**3.** *Bacillus sphaericus*

to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one *Ae. sticticus* population. This study facilitated information of susceptibility status to individual Cry toxins of *B.t.i*. Bioassays with individual Cry toxins allow a more sensitive monitoring of *B.t.i.* resistance in the field [21]. When individual Cry toxins were tested against the LiTOX strain that carried low-level resistance to *B.t.i.*, increased resistance of 68-, 9-, and 9-fold to Cry4Aa, Cry4Ba, and

Exposures to individual toxins of *B.t.i.* are conducive to resistance development, where *Cx. quinquefasciatus* developed high resistance to individual toxins in absence of Cyt1A toxin. Resistance became evident in the mosquitoes that were selected with a single toxin (CryIVD), reaching the highest level of 913-fold. Resistance developed at a slower pace and reached a lower level when being selected with CryIVA + CryIVB. Resistance levels were further suppressed when being selected with CryIVA + CryIVB + CryIVD or full combination of all four toxins. These results highlighted the importance of the full combination of toxins found in wild *B.t.i.* in resistance management [23]. This fact, i.e., target species rapidly develops resistance to individual toxins but not to the toxin complex from the wild strain, also exists in other *B.t.* subspecies studied, for instance, *B. thuringiensis* subsp *jegathesan* [24]. Crossresistance occurs among the Cry toxin of *B.t.i*. Resistance was generally highest toward the toxin(s) that were used in the selections. The strain that was selected with CryIVD demon‐ strated significant cross-resistance to CryIVA + CryIVB, and vice versa. Strain that was selected with naturally occurring insecticidal toxin mixture in *B.t.i.* only showed a low level of resist‐ ance to this mixture, but much higher levels of resistance occurred to individual CryIV toxins and also to combinations of 2 or 3 CryIV toxins. The fact that all of the selected populations stayed susceptible to the natural toxin mixture in *B.t.i.* suggested that the CytA toxin with different sequence and mode of action from the CryIV toxins plays an important role in suppressing resistance to CryIV toxins [25]. Cross-resistance among Cry toxins can be extended to other *B.t.* subspecies. *Cx. quinquefasciatus* that are resistant to *B.t.i.* Cry toxins also show crossresistance to Cry11B from *B.t. jegathesan* [26], but not to Cry19A from the same species [27]. Cyt1A, a cytolytic endotoxin of *B. thuringiensis*, does not possess significant larvicidal activity alone [28]. However, it plays a critical role in overcoming, preventing, and delaying resistance

The high levels of resistance to CryIV in *Cx. quinquefasciatus* was reduced remarkably by combining CryIV with sublethal quantities of CytA. A 127-fold resistance to a combination of CryIVA, B, and D was completely suppressed by combining CytA in a 1:3 ratio with CryIVA, CryIVB, and CryIVD. Combining the CytA with CryIVA and CryIVB also completely sup‐ pressed 217-fold resistance to the latter toxins, whereas the combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from >1000-fold to < 8-fold [29]. The same species of mosquitoes were subjected to selection for 20 generations using the recombi‐ nant strains of *B.t.* that produced Cyt1Aa, Cry11Aa, or a 1:3 mixture. The resistance in the Cry11Aa strain- and Cyt1Aa strain-selected populations reached 1,237-fold and 242-fold, respectively. On the other hand, the resistance only reached 8-fold in the population that was selected with the Cyt1Aa and Cry11Aa (1:3) strain. Mosquitoes that were selected by Cyt1Aa-Cry11Aa for 48 generations only developed 9.3-fold resistance to Cry11Aa. Obviously, resistance to Cry11Aa developed at a much slower pace in the presence than in the absence of

Cry11Aa protoxins, respectively was revealed [22].

138 Insecticides Resistance

development to Cry toxins.

The mosquitocidal activity of some strains of *B. sphaericus* has long been recognized. Up to date, 49 serotypes over 300 strains of *B. sphaericus* have been identified, among which 9 serotypes 16 strains showed activity against mosquito larvae at different extent. 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* (Diptera: Simuliidae) in Nigeria. Recently, *B. sphaericus* was renamed as *Lysinibacillus sphaeri‐ cus* Meyer and Neide [35]. Active strains produce parasporal inclusions during sporulation, which contains crystal binary toxins. Some strains also synthesize noncrystal Mtx. The mode of action of the binary toxins is somewhat similar in general to *B.t.i*. toxins with detail differ‐ ences at molecular levels. The receptor of the binary toxins is a 60-kDa alpha-glucosidase, which is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor. As *B.t.i*., *B. sphaericus* also belongs to IRAC Group 11. Compared with *B.t.i*., the target species spectrum is narrower, some *Aedes* spp., for example, *Ae. aegypti* is much less susceptible than *Culex* spp. to *B. sphaericus*. During the past decades, numerous products have been developed using various strains and applied to control *Culex* spp. worldwide.

