# The Perpetual Vector Mosquito Threat and Its Eco-Friendly Nemeses
## 5 Eco-Friendly _Lysinibacillus sphaericus_ and _Bacillus thuringiensis_ subsp. _israelensis_
### Brief History of Lysinibacillus (Formerly Bacillus) sphaericus (Ls)
#### 5.2.4 Mechanism of Toxicity of Bti's Larvicidal Proteins  
Numerous studies and reviews have been published on the mechanisms involved in the toxicity of larvicidal Cry proteins [272; 273; 274; 275; 276]. In summary, as with Ls binary toxin, the protoxins that compose the crystalline inclusions of Bti's PILO are solubilized and proteolytically activated in the alkaline midgut of mosquito larvae. The activated Cry toxins disrupt the membrane integrity and osmotic balance by creating pores through interactions with midgut microvillar membrane receptors and binding to plasma membrane adhesion proteins. Several receptors have been identified and include cadherins, aminopeptidase N, alkaline phosphatases, and \(\alpha\)-amylase.  
Unlike Bti's Cry4 Aa1, Cry4 Ba1, and Cry11 Aa1, which elicit cytotoxicity through a receptor-mediated mechanism, specific membrane-associated receptors have not been identified for Cyt1 Aa1. Nonetheless, activated Cyt1 Aa1 is intrinsically highly lipophilic and preferentially binds unsaturated fatty acids. To prevent the destruction of the plasma membrane in vivo, the synthesis of Cyt1 Aa1 requires a 20-kDa helper \"chaperone\" protein that is also encoded by pBtoxis, where the corresponding gene is a component of the _cry11 Aa1_ operon [277; 278; 279]. It is thought that binding of activated Cyt1 Aa1 to lipids directly perturbs and destabilizes the plasma membrane integrity [271; 279; 280; 281].  
Interestingly, Cyt1 Aa1 is considerably less toxic than Cry4 Aa1, Cry4 Ba1, and Cry11 Aa1. Previous studies with Cyt1 Aa1 showed that the LC\({}_{50}\) and LC\({}_{95}\) for _Culex quinquefasciatus_ SLAB were 47,370 ng/mL and 155,050 ng/mL, respectively; for _Aedes aegypti_ were 4219 ng/mL and 22,765 ng/mL, respectively; for _Anopheles gambiae_ were 46,557 ng/mL and 129,979 ng/mL, respectively; and for _Anopheles stephenesi_ were 7780 ng/mL and 13,772 ng/mL, respectively [282]. In contrast, individual Cry toxins against _Culex quinquefasciatus_, for example, are significantly \(<\) 500 ng/mL, with Cry 11 Aa1 (previously CryIVD) being the most toxic, i.e., 86 ng/mL (LC\({}_{50}\)) and 93 (LC\({}_{95}\)) ng/mL [283]. Nevertheless, Cyt1 Aa1 is indispensable to the robust larvicidal activity of Bti. Cyt1 Aa1 synergistically interacts with these three Cry proteins against a wide range of mosquito and blackfly species, amplifying their larvicidal activities while delaying and preventing the development of resistance to these toxins [284; 285; 283], presumably by functioning as a surrogate receptor for these toxins [286; 230; 287].  
The synergistic effect of Cyt1 Aa1 extends to Ls's Tpp1 Aa1/Tpp2 Aa1 against many mosquito species, including _Aedes aegypti_, which is normally refractive to the binary toxin; the cytotoxain also restores the toxicity of _Culex quinquefasciatus_, which developed resistance to Ls [288; 230; 289; 290]. Interestingly, as _Aedes aegypti_ lacks a GPI-anchored amylomalatseortholog receptive to the Tpp2 Aa1 ligand, and as the binary toxin elicits its effect intracellularly, exactly how Cyt1 Aa1 facilitates the activity of the binary toxin or the toxin domain, Tpp1 Aa1, in this species remains to be resolved. Translocation of the binary toxin into the cytoplasm mediated through irregular membrane perturbations where Cyt1 Aa1 interacts with molecules bound to, but not inserted, into the membrane (\"detergent-like model\"), or forming membrane pores of 6-20 angstroms in diameter (\"pore-forming model\"), have been suggested [291; 292; 293; 294; 295; 296]. More recent structural insights by [PERSON] et al. [271] opposed the pore-forming and detergent-like models and suggested that interactions of non-porous Cyt1 Aa1 oligomers, called membrane-bound aggregates MBA and MBA protomers, can lead to the formation of large pores of \(>\)54 nm in diameter that can accommodate the translocation of large molecules, such as the 42 kDa Tpp1 Aa1, or perhaps Tpp1 Aa1/Tpp2 Aa1.