BACKGROUND Chitin is a major component in the extracellular matrix of insects, and its metabolism largely affects insect development and molting. As essential degradative enzymes, chitinases are encoded by multiple genes that differ in size, expression pattern and function in insects. However, our limited knowledge on the functions of different chitinases in Agrotis ipsilon has prevented our application of new technologies to target these genes as new pest management strategies. RESULTS We revealed 11 full‐length complementary DNA sequences of chitinase genes (AiChts) from A. ipsilon transcriptome. Although the domain architecture of these chitinases varied greatly, they all contained at least one chitinase catalytic domain. Developmental stage‐ and tissue‐dependent expression profiles showed that most AiChts had the highest expression in the pupal stage. Furthermore, AiCht2, AiCht6, AiCht7 and AiCht10 were mainly expressed in the integument, whereas AiCht8 and AiCht‐h had the highest expression in the midgut. The RNA interference (RNAi) experiment revealed that knockdown of AiCht10 or the imaginal disc growth factor gene (AiIDGF) induced high larval mortality. Larvae failed to shed the old cuticle during molting after the injection of double‐stranded RNA targeting AiCht10 (dsAiCht10), whereas the larval bodies shrunk and blackened after the injection of dsRNA targeting AiIDGF (dsAiIDGF). CONCLUSION Our results revealed for the first time the important functions of AiCht10 and AiIDGF in A. ipsilon. These genes are essential for larval development, and can potentially serve as new targets for RNAi‐based pest management. © 2022 Society of Chemical Industry.
Hawthorn spider mite, Amphitetranychus viennensis Zacher, 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. viennensis to 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 3 for avermectin, 2) GAPDH, RPS9, and 28S for bifenazate, 3) Actin 3, V-ATPase B, and α-tubulin for spirodiclofen, and 4) Actin 3, α-tubulin, and V-ATPase A for fenpropathrin. Although unique sets of genes are proposed for each acaricide, α-tubulin, EF1A, and GAPDH are the most consistently stably expressed reference genes when A. viennensis was 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.
Black cutworm (BCW), Agrotis ipsilon (Hufnagel), is an occasional pest of maize that can cause considerable economic loss and injury to corn seedlings. This research mainly assessed the susceptibility of BCW neonates to 11 Bt toxins (Cry1Ab, Cry1Ac, Cry1Ah, Cry1F, Cry1Ie, Cry1B, Cry2Aa, Vip3_ch1, Vip3_ch4, Vip3Ca2, Vip3Aa19) by exposing neonates to an artificial diet containing Bt toxins and evaluated the efficacy of three transgenic maize events (C008, C009, C010) expressing Vip3Aa19 toxin against BCW. The toxin-diet bioassay data indicated that Vip3Aa19 protein (LC50 = 0.43 μg/g) was the most active against BCW. Chimeric protein Vip3_ch1 (LC50 = 5.53 μg/g), Cry1F (LC50 = 83.62 μg/g) and Cry1Ac (LC50 = 184.77 μg/g) were less toxic. BCW was very tolerant to the other Bt toxins tested, with LC50 values more than 200 μg/g. Greenhouse studies were conducted with artificial infestations at the whorl stage by placing second-instar BCW larvae into whorl leaf and the fourth-instar larvae at the base of maize seedings. These results suggest that these transgenic maize events expressing Vip3Aa19 can provide effective control for BCW.
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. ipsilon management 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 synthases to control A. ipsilon. As a result, two chitin synthases, AiCHS1 and AiCHS2, were identified and cloned from A. ipsilon. The temporal-spatial distribution study showed that AiCHS1 was predominantly expressed at the pupal stage and most abundant among tissues of head capsule and integument, while AiCHS2 was 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. ipsilon responded with an elevated expression in AiCHS1 and AiCHS2, while larvae showed difficulty in shedding old cuticle, and a cumulative mortality of 69.24% at 48 h. In summary, chitin synthases are crucial for chitin biosynthesis in A. ipsilon and can be targeted for the control (e.g., RNAi-based biopesticides) of this devastating insect pest.
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