Characterization of four mitochondrial genomes of Crambidae (Lepidoptera, Pyraloidea) and phylogenetic implications.

Loxostege turbidalis, Loxostege aeruginalis, Pyrausta despicata, and Crambus perlellus belong to Crambidae, Pyraloidea. Their mitochondrial genomes (mitogenomes) were successfully sequenced. The mitogenomes of L. turbidalis, L. aeruginalis, P. despicata, and C. perlellus are 15 240 bp, 15 339 bp, 15 389 bp, and 15 440 bp. The four mitogenomes all have a typical insect mitochondrial gene order, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and one A + T rich region (control region). The PCGs are initiated by the typical ATN codons, except CGA for the cox1 gene. Most PCGs terminate with common codon TAA or TAG, the incomplete codon T is found as the stop codon for cox2, nad4, and nad5. Most tRNA genes exhibit typical cloverleaf structure, except trnS1 (AGN) lacking the dihydrouridine (DHU) arm. The secondary structure of rRNA of four mitogenomes were predicted. Poly-T structure and micro-satellite regions are conserved in control regions. The phylogenetic analyses based on 13 PCGs showed the relationships of subfamilies in Pyraloidea. Pyralidae, and Crambidae are monophyletic, respectively. Pyralidae comprises four subfamilies, which form the following topology with high support values: (Galleriinae + ((Pyralinae + Epipaschiinae)+ Phycitinae)). Crambidae includes seven subfamilies and is divided into two lineages. Pyraustinae and Spilomelinae are sister groups of each other, and form the "PS clade." Other five subfamilies (Crambinae, Acentropinae, Scopariinae, Schoenobiinae, and Glaphyriinae) form the "non-PS clade" in the Bayesian inference tree. However, Schoenobiinae is not grouped with the other four subfamilies and located at the base of Crambidae in two maximum likelihood trees.

Identification and Functional Characterization of Two Chitin Synthases in the Black Cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae).

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.

Identification and RNAi-based functional analysis of chitinase family genes in Agrotis ipsilon.

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.

Complete mitochondrial genome of Dioryctria yiai (Lepidoptera: Pyralidae).

The Dioryctria yiai belongs to Pyralidae in Lepidoptera. The complete mitogenome of D. yiai was described in this study, which is typical circular duplex molecules and 15,430 bp in length, containing the standard metazoan set of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and an A + T-rich region. The gene order is same with other lepidopterans. Except for cox1 started with CGA, all other PCGs started with the standