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The black cutworm, Agrotis ipsilon(Hufnagel), a seasonal migrant and a prolific generalist, can feed on nearly all vegetables and grain crops, causing considerable economic impacts on a global scale. Given its cryptic nature, A. ipsilonmanagement has been extremely challenging. Chitin synthase(CHS), a key enzyme involved in chitin biosynthetic pathway and crucially important for the growth and development of insects, is the molecular target of chitin synthesis inhibitors, a group of broad-spectrum insecticides that is compatible with Integrated Pest Management practices. In this study, we investigated the potential of targeting chitin synthasesto control A. ipsilon. As a result, two chitin synthases, AiCHS1and AiCHS2, were identified and cloned from A. ipsilon. The temporal-spatial distribution study showed that AiCHS1was predominantly expressed at the pupal stage and most abundant among tissues of head capsule and integument, while AiCHS2was mainly expressed at the sixth instar larval stage and tissues of foregut and midgut. RNAi-based functional study confirmed gene silencing caused significant reduction in the expression levels of the corresponding mRNA, as well as resulted in abnormal pupation and mortality, respectively. Furthermore, under the treatment of lufenuron, a chitin synthesis inhibitor, A. ipsilonresponded with an elevated expression in AiCHS1and AiCHS2, while larvae showed difficulty in shedding old cuticle, and a cumulative mortality of 69.24% at 48 h. In summary, chitin synthasesare crucial for chitin biosynthesis in A. ipsilonand can be targeted for the control (e.g., RNAi-based biopesticides) of this devastating insect pest.

Hawthorn spider mite, Amphitetranychus viennensisZacher, one of the most damaging arthropod pests for Rosaceaous fruit trees and ornamentals, has developed resistance to most of the commercially available acaricides. To understand the molecular basis of acaricide resistance, a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) following the MIQE (minimum information for publication of quantitative real time PCR experiments) guidelines is needed. In this study, we screened for the internal references in A. viennensisto study in acaricide resistance. In total, 10 candidate reference genes, including EF1A, 28S rRNA, 18S rRNA, α-tubulin, Actin3, RPS9, GAPDH, V-ATPase B, RPL13, and V-ATPase A, were assessed under the treatments of four commonly used acaricides with distinct mode-of-actions (MOAs). Based on the Insecticide Resistance Action Committee MOA classification, avermectin, bifenazate, spirodiclofen, and fenpropathrin belong to group 6, 20D, 23, and 3A, respectively. The expression profiles of these candidate genes were evaluated using geNorm, Normfinder, BestKeeper, and ∆Ct methods, respectively. Eventually, different sets of reference genes were recommended for each acaricide according to RefFinder, a comprehensive platform integrating all four above-mentioned algorithms. Specifically, the top three recommendations were 1) 28S, V-ATPase A, and Actin 3for avermectin, 2) GAPDH, RPS9, and 28Sfor bifenazate, 3) Actin 3, V-ATPase B, and α-tubulinfor spirodiclofen, and 4) Actin 3, α-tubulin, and V-ATPase Afor fenpropathrin. Although unique sets of genes are proposed for each acaricide, α-tubulin, EF1A,and GAPDHare the most consistently stably expressed reference genes when A. viennensiswas challenged chemically. Our findings lay the foundation for the study of acaricide resistance in the phytophagous mites in general, and in the hawthorn spider mite, A. viennensis, in particular.