Bt Research 2025, Vol.16, No.5, 224-233 http://microbescipublisher.com/index.php/bt 228 4.3 Effect of exogenous environment on plasmid stability The stability of Bt plasmid in the strain is also affected by many factors in the external environment. Growth matrix and nutrient levels are important factors. In an environment rich in available carbon and nitrogen sources, plasmid-free cells grow slightly faster, and long-term co-culture may lead to gradual loss of plasmids. Experimental evolutionary studies show that the Bt strain was repeatedly passed in culture medium without insects, and the virulence plasmid of the strain was lost after tens of generations, while the plasmid was retained in the passages containing the insect infection cycle. This shows whether there are advantages of plasmid-assigned functional requirements in the outside world, which will directly affect plasmid stability (Sheppard et al., 2016). In addition, population density and competition among strains also have an effect. When population density is high, Bt strains compete fiercely. If plasmid-free mutant strains obtain a slight proliferation advantage by reducing the metabolic burden, they may win in the competition, which will reduce the population plasmid carrier rate. Environmental stress can also affect plasmids (Wang and Huang, 2025), such as violent pH fluctuations and the presence of heavy metals may interfere with plasmid replicase or structural stability. 5 Effects of Plasmid Loss on Bt Virulence 5.1 Toxin gene loss and expression decrease Plasmid loss directly leads to the loss of toxin genes in Bt strains or the expression level decreases, thereby weakening its insecticidal virility. This is the most intuitive aspect of the impact of plasmid loss on Bt virulence. When the plasmid carrying the main virulence gene is lost, the corresponding Cry or Vip toxin will no longer be generated, and the strain's pathogenicity to the target insect will be significantly reduced. For example, after the pBtoxis plasmid of the Bt subspecies Israeli strain was removed, the strain no longer produced mosquitoxins such as Cry4 and Cry11, and the mortality rate of Aedes larvae dropped to almost zero (Figure 2) (Chen et al., 2022). After the pBtoxis plasmid of the Bt QBT220 strain reported by Wang et al. (2020) lost the cry10A and cyt1C genes, although other toxin genes are still present, the insecticidal spectrum has changed and the toxic activity on certain targets has been reduced. Plasmid loss may also disrupt the regulation of toxin expression. Some plasmids have lost promoters or regulatory elements of toxin genes, and the remaining gene expression levels may also decrease. Strains with plasmid loss often experience reduced crystal protein yield and imbalance in spore-to-crystal ratios. Compared with wild type, Cry protein production in plasmid-free mutant strains can be reduced by more than 30% (Wang et al., 2020). Therefore, the primary effect of plasmid loss on Bt virility is to cause the strain to lose some or even all of the insecticidal toxins, or the toxins cannot be expressed normally, which directly leads to a decrease in the strain's insecticidal rate and lethal effect. 5.2 Indirect effect on the growth and adaptation of Bt strain Plasmid loss not only reduces toxin genes, but also indirectly affects the growth characteristics and environmental adaptability of Bt strains, thereby affecting its overall virulence performance. On the one hand, plasmid loss is usually accompanied by a reduction in metabolic burden, and plasmid-free strains tend to grow faster under nutritious conditions. This may lead to an increase in its proportion in the bacterial population, but rapid growth does not mean an increase in virility. Experimental evolution results of Dimitriu et al. (2023) show that Bt clones that lose virulence plasmid have higher competitive fitness in vitro compared with ancestral strains, but have significantly decreased virulence (LC50) against the insect strain Diamond moth (Dimitriu et al., 2023). On the other hand, plasmid loss may weaken the adaptability of Bt in the host environment. Bt-infected insects need to resist host immunity and compete with other microorganisms. Plasmids often carry some cofactors to help strain colonize in the insect's blood cavity. Strains with lost plasmids may be more susceptible to being cleared by the host immune system or competition failed. Some studies compared the survival of a certain Bt strain and its plasmid-free mutant strain in insect hemolymph, and found that the proliferation of plasmid-free strains was inhibited, which was speculated that it was due to the lack of antibacterial substances encoded on the plasmid. 5.3 Changes to insect mortality and toxicity spectrum Plasmid loss often causes significant changes in the mortality and toxicity profile of Bt strains on target insects. With the deletion of the toxin gene, the lethality rate of the strain on the original target insect was significantly
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