#### **3.1. Field occurrence**

The earliest resistance in field populations was reported in *Cx. pipiens*in southern France where the resistance ratio at LC50 was 70-fold as a result of extended field applications [36]. A fieldcollected population of *Cx. quinquefasciatus* larvae from an urban area of Recife in Brazil, which has been treated for 2 years with *B. sphaericus*, was found to be about 10-fold less susceptible than the untreated control field populations [37]. The field resistance to strain 1593 was reported in Kochi, India, in the same year. The larvae of *Cx. quinquefasciatus* from the sprayed area showed LC50 and LC90 values that were 146 and 180 times higher than corresponding values for a susceptible strain from an unsprayed area after 35 rounds of application over 2 years. The subsequent laboratory selection of the collection from the treated area resulted in much higher levels of resistance, 6,223- and 31,325-fold at LC50 and LC90, respectively [38]. The similar situation also happened to strain B101 in the same mosquito species where low levels of resistance occurred in response to field applications; the population reached 52,000-fold resistance after selection for 6 generations in the laboratory [39]. Field *Cx. pipiens* mosquitoes that were collected after a control failure with Spherimos in southern France developed high resistance (>10,000-fold) after <8 generations of laboratory selection [40]. In southern China, a flowable formulation of strain C3-41 was continuously used for 8 years to control *Cx. quin‐ quefasciatus* larvae. The resistance of field-collected larvae at LC50 was 22,672-fold [41]. More occurrences on resistance to strain 2362 were reported later in France (5,958-fold) [42] and Tunisia (750-fold) [43]. Declined efficacy and control failure of *B. sphaericus* was noticed within 4 months after 5 treatments using VectoLex WDG at the dosages of 50–200 mg/m2 to control *Cx. quinquefasciatus* in Thailand [44]. A high level of *B. sphaericus* resistance was documented in this population. The resistance levels at LC50, depending on reference colonies, were 21,100 to 28,100-fold against VectoLex WDG (650 ITU/mg) or >125,000- to 200,000-fold against *B. sphaericus* technical-grade material (2000 ITU/mg) [45]. Between 1990 and 1993, the suscepti‐ bility of *Cx. pipiens* complex to *B. sphaericus* was determined in 31 collections from California, before the registration of this agent. Variation was about 5-fold at the LC50 and LC95 [16]. Similar results were obtained for *Culex* spp. breeding in dairy lagoons in southern California soon after *B. sphaericus* was registered and applied in California [46]. No case on resistance to *B. sphaeri‐ cus* in the USA has ever been reported in wild mosquito populations thus far regardless of the substantial amount of *B. sphaericus* products that has been applied, particularly since the invasion of the West Nile virus. The resistance development in response to the field application of *B. sphaericus* products varies greatly, depending on prior exposure to naturally existing strains, population genetic background, and gene exchange with untreated populations, as well as product application strategies.

#### **3.2. Laboratory studies**

Resistance to *B. sphaericus* in laboratory colonies of *Cx. pipiens* complex has been reported in different countries since 1994. Larvae of laboratory colony and field-collected (southern California) *Cx. quinquefasciatus* developed moderate level of resistance to strain 2362 (27- to 37 fold) in response to selection at LC80 for 80 generations [47]. This moderate level of resistance to strain 2362 in laboratory colony of the same species was reconfirmed by later studies on resistance management tactics [48, 49]. A previously untreated field population of *Cx. quinquefasciatus*, collected near Bakersfield, California, survived the LC50 of *B. sphaericus* that was 7,000 times higher than in the susceptible reference colony after 12 generations of selection at LC95. Late and early instar larvae in this study showed the similar levels of resistance [50]. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a fieldcollected low-level-resistant colony of *Cx. quinquefasciatus* developed >144,000-fold and 46.3 fold resistance, respectively. A field-collected susceptible colony was selected with strain 2362 and IAB59 for 46 and 12 generations and reached >162,000-fold and 5.7-fold resistance to the two agents, respectively. The slower evolution of resistance against strain IAB59 may be attributable to the presence of another larvicidal factor [51]. However, in another study, selection by treating about 15,000 of 3rd and 4th instar larvae at each generation at LC70 of IAB59 resulted in 40,000-fold resistance after a much longer selection for 72 generations [52]. The resistance development to selection depends on genetic background, size of population used, selection pressure, length of selection, etc. Resistance ratio is also dependent on the susceptibility levels of the reference population. The resistance to *B. sphaericus* is fairly stable in absence of selection pressure. For instance, bioassays showed that the frequency of resistant larvae did not decrease throughout 11 generations after interruption of selection, and it was associated with a similar frequency of larvae lacking the Cqm1alpha-glucosidase receptor. The frequency of the cqm1 (REC) allele remained stable throughout 11 generations without further selection [53].

