# Morphological Study of _Bacillus thuringiensis_ Crystals and Spores
## 4 Discussion  
In this study, the morphologies and size distributions of the crystals and spores of seven different strains of _Bacillus thuringiensis_ were analyzed, with an emphasis on the two most used and studied serovars: the anti-lepidopteran reference serovar, _kurstaki_, and the anti-dipteran one, _israelensis_, in addition to a strain with anti-dipteran activity, but that is phylogenetically different from the latter group. On one hand, we found that all _kurstaki_ strains synthesize bipyramidal crystals with sizes that are statistically different. HD1 produces the most voluminous and shortest bipyramidal crystals, and LIPMKA produces the least voluminous ones with a more stretched form than the other two--a characteristic related to the way that the crystal proteins are associated together. As for the cubic ones, a significant variation was observed in crystal size between strains, with BLB1 showing the largest cubic crystal and LIP the smallest one. Composition of the bipyramidal and cubic crystal protein coding genes of the three _B. thuringiensis_ var. _kurstaki_ strains HD1, LIPMKA and BLB1 showed little alteration in the coding DNA sequences and the promotor regions. In fact, these strains, being from the same serovar, showed high DNA sequence similarity in their toxin-carrying plasmids. However, the level of expression of the toxin coding genes, particularly those encoding proteins of the Cry2 family and producing cubic crystals, needs further investigation to elucidate the reason behind the larger size of the BLB1 cubic crystals. As for the _B. thuringiensis_ var. _israelensis_ strains, it is remarkable that AM65-52 crystal showed a larger volume than those of AR23 and BUPM98, despite having one less Cry4B protein, thanks to a pseudogeneized version of a _cry4 Ba_ gene in AM65-52, vs. a functional one in AR23 and BUPM98 [15]. This pseudogeneized _cry4 Ba_ gene in AM65-52 means that the corresponding protein is absent from this strains' crystal, but is present in those of AR23 and BUPM98, as shown in previous studies [15; 28]. In this case as well, a difference in the expression rate and copy numbers of the protein variants in the crystal, found in all three strains, cannot be excluded and requires further transcriptomic and proteomic analysis. Furthermore, the H3 strain synthesizes larger spherical crystals than the _israelensis_ strains. This is most likely due to a different crystal protein composition and the fact that H3 belongs to a different phylogenetic cluster [32]. Nevertheless, we must keep in mind the fact that a change of culture medium does affect the production of _B. thuringiensis_ crystals and their size [37]. Hence, this morphological analysis should be conducted after production in various culture media in order to select the optimal one for the best crystal yield. On the other hand, in this study, we took a closer look at the intra- and inter-servorar differences of the size and shape (length, width, volume and aspect ratio) of _B. thuringiensis_ spores, whereas previous studies focused on the spore size distribution among various _Bacillus_ species [35; 38; 39]. Nonetheless, the variations of morphological attributes of spores among _B. thuringiensis_ serovars were yet to be elucidated, and that was the focus of our study. Our results did show variations within all these parameters, proving that the spores of the various strains within the same serovar are not identical. Therefore, we first calculated the aspect ratios of the spores of the various strains, allowing us to show that H3 spores are more circular than all the others. As for the intra-servorar variability, HD1 and AM65-52 produce spores that are less ellipsoidal than their fellow Lebanese and Tunisian _kurstaki_ and _israelensis_ strains, respectively. In addition, the spores of the reference _B. thuringiensis_ var.