# 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)
#### 5.2.5 Prospects for Engineering More Robust Strains of Bi  
As mentioned above, a unique strategy has evolved in Bi to package and deliver its Cry4 Aa1, Cry4 Ba1, Cry1 Aa1, and Cyt1 Aa1 larvicidal toxins as a single composite unit, i.e., the prokaryotic insect larvicidal organelle, PILO [127]. This is the essential reason why commercial formulations of the natural strain of Bi\(\grave{\text{t}}\) are successful and robust in the field. In particular, the \"built-in\" Cyt1 Aa1-based synergism strategy integral to the PILO is key to preventing or delaying the development of resistance to the Cry proteins in natural populations of vector mosquitoes. Indeed, resistance to Bi\(\grave{\text{t}}\) in _Aedes_, _Anopheles_, and _Culex_ mosquitoes has not been reported, even after 40 years of use. With regard to _Aedes aegypti_, the long-term efficacy of Bi\(\grave{\text{t}}\) has even been demonstrated under laboratory conditions where this prolific vector was exposed to 0.5 mg/L of VectoBac throughout 30 generations, and where the offspring of survivors remained susceptible to Bi\(\grave{\text{t}}\)'s composite PILO, with LC\({}_{50}\) and LC\({}_{95}\) ranging from 0.013 to 0.022 mg/L and from 0.030 to 0.049 mg/L, respectively [297]. As Bi\(\grave{\text{t}}\) remains a safe and highly efficacious biopesticide, there have been few innovations to warrant the release of a competitive engineered strain for applied purposes. Regardless, a number of studies have been conducted to determine the molecular mechanisms responsible for the expression and synthesis of larvicidal component systems in Bi\(\grave{\text{t}}\)[221], but the knowledge gained from such studies has rarely been used to manipulate Bi\(\grave{\text{t}}\) for practical commercial purposes. To date, there has only been one report clearly demonstrating that a recombinant strain of Bi\(\grave{\text{t}}\) IPS-82 can be impressively more toxic than parental Bi\(\grave{\text{t}}\) or Ls 2362, with 21-fold and 32-fold more toxicity, respectively [298]. The Bi\(\grave{\text{t}}\) IPS-82 recombinant was manipulated to express its native composite PILO, and the Ls Tpp1 Aa1/Tpp2 Aa1 operon under control of strong sporulation-dependent promoters of Cyt1 Aa1 [299] and the mRNA stabilizing sequence (STAB-SD) of Cry3A [300; 301]. The engineered strain produced \(\sim\)472 g/mL of Cry4 Aa1, Cry4 Ba1, Cry1 Aa1, Cyt1 Aa1, and Tpp1 Aa1/Tpp2 Aa1 compared to 250 g/mL for parental Bi-IPS82 (190% increase), and over 450% more Tpp1 Aa1/Tpp2 Aa1 than Ls 2362. [PERSON] et al. [302] also engineered Bi\(\grave{\text{t}}\) IPS-82 to produce the components of its PILO and Cry11B, but the Bi\(\grave{\text{t}}\) IPS-82/Cry11B recombinant was only twice as toxic as the parental strain. Other approaches have been attempted to develop more commercially competitive Bi\(\grave{\text{t}}\) strains using genes that code for non-larvicidal toxins, including chitinases. The intent of the latter is that the enzyme, once released in the midgut, could weaken the chitin-containing peritrophic membrane, thereby decreasing the time required while concomitantly increasing access for activated toxins to bind to membrane receptors to induce larval lethality. Toward this end, [PERSON] et al. [303] engineered Bi\(\grave{\text{t}}\) to produce its PILO and inclusions of a chitinase, ChiA74. Despite developing a method to produce normally soluble ChiA74 as stable inclusions in Bi\(\grave{\text{t}}\) during sporulation, and demonstrating that the enzyme was indeed active at pH 6-8, the recombinant was only twice as toxic to _Aedes aegypti_ compared to parental Bti (LC\({}_{50}\) 9 ng/mL versus 19.8 ng/mL). Whether or not the narrow pH range for ChiA74 limited its intended effect in the alkaline larval midgut is not clear. Nonetheless, the study shed light on the fact that additional toxins, enzymes, or other biomoleculesproduced in Bti during sporulation should ideally be particulate; if soluble, they will likely be eliminated during the production and commercial formulation process.