β-Glucosidases play an important role in the chemical defense of many insects by hydrolyzing and thereby activating glucosylated pro-toxins that are either synthesized de novo or sequestered from the insect's diet. The horseradish flea beetle, Phyllotreta armoraciae, sequesters pro-toxic glucosinolates from its brassicaceous host plants and possesses endogenous β-thioglucosidase enzymes, known as myrosinases, for glucosinolate activation. Here, we identify three myrosinase genes in P. armoraciae (PaMyr) with distinct expression patterns during beetle ontogeny. By using RNA interference, we demonstrate that PaMyr1 is responsible for myrosinase activity in adults, whereas PaMyr2 is responsible for myrosinase activity in larvae. Compared to PaMyr1 and PaMyr2, PaMyr3 was only weakly expressed in our laboratory population, but may contribute to myrosinase activity in larvae. Silencing of PaMyr2 resulted in lower larval survival in a predation experiment and also reduced the breakdown of sequestered glucosinolates in uninjured larvae. This suggests that PaMyr2 is involved in both activated defense and the endogenous turnover of sequestered glucosinolates in P. armoraciae larvae. In activity assays with recombinant enzymes, PaMyr1 and PaMyr2 preferred different glucosinolates as substrates, which was consistent with the enzyme activities in crude protein extracts from adults and larvae, respectively. These differences were unexpected because larvae and adults sequester the same glucosinolates. Possible reasons for different myrosinase activities in Phyllotreta larvae and adults are discussed.

β-葡萄糖苷酶通过水解并从而激活从头合成或从昆虫饮食中隔离的糖基化原毒素，在许多昆虫的化学防御中发挥重要作用。辣根跳甲虫 ( Phyllotreta armaciae ) 从其芸苔科寄主植物中隔离前毒性芥子油苷，并拥有内源性β-硫代葡萄糖苷酶（称为黑芥子酶），用于激活芥子油苷。在这里，我们在甲虫个体发育过程中鉴定了三种黑芥子酶基因，它们在甲虫个体发育过程中具有不同的表达模式。通过使用 RNA 干扰，我们证明PaMyr1负责成虫中的黑芥子酶活性，而PaMyr2负责幼虫中的黑芥子酶活性。与PaMyr1和PaMyr2相比，PaMyr3在我们的实验室群体中仅弱表达，但可能有助于幼虫中的黑芥子酶活性。在捕食实验中， PaMyr2的沉默导致幼虫存活率降低，并且还减少了未受伤幼虫中隔离的芥子油苷的分解。这表明 PaMyr2 参与P. armaraciae幼虫中隔离的芥子油苷的激活防御和内源周转。在重组酶活性测定中，PaMyr1和PaMyr2优选不同的芥子油苷作为底物，这分别与成虫和幼虫粗蛋白提取物中的酶活性一致。这些差异是出乎意料的，因为幼虫和成虫吸收相同的芥子油苷。讨论了叶甲幼虫和成虫中黑芥子酶活性不同的可能原因。