# Morphological Study of _Bacillus thuringiensis_ Crystals and Spores
## 3 Results  
### Anti-Lepidopteran Strains: Collection of Bipyramidal and Cubic Crystals  
The following depicts the distribution of crystal sizes of the _B. thuringiensis_ var. _kurstaki_ strains in histograms plotted according to the normal distribution.  
The size distribution of the bipyramidal height was plotted for bipyramidal crystals and the size distribution of square side for the cubic crystals. The x-axis corresponds to the size ranges in micrometers, the left-hand y-axis represents the percentage of crystals and the right-hand y-axis represents the cumulative percentage, as shown in Figures 2-4 for _B. thuringiensis_ var. _kurstaki_ strains HD1, LIPMKA and BLB1, respectively. These histograms show a Gaussian distribution for the sizes of bipyramidal and cubic crystals. The average heights of the bipyramidal crystals were 1.508 \(\pm\) 0.009 \(\upmu\)m for the reference _B. thuringiensis_ var. _kurstaki_ strain HD1, 1.549 \(\pm\) 0.016 \(\upmu\)m for the Lebanese LIPMKA strain and 1.531 \(\pm\) 0.014 \(\upmu\)m for the Tunisian BLB1 strain. For the cubic crystals, the average cube edges were 0.573 \(\pm\) 0.009 \(\upmu\)m for the HD1 strain, 0.478 \(\pm\) 0.014 \(\upmu\)m for the LIPMKA strain and 0.614 \(\pm\) 0.006 \(\upmu\)m for the BLB1 strain.  
Figure 2: **(a)** SEM image of the HD1 purified crystals. **(b,c)** Graphs representing the size distribution in terms of frequency, along with the cumulative percentage of the bipyramidal height for the bipyramidal crystals and the cubic size of the cubic crystals.  
Figure 4: **(a)** SEM image of the BLB1 purified crystals. (**b,c**) graphs representing the size distribution in terms of frequency, along with the cumulative percentage of the bipyramidal height for the bipyramidal crystals and the cubic size of the cubic crystals.  
Figure 3: **(a)** SEM image of the LIPMKA purified crystals. (**b,c**) Graphs representing the size distribution in terms of frequency, along with the cumulative percentage of the bipyramidal height for the bipyramidal crystals and the cubic size of the cubic crystals.  
Morphological data for the three _kurstaki_ crystals, and their volumes and aspect ratios are shown in Table 2.  
ANOVA univariate analysis revealed significant differences between the three morphological characteristics of bipyramidal crystals with \(p\)-values lower than 0.05. The null hypothesis was therefore rejected. Although bipyramidal crystals have roughly the same bipyramidal height (2 h) for the three _kurstaki_ strains, the HSD test revealed a statistically significant difference only between the LIPMKA height value and the HD1 one. As for the volume and the aspect ratio that reflects the shape of bipyramid, the post-hoc test highlighted statistically significant differences between the three _kurstaki_ strains. The bipyramidal crystals of LIPMKA were the most stretched and the least voluminous crystals of the three strains, and those of HD1 were the shortest and the most voluminous (Table 2a). Finally, for cubic crystals, the null hypothesis was also rejected (\(p\) = 0), and the HSD test displayed significant differences among the three _kurstaki_ strains: BLB1 synthesized the most voluminous cubic crystals and LIPMKA the least voluminous ones.  
These three strains carry Cry protein coding genes from the same families, with small variations in the amino acid compositions of the proteins. This includes four Cry1 coding genes, whose produced proteins are responsible for the bipyramid-shaped crystals, and two Cry2 coding genes, producing a cubic crystal [9; 20].