar CrossRef Search ADS   Zhu, F., Lavine L., O’Neal S., Lavine M., Foss C., and Walsh D.. 2016. Insecticide resistance and management strategies in urban ecosystems. Insects  7: 2. Google Scholar CrossRef Search ADS   Wirth, M. C. 2010. Mosquito resistance to bacterial larvicidal toxins. OpenToxinol. J . 3: 126– 140. Google Scholar CrossRef Search ADS   Wirth, M. C., Ferrari J. A., and Georghiou G. P.. 2001. Baseline susceptibility to bacterial insecticides in populations of Culex pipiens complex (Diptera: Culicidae) from California and from the Mediterranean Island of Cyprus. J. Med. Entomol . 94: 920– 928. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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Journal of Medical EntomologyOxford University Press

Published: Mar 1, 2018

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