Furthermore, once mosquitoes develop resistance to a given strain of *B. sphaericus*, they are also often resistant to other strains because of the similarity of the binary toxins in most strains. Fortunately, mosquitoes that have developed resistance to various strains of *B. sphaericus* remain susceptible to *B.t.i.* [38, 39, 44, 45, 48–52, 55–59]. The cross-resistance among different strains is mild between the strains that also produce Mtx toxins. For example, cross-resistance in strain 2362-resistant *Cx. quinquefasciatus* was detected toward strains 1593 and 2297, but little or no cross-resistance was observed toward strains IAB59 or ISPC5 [50]. The resistant colonies resulted from the selection with C3-41 or 2362 showed very high levels of cross-resistance to strains 2362, C3-41, 1593, and 2297 but only low-level cross-resistance to IAB59, LP1-G, and 47-6B, which all produce a major 49-kDa protein, another mosquitocidal factor. On the other hand, the IAB59 selected colonies showed high cross-resistance to both strains C3-41 and 2362 [57].

#### **3.3. Resistance mechanism**

collected population of *Cx. quinquefasciatus* larvae from an urban area of Recife in Brazil, which has been treated for 2 years with *B. sphaericus*, was found to be about 10-fold less susceptible than the untreated control field populations [37]. The field resistance to strain 1593 was reported in Kochi, India, in the same year. The larvae of *Cx. quinquefasciatus* from the sprayed area showed LC50 and LC90 values that were 146 and 180 times higher than corresponding values for a susceptible strain from an unsprayed area after 35 rounds of application over 2 years. The subsequent laboratory selection of the collection from the treated area resulted in much higher levels of resistance, 6,223- and 31,325-fold at LC50 and LC90, respectively [38]. The similar situation also happened to strain B101 in the same mosquito species where low levels of resistance occurred in response to field applications; the population reached 52,000-fold resistance after selection for 6 generations in the laboratory [39]. Field *Cx. pipiens* mosquitoes that were collected after a control failure with Spherimos in southern France developed high resistance (>10,000-fold) after <8 generations of laboratory selection [40]. In southern China, a flowable formulation of strain C3-41 was continuously used for 8 years to control *Cx. quin‐ quefasciatus* larvae. The resistance of field-collected larvae at LC50 was 22,672-fold [41]. More occurrences on resistance to strain 2362 were reported later in France (5,958-fold) [42] and Tunisia (750-fold) [43]. Declined efficacy and control failure of *B. sphaericus* was noticed within

4 months after 5 treatments using VectoLex WDG at the dosages of 50–200 mg/m2

well as product application strategies.

**3.2. Laboratory studies**

140 Insecticides Resistance

*Cx. quinquefasciatus* in Thailand [44]. A high level of *B. sphaericus* resistance was documented in this population. The resistance levels at LC50, depending on reference colonies, were 21,100 to 28,100-fold against VectoLex WDG (650 ITU/mg) or >125,000- to 200,000-fold against *B. sphaericus* technical-grade material (2000 ITU/mg) [45]. Between 1990 and 1993, the suscepti‐ bility of *Cx. pipiens* complex to *B. sphaericus* was determined in 31 collections from California, before the registration of this agent. Variation was about 5-fold at the LC50 and LC95 [16]. Similar results were obtained for *Culex* spp. breeding in dairy lagoons in southern California soon after *B. sphaericus* was registered and applied in California [46]. No case on resistance to *B. sphaeri‐ cus* in the USA has ever been reported in wild mosquito populations thus far regardless of the substantial amount of *B. sphaericus* products that has been applied, particularly since the invasion of the West Nile virus. The resistance development in response to the field application of *B. sphaericus* products varies greatly, depending on prior exposure to naturally existing strains, population genetic background, and gene exchange with untreated populations, as

