Synergistic Insecticidal Activity Against Hyphantria cunea by Cry9Aa3 Mutants and Cry1Ah Combinations

The larvae of <i>Hyphantria cunea</i> feed on plant leaves, causing significant losses to forestry and agricultural production. At present, <i>cry1</i> genes such as <i>cry1Ac</i> and <i>cry1Ah</i> are mainly used to control <i>H. cunea</i>. To delay the problem of pest resistance induced by a single insecticidal gene, it is crucial to discover and develop new insecticidal genes or gene combinations. This study found <i>cry9Aa3</i> and <i>cry9Aa4</i> showed insecticidal activity against <i>H. cunea</i>. The toxicity of 14 mutants of Cry9Aa3 was analyzed and the LC₅₀ of the triple-amino-acid substitution mutant 316LRG318AAA was 3.69 μg/g, which represents a 1.49-fold increase in insecticidal activity compared to Cry9Aa3. Additionally, enhanced stability of this mutant was detected in the midgut juice of <i>H. cunea</i>. Cry9Aa3 and 316LRG318AAA, in combination with Cry1Ah, demonstrated synergistic effects against <i>H. cunea</i>, with synergistic factors of 4.76 and 8.33, respectively. This study has identified the mutant 316LRG318AAA and its combination with Cry1Ah as exhibiting high toxicity against <i>H. cunea</i>, providing valuable genetic resources for the development of transgenic poplars and holding significant importance for delaying resistance in this pest.