**2.** *Bacillus thuringiensis* **subsp** *israelensis* **(***B.t.i.***)**

The entomopathogenic *Bacillus* was first discovered in 1901 by Japanese biologist Ishiwata Shigetane, who was investigating the cause of the sotto disease (sudden-collapse disease) that was killing large populations of silkworms. Shigetane named the bacterium *Bacillus sotto*, but the name was later ruled invalid. In 1911, this pathogen was rediscovered in Germany by Ernst Berliner, who isolated it from Mediterranean flour moth *Anagasta kuehniella* (Zeller, 1879) caterpillars that suffered *Schlaffsucht.* He named it *Bacillus thuringiensis*, after the German town Thuringia where the moth was found [1]. Up to date, at least 70 serotypes, more than 80 subspecies have been identified, among which 14 serotypes and 16 subspecies show lethal activities against mosquito larvae. *Bacilllus thuringiensis israelensis* (*B.t.i*.), a subspecies belong‐ ing to serotype H-14, was discovered from a natural mosquito habitat located in Israel desert in 1976 [2, 3]. Four synergistic endotoxins including Cry4A, Cry4B, Cry 11A, and Cyt1A are produced during sporulation of *B.t.i.* [4–7]. These protoxins are activated by enzymatic proteolysis activities under high pH environment in mosquito midgut. The activated toxins combine with the receptors located at the epithelium cells in midgut brush border and cause subsequent pathological consequences and death of the target species. Toxins of *B.t.i.* are toxic to species in nematoceran group including culicidae, simuliidae [8, 9], chironomidae [10–12], and some fungus gnats. *B.t.i.* is categorized as Group 11 pesticide, i.e., microbial disruptor of insect midgut membranes by the Insect Resistance Action Committee (IRAC). During the past 35 years or so, *B.t.i.* products have been extensively used to control mosquitoes and blackflies and occasionally chironomid midges worldwide.

#### **2.1. Field occurrence**

**1. Introduction**

136 Insecticides Resistance

implemented.

**2.** *Bacillus thuringiensis* **subsp** *israelensis* **(***B.t.i.***)**

and occasionally chironomid midges worldwide.

Mosquitoes and mosquito-borne diseases remain one of the leading public health concerns and socioeconomic burdens of mankind globally, particularly in tropical and subtropical regions. Nowadays, human and animal population movement, freight exchange, fast demo‐ graphic growth, economic development, and subsequent environmental impact further elevate the scope and magnitude of the problem. Mosquito control is often the only or most effective way of the integrated management to combat mosquito-borne illnesses. Considering the strict governmental regulations, high environmental vulnerability, and increasing demand of mosquito control upon emergence, and spreading of mosquito-borne diseases, ecologically friendly management approaches based on microbial and insect growth regulator larvicides have been the great promise for their high activity and efficacy, target specificity, and envi‐ ronmental and nontarget safety profile. However, the development of resistance in the mosquito populations to these biorational larvicides has been reported since the past decades. In order to maintain the sustainability of mosquito control, susceptibility monitoring and resistance management tactics toward these available control tools must be developed and

The entomopathogenic *Bacillus* was first discovered in 1901 by Japanese biologist Ishiwata Shigetane, who was investigating the cause of the sotto disease (sudden-collapse disease) that was killing large populations of silkworms. Shigetane named the bacterium *Bacillus sotto*, but the name was later ruled invalid. In 1911, this pathogen was rediscovered in Germany by Ernst Berliner, who isolated it from Mediterranean flour moth *Anagasta kuehniella* (Zeller, 1879) caterpillars that suffered *Schlaffsucht.* He named it *Bacillus thuringiensis*, after the German town Thuringia where the moth was found [1]. Up to date, at least 70 serotypes, more than 80 subspecies have been identified, among which 14 serotypes and 16 subspecies show lethal activities against mosquito larvae. *Bacilllus thuringiensis israelensis* (*B.t.i*.), a subspecies belong‐ ing to serotype H-14, was discovered from a natural mosquito habitat located in Israel desert in 1976 [2, 3]. Four synergistic endotoxins including Cry4A, Cry4B, Cry 11A, and Cyt1A are produced during sporulation of *B.t.i.* [4–7]. These protoxins are activated by enzymatic proteolysis activities under high pH environment in mosquito midgut. The activated toxins combine with the receptors located at the epithelium cells in midgut brush border and cause subsequent pathological consequences and death of the target species. Toxins of *B.t.i.* are toxic to species in nematoceran group including culicidae, simuliidae [8, 9], chironomidae [10–12], and some fungus gnats. *B.t.i.* is categorized as Group 11 pesticide, i.e., microbial disruptor of insect midgut membranes by the Insect Resistance Action Committee (IRAC). During the past 35 years or so, *B.t.i.* products have been extensively used to control mosquitoes and blackflies

