# 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)
#### 5.2.3 Structural Characteristics of Bit's Larvicidal Proteins  
As mentioned above, the main larvicidal components of the Bit's prokaryotic insect larvicidal organelle (PILO) are crystalline inclusions of Cry4 Aa1, Cry4 Ba1, Cry11 Aa1, and Cyt1 Aa1 (Figure 1) [127].  
Figure 1: Structural features of _Bacillus thuringiensis_ subsp. _isnelensis_ parasporal body (PB), a unique prokaryotic insect larvicidal organelle (PILO). Sodium dodecyl polyacrylamide gel electrophoresis protein profile showing that Cry4 Aa1 (135 kDa), Cry4 Ba1 (128 kDa), Cry11 Aa1 (65 kDa), and Cyt1 Aa1 (27 kDa) (**A**) are the major mosquito larvicidal proteins of the PILO (**B**). Note that crystals of Cry4 Aa1/Cry4 Ba1, Cry11 Aa1, and Cyt1 Aa1 are each enveloped by a multilamellar fibrous matrix (MFM; arrowheads) that is also found in the peripheral composite structure. Purified MFM derived from the PILO treated with alkaline to dissolve and remove the Cry and Cyt proteins; multiple layers of the MFM are observed (arrowheads) (**C**). Brightfield and corresponding fluorescence confocal microscopy showing the progressive formation of [PERSON]’s PILO; GFP-labeled Bt0152, a pBtoxis-coded protein, known to specifically bind to the MFM [266], associates with this structure as early as 9 h, well before the three crystalline inclusions are observed at 12-20 h (**D**). Ultrastruct analysis of the MFM showing that it contains hexagonal pores (**E**) and discrete particles thought to be ribosomes (arrowheads) attached to the MFM (**F**), suggesting that the Cry and Cyt proteins are synthesized through the pores and are concentrated and crystallized in their respective compartment in the PILO. GFP-labeled Bt075, a protein also coded for by pBtoxis and which is structurally similar to phage capsid and encapsulin shell proteins, is also a PILO-specific component that associates with the MFM as early as 6 h before distinct crystals are observed; phase (p), phase-fluorescence overlay (o), and fluorescence (f) in sporulating cells at 20 h (**G**); and cells that have autolyzed at 48 (**H**) to release spores (s) and the PILO are shown. Bar (**B**,**C**,**F**) = 0.2 mm. (Adapte  
Figure 2: Structural characteristics of [PERSON]’s Larvicidal ProteinsThe genes coding for these proteins are harbored on plasmid pBtoxis [267]. The crystallographic structures of the four toxins have been determined. Cry4 Aa1, Cry4 Ba1, and Cry11 Aa1 contain the typical three-domain structure of Cry toxins. Domain I, located in the N-terminal, is an amphipathic \(\alpha\)-helical bundle responsible for oligomerization, membrane insertion, and pore formation, whereas domain II is formed by antiparallel \(\beta\)-strands harboring loops that participate in receptor binding and specificity. Domain III is a sandwich of two antiparallel \(\beta\)-sheets involved in receptor binding and protection of the toxin's structural integrity. Although the three domains among Bt larvicidal toxins are very similar, they possess structural differences, but in comparison to domains I and III, domain II is the most divergent, which supports its role in toxin specificity. It is interesting that Cry4 Ba1, a diptera-specific protein, is more closely related to Cry1A (lepidopteran-specific) than Cry3 Aa (coleoptera-specific), but is less closely related to Cry2 Aa (lepidoptera/diptera specificity) [268; 269; 270].  
The 3D structure of Cyt1 Aa1 was elucidated at 2.2 A resolution. The toxin has a cytolysis fold with central \(\beta\)-sheets surrounded by two outer \(\alpha\)-helical layers that can undergo conformational changes. The \(\alpha\)-helical layers swing away to allow the exposed \(\beta\)-sheets to insert into the membrane. Lipid-binding moieties have been identified between the \(\beta\)-sheets and the \(\alpha\)-helical layers and highlight their affinity for membrane lipids once activated in the larval alkaline midgut [271].