Resistance to *B. sphaericus* in laboratory colonies of *Cx. pipiens* complex has been reported in different countries since 1994. Larvae of laboratory colony and field-collected (southern California) *Cx. quinquefasciatus* developed moderate level of resistance to strain 2362 (27- to 37 fold) in response to selection at LC80 for 80 generations [47]. This moderate level of resistance to strain 2362 in laboratory colony of the same species was reconfirmed by later studies on resistance management tactics [48, 49]. A previously untreated field population of *Cx. quinquefasciatus*, collected near Bakersfield, California, survived the LC50 of *B. sphaericus* that was 7,000 times higher than in the susceptible reference colony after 12 generations of selection at LC95. Late and early instar larvae in this study showed the similar levels of resistance [50].

to control

It is mostly believed that recessive genetic mechanism is involved in resistance to *B. sphaeri‐ cus* [20, 42, 43, 50, 52, 59]. Among the multiple theories, the predominant one is the lack of specific binding of binary toxins to alpha-glucosidase, which act as midgut receptors [37, 53, 59–63]. The main reason leading the lack of specific binding is related to the deletions of gene encoding the receptor alpha-glucosidase [64–66], where the integrity of the receptor is compromised. However, the resistance in field *Cx. pipiens* mosquitoes after a control failure of Spherimos in southern France is not associated with any loss of binding affinity between brush border membrane fractions and toxins [40]. The similar results were also seen in Brazil, with additional findings of slight declined receptor density [37]. Behavioral modifications such as reduced ingestion on toxins [67] and other unknown mechanisms [40, 42] are also involved.

#### **3.4. Resistance management**

*B.t.i.* can be used as a powerful tool to mitigate resistance to *B. sphaericus* in mosquitoes. The susceptibility to *B. sphaericus* was partially restored by the selection of previously resistant colony with *B.t.i.* alone for 10 generations. After this colony was reexposed to *B. sphaericus* for 20 generations, resistance to *B. sphaericus* surged back to a stable level. Selections of *B. sphaer‐ icus*-resistant colonies with *B.t.i.* and *B. sphaericus* in rotation or mixture lead to steady decline of resistance over 30 generations [48]. Resistance to *B. sphaericus* can be delayed or prevented by the mixture of *B.t.i.* and *B. sphaericus* because of the synergistic action among 4 toxins, particularly the presence of Cyt1A [49, 68–71]. While *B. sphaericus* resistance increased after F15 in response to the selection using *B. sphaericus* alone, the rotation of *B. sphaericus* and *B.t.i.* surprisingly resulted in much higher level and faster emergence of resistance to *B. sphaericus*. However, selection with mixtures of *B.t.i.* and *B. sphaericus* for 36 generations showed no emergence of resistance to *B. sphaericus* [49]. Recently, the recombinant that produces toxins from both *B.t.i.* and *B. sphaericus*, even at greater amount than the wild type of bacteria [72– 74], provides another path for not only mitigation of resistance but also enhancement of larvicidal activity and efficacy. The combination of *B. sphaericus* with botanical pesticides such as azadirachtin from the neem oil is also considered as an alternative to mitigate resistance development to *B. sphaericus* in mosquitoes [75].

Efforts were made to find practical strategies for controlling resistant mosquitoes and to prevent or delay the development of resistance in wild mosquito populations. In Nonthaburi Province, Thailand, the larvae of *Cx. quinquefasciatus* that were highly resistant (>125,000-fold) to *B. sphaericus* strain 2362 were successfully controlled with applications of *B.t.i.* alone or in combination with *B. sphaericus*. In order to elucidate resistance management strategy in the field, one selected site was treated with *B. sphaericus* 2362 alone and the other treated with a mixture of *B. sphaericus* 2362 and *B.t.i*. Moderate resistance was detected after the 9th treatment and almost complete control failure occurred by the 17th treatment in the site that was treated with *B. sphaericus* 2362 alone. However, no noticeable change in susceptibility to *B. sphaericus* was detected after 9 treatments with the mixture at another site. During this period, the site treated with *B. sphaericus* alone required 19 treatments, whereas the site treated with mixtures only took 9 treatments because of comparatively slower resurgence of larval populations [44]. In this resistance population, the resistance levels to the mixtures of *B. sphaericus* + *B.t.i.* increased steadily upon the increase of *B. sphaericus* ratios in the mixtures from 50%, 75%, 90%, 95%, to 99%. The resistance levels to the mixtures with various ratios of *B. sphaericus* and *B.t.i*., however, were substantially lower than that in *B. sphaericus* alone, suggesting that the addition of *B.t.i.* to *B. sphaericus* enhanced the mosquitocidal activity (synergism) against these highly *B. sphaericus*-resistant *Cx. quinquefasciatus*. Moderate tolerance and low levels of resistance to *B. sphaericus*/*B.t.i.* recombinant (RR 7.29–12.75 at LC50 and 5.15–13.40 at LC90) were also noted in this *B. sphaericus*-resistant population [45]. The similar success was achieved in southern China. After 6 months of treatment with *B.t.i*. in the *B. sphaericus*-resistant popula‐ tions, their susceptibility to *B. sphaericus* C3-41 recovered, with the resistance ratio of fieldcollected larvae declining from 22,672-fold to 5.67-fold [41]; the gene exchange with populations in surrounding untreated areas may also have contributed to the rapid decline of resistance levels. There is a lack of cross-resistance between binary toxins and Mtx toxins [76], indicating that Mtx could be a potential tool to manage resistance to binary toxins in the future. It was suggested that once resistance to *B. sphaericus* is detected in the field, its use should be discontinued until the mosquito population becomes susceptible again because of the decline in number of resistant individuals [77].

#### **3.5. Fitness cost of resistance**

colony with *B.t.i.* alone for 10 generations. After this colony was reexposed to *B. sphaericus* for 20 generations, resistance to *B. sphaericus* surged back to a stable level. Selections of *B. sphaer‐ icus*-resistant colonies with *B.t.i.* and *B. sphaericus* in rotation or mixture lead to steady decline of resistance over 30 generations [48]. Resistance to *B. sphaericus* can be delayed or prevented by the mixture of *B.t.i.* and *B. sphaericus* because of the synergistic action among 4 toxins, particularly the presence of Cyt1A [49, 68–71]. While *B. sphaericus* resistance increased after F15 in response to the selection using *B. sphaericus* alone, the rotation of *B. sphaericus* and *B.t.i.* surprisingly resulted in much higher level and faster emergence of resistance to *B. sphaericus*. However, selection with mixtures of *B.t.i.* and *B. sphaericus* for 36 generations showed no emergence of resistance to *B. sphaericus* [49]. Recently, the recombinant that produces toxins from both *B.t.i.* and *B. sphaericus*, even at greater amount than the wild type of bacteria [72– 74], provides another path for not only mitigation of resistance but also enhancement of larvicidal activity and efficacy. The combination of *B. sphaericus* with botanical pesticides such as azadirachtin from the neem oil is also considered as an alternative to mitigate resistance

Efforts were made to find practical strategies for controlling resistant mosquitoes and to prevent or delay the development of resistance in wild mosquito populations. In Nonthaburi Province, Thailand, the larvae of *Cx. quinquefasciatus* that were highly resistant (>125,000-fold) to *B. sphaericus* strain 2362 were successfully controlled with applications of *B.t.i.* alone or in combination with *B. sphaericus*. In order to elucidate resistance management strategy in the field, one selected site was treated with *B. sphaericus* 2362 alone and the other treated with a mixture of *B. sphaericus* 2362 and *B.t.i*. Moderate resistance was detected after the 9th treatment and almost complete control failure occurred by the 17th treatment in the site that was treated with *B. sphaericus* 2362 alone. However, no noticeable change in susceptibility to *B. sphaericus* was detected after 9 treatments with the mixture at another site. During this period, the site treated with *B. sphaericus* alone required 19 treatments, whereas the site treated with mixtures only took 9 treatments because of comparatively slower resurgence of larval populations [44]. In this resistance population, the resistance levels to the mixtures of *B. sphaericus* + *B.t.i.* increased steadily upon the increase of *B. sphaericus* ratios in the mixtures from 50%, 75%, 90%, 95%, to 99%. The resistance levels to the mixtures with various ratios of *B. sphaericus* and *B.t.i*., however, were substantially lower than that in *B. sphaericus* alone, suggesting that the addition of *B.t.i.* to *B. sphaericus* enhanced the mosquitocidal activity (synergism) against these highly *B. sphaericus*-resistant *Cx. quinquefasciatus*. Moderate tolerance and low levels of resistance to *B. sphaericus*/*B.t.i.* recombinant (RR 7.29–12.75 at LC50 and 5.15–13.40 at LC90) were also noted in this *B. sphaericus*-resistant population [45]. The similar success was achieved in southern China. After 6 months of treatment with *B.t.i*. in the *B. sphaericus*-resistant popula‐ tions, their susceptibility to *B. sphaericus* C3-41 recovered, with the resistance ratio of fieldcollected larvae declining from 22,672-fold to 5.67-fold [41]; the gene exchange with populations in surrounding untreated areas may also have contributed to the rapid decline of resistance levels. There is a lack of cross-resistance between binary toxins and Mtx toxins [76], indicating that Mtx could be a potential tool to manage resistance to binary toxins in the future. It was suggested that once resistance to *B. sphaericus* is detected in the field, its use should be discontinued until the mosquito population becomes susceptible again because of the decline