Generally, the risk of resistance development to wild-type *B.t.i*., i.e., the natural toxin complex, is very low. For example, *B.t.i.* products were widely used to control floodwater mosquitoes *Aedes vexans* (Meigen) over an area of approximately 500 km2 for more than 10 years in Rhine River area in Germany; no reduction in susceptibility was noticed [13]. One report, however, from New York, USA, showed low-level resistance in wild population of *Culex pipiens* L. Briefly, collections from Syracuse and Albany showed 33- to 41-fold and 6- to 14-fold resistance to *B.t.i*. Based on the considerable difference in resistance levels between the populations from Syracuse and Albany, it seems that there was lack of gene flow between these populations of *Cx. pipiens*, resulting in aggregation of resistant individuals [14]. *Culex pipiens* populations from Cyprus (2002–2008) showed dose–response values ranging approximately 8-fold to *B.t.i*., but no resistance was detected after years of application [15]. Between 1990 and 1993, the suscept‐ ibility of *Cx. pipiens* complex to *B.t.i.* was determined in 31 collections from California, USA. The samples were collected before the widespread use in California. Seven collections from the Mediterranean island of Cyprus, where no microbial insecticides have ever been used, also were tested. The collections from California during 1990–1991 exhibited 3- to 4-fold variations in susceptibility at LC50 and LC95. The collections from Cyprus in 1993 exhibited both higher mean LC values, and greater variability in those values, than the California collections. No significant geographic variation in susceptibility was observed among regions within Califor‐ nia [16].

#### **2.2. Laboratory studies**

Multiple attempts to select resistance in laboratory colonies of *Cx. pipiens* complex or *Aedes aegypti* L. for various generations only resulted in low-level and unstable resistance. Four populations of *Cx. quinquefasciatus* Say collected from Southern California were subjected to different levels of selection pressure for 11–60 generations; 4.1- to 16.5-fold resistance was achieved. The resistance tended to decline in absence of selection pressure [17]. One laboratory and two wild populations of *Ae. aegypti* were used in selection for resistance to *B.t.i.* After 14 generations of selection at LC50, a statistically significant decline (2-fold) in susceptibility was observed in the F15 of one wild strain only. Regression lines, LC50 values, and slopes of parental populations between strains did not differ significantly [18]. When larvae of *Cx. pipiens* were subjected repeatedly to selection pressure with *B.t.i*. in the laboratory, only 2.78-fold tolerance was induced as a result of 20 generations of selection, which decreased by about 58% after the selection was withdrawn for 3 generations. Larval selection with *B.t.i.* caused a reduction in the reproductive potential in resulted survivors [19]. Another study revealed 2- to 3-fold increase in the LC50 or LC90 values to *B.t.i.* preparation in the larvae of *Cx. quinquefasciatus* after 20 generations of selection, and these values fluctuated in different generations during the selection [20]. The laboratory selection with field-persistent *B.t.i.* toxins led to a 3.5-fold resistance to VectoBac WG in *Ae. aegypti* after 30 generations, but to relatively high levels of resistance to individual Cry toxins. Bioassay procedure was developed using each Cry toxin to detect cryptic *B.t.i.* resistance that evolved in field mosquito populations. Although no resistance to *B.t.i.* was detected in three *Aedes* mosquito species tested, an increased tolerance 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 Cry11Aa protoxins, respectively was revealed [22].

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 development to Cry toxins.

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 Cyt1Aa [30]. Studies on the mechanism of activity enhancement and resistance prevention of Cry toxins by Cyt1A indicated that Cyt1A functions as a receptor of Cry11A. Cyt1Aa binding to *Ae. aegypti* brush border membrane vesicles enhanced the binding of Cry11Aa. Two exposed regions in Cyt1Aa, namely, loop beta6-alphaE and part of beta7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop alpha8 and beta-4, which are also involved in midgut receptor interaction. The key residues involved in the interaction and synergism between Cry11Aa and Cyt1Aa were S259 and E266 in Cry11Aa and K198, E204 and K225 in Cyt1Aa [31]. Further studies revealed that binding of Cry11A to Cyt1A facilitates the formation of a Cry11A prepore oligomeric structure that is capable of forming pores in membrane vesicles [32].

It was discovered recently that the mosquitocidal toxins (Mtx) from some *B. sphaericus* strains not only enhance the larvicidal activity of *B.t.i*. Cry toxins but also mitigate resistance devel‐ opment to Cry toxins. Mtx-1 and Mtx-2 were active against mosquitoes that were susceptible or resistant to Cry toxins. A mixture of Mtx-1 or Mtx-2 with different Cry toxins in the ratio of 1:1 showed moderate synergism. Some combinations of Mtx and Cry toxins were highly active to kill resistant larvae and also suppressed resistance to Cry toxins [33]. There is a lack of crossresistance to the wild type of other *B.t.* subspecies such as *B.t. jegathesan, B.t. kyushuensis*, and *B.t. fukuokuensis* in *Cx. quinquefasciatus* that are resistant to individual toxins from *B.t.i*. [34]. It is advised not just to express Cry toxins of *B.t.i*. in transgenic microbial organisms or algae from the perspectives of resistance prevention in target species.
