There have been many alternatives used to control mosquitoes, the vectors of important diseases that cause the death of many people around the world annually (McGregor and Connelly 2021; Şengül and Canpolat 2022). However, although important progress has been made, it is still necessary to develop more efficient, sustainable, and environmentally friendly comprehensive strategies. The existing biodiversity on the planet offers the possibility of finding alternatives for development and use in the solution of this important problem or at least significantly reducing the negative effect of transmission by these vectors of serious diseases.
Since its first isolation in 1902 by Ishiwata, B. thuringiensis has been isolated from various ecologies and has been widely studied. In spite of this worldwide exploration, the research on this bacterium is still incomplete because of the many ecologies that still remain unexplored and the fact that B. thuringiensis-based insecticides have not been able to completely replace the harmful chemical insecticides in the market (Melo et al. 2016).
This paper addresses the isolation, identification, characterization, and use of soil microorganisms for vector control, specifically, through the use of B. thuringiensis for the control of mosquito larvae and adults. Although 235 soil samples from three different regions were analyzed, the number of B. thuringiensis-related isolates was low. Among the bacterial isolates from the soil, eight isolates were identified as B. thuringiensis producing parasporal crystals during sporulation. Molecular analyses of the 16S ribosomal DNA region allowed the identification of B. thuringiensis in only eight colonies. On the other hand, through the light and scanning electron microscope, it was possible to appreciate the different characteristics of the spores and endotoxins produced by B. thuringiensis. As for the endotoxins, different forms were observed.
An important aspect in the characterization of B. thuringiensis isolates was the determination of the frequency and number of cry and cyt genes. This allows an approximation of the possible toxicity of the isolates on the different species of existing mosquitoes. Sometimes, a greater number of cry and cyt genes in a B. thuringiensis isolate is not associated with greater toxicity. However, in other cases, the presence of a greater number of cry and cyt genes could be related to a greater diversity of production of Cry and Cyt proteins, which can help to avoid insect resistance and increase the spectrum of action against various species of mosquitoes.
Different profiles of toxic cry and cyt genes were detected among the evaluated B. thuringiensis isolates, and six genes were not detected in the B. thuringiensis isolates analyzed. This diversity of profiles could be attributed to the high frequency of genetic information exchange between B. thuringiensis bacteria, enabling variation in the genes within a single isolate (Bravo et al. 1998). These new bacterial gene combinations can give rise to isolates with gene compositions specific to different groups of insects, and such a broad spectrum of action against different groups may be the reason why B. thuringiensis is successful in insect control (Ben-Dov et al. 1997; Ben-Dov 2014; Bravo et al. 2011). In addition, the genetic diversity of the new B. thuringiensis isolates between the three biomes where they were collected corroborated the results of other studies (Baig and Mehnaz 2010; Mahalakshmi et al. 2012; Shishir et al. 2014) that showed the genetic diversity in Cry and Cyt toxins between the samples of isolates collected from different ecosystems.
None of the B. thuringiensis isolates harbored all of the investigated cry and cyt genes, but this result is perhaps expected, since none of the known B. thuringiensis species or subspecies presents all of the potential genes. However, it should be emphasized that some isolates may present high toxicity to susceptible insects even with a synthetic composition of cry-cyt genes (Costa et al. 2010). The number of genes in a single isolate does not appear to be the factor determining the immediate death of the susceptible insect per se, but it may affect the toxic activity of bacteria when they encounter optimal conditions in the gut of the insect.
The δ-endotoxins are classified into two families of proteins, namely, Cry and Cyt, that can frequently be produced by B. thuringiensis subspecies. The combination of Cry and Cyt in the same subspecies is frequent in strains with action against Diptera. The Cry family of proteins comprises 74 types that are encoded by 770 different cry genes, whereas the Cyt family is grouped into three types that are encoded by 38 cyt genes (Crickmore 2016). The specificity of the quantity of and the synergy between the toxic genes in each isolate, together with the susceptibility of the larvae and the highly alkaline pH of the intestine, favors the solubilization and the release of the protein crystals (Boonserm et al. 2005; Lacey 2007), all of which are important factors that determine the effectiveness of the bacterial toxicity against larvae.
The number of genes in B. thuringiensis isolates has also been analyzed by other authors, such as Ejiofor and Johnson (2002), Baig and Mehnaz (2010), Khojand et al. (2013), and Soares-da-Silva et al. (2015), who reported the lack of a correlation between the number of amplified genes and the intensity of the toxic activity in the mosquito gut. Among the twenty genes evaluated in this study, including fifteen in the Cry family and five in the cyt family, the most frequent were the cry1, cry11Ba, cry4Aa, cry10Aa, cry11Aa, cry10, and cry11 genes of the new B. thuringiensis isolates. The cry1 and cry10 genes were more frequent in the B. thuringiensis isolates previously described (Jouzani et al. 2008; Salekjalali et al. 2012; Khojand et al. 2013; El-Kersh et al. 2014). The combination of the Cry4, Cry10, Cry11, and Cyt toxins is known to be very potent against mosquitoes (Ibarra et al. 2003).
Local B. thuringiensis isolates exhibited larvicidal activity. Several B. thuringiensis strains with good mosquitocidal activity have been isolated from various parts of the world (Nair et al. 2020; Viana et al. 2021; Salamun et al. 2021; da Costa Fernandes et al. 2022). Amongst the B. thuringiensis isolates with significantly enhanced larvicidal activity identified in this study, isolate A4 was the most toxic with bioactivity against C. pipiens larvae. Notably, it was the result related to the control of B. thuringiensis in adult mosquitoes, mainly by the formulation based on extracts of natural odorant compounds. The biological effect of B. thuringiensis on mosquito larvae is well known; however, this effect is not the same when the mosquito is in the adult phase. The crystals must be soluble, and there must be compounds that attract the adult.
The new isolates of B. thuringiensis from Rizhao soil are promising for the biological control of C. pipiens larvae, which can be evidenced by the biological activity in larvae caused by the Cry and Cyt toxins, making these isolates highly toxic and enabling them to be safely used as a biotechnological tool for the manufacture of biological larvicide with different combinations of toxins from those currently used. We believe that the discovery of the A4 strain may prove crucial for future bioinsecticide production against mosquito vectors. This is due to its greatly enhanced mosquitocidal activity against C. pipiens, combined with its possible economic and environmental advantages.