development to *B. sphaericus* in mosquitoes [75].

142 Insecticides Resistance

in number of resistant individuals [77].

In a laboratory studies [77], the resistant strains showed some disadvantages such as lower fecundity and fertility, but higher survival rates were observed at the same time. The immature stages of the females from the resistant population developed slightly faster as compared with those of the susceptible strains, which could result in a shorter generation time. The similar findings are that the resistant colony showed lower fecundity and fertility and slower devel‐ opment than the susceptible colony [78]. However, the opposite results were achieved in another study where the resistant colony did not display biological costs regarding fecundity, fertility, and pupal weight [53].

#### **4. Spinosyns**

Spinosad, a biopesticide consisting of spinosyn A (C41H65NO10) and D (C42H67NO10), is pro‐ duced by a naturally occurring, soil-dwelling bacterium, *Saccharopolyspora spinosa* Mertz and Yao. As a new class of polyketide-macrolide insecticide that acts as nicotinic acetylcholine receptor (nAChR) allosteric modulator, spinosad is categorized as Group 5 insecticide by IRAC. Spinosad exerts pesticidal activity after ingestion and cuticle absorption against a broad spectrum of susceptible insect species, by stimulating nACh and γ-aminobutyric acid (GABA) receptors and causing rapid excitation of the insect nervous system. The application of spinosad products for mosquito control is relatively new; studies to evaluate resistance development risk and resistance management strategy are rather rare. The first attempt was made in *Cx. quinquefasciatus*. Surviving late instars and pupae were collected from a semifield evaluation on Natular® XRG (2.5% spinosad), and a laboratory colony was established. Selection pressure was applied at LC70– 90 levels to 10,000–15,000 of the late 3rd and the early 4th instar larvae of each generation after initial lethal levels of Natular XRG against this colony were determined. Susceptibility changes upon selection were determined every other gener‐ ation. The susceptibility to spinosad in this selected population gradually and steadily declined from generation F1 to F35. From generations F37 to F45, the susceptibility decreased at a much faster pace. For reference purposes, the susceptibility of freshly collected wild populations as well as a laboratory reference colony of the same species was also determined concurrently. By comparing with the wild populations and laboratory reference colony for resistance ratio calculation, spinosad tolerance was observed from the first 9 generations. Resistance increased gradually from generations F11 to F35 and elevated significantly from generations F37 to F45, when resistance ratios reached 1415.3- to 2229.9-fold at LC50 and 9,613.1- to 17,062.6-fold at LC90. The exponential elevation of resistance levels throughout selection indicated that a recessive mechanism might have been involved during resistance development to spinosad [79, 80]. The spinosad-resistant *Cx. quinquefasciatus* with various levels of resistance was found not to be cross-resistant to *B.t.i.*, a combination of *B.t.i.* and *B. sphaericus*, methoprene, pyri‐ proxyfen, diflubenzuron, novaluron, temephos, or imidacloprid. However, it showed various levels of cross-resistance to *B. sphaericus*, spinetoram, abamectin, and fipronil. On the other hand, a long-term laboratory colony of *Cx. quinquefasciatus* that is highly resistant to *B. sphaericus* [50] was as susceptible as laboratory reference colony to spinosad and spinetoram. Field-collected and laboratory-selected *Cx. quinquefasciatus* that were resistant to methoprene did not show cross-resistance to spinosad and spinetoram. The presence and absence of crossresistance to other pesticides in spinosad-resistant mosquitoes seemed to be related to their modes of actions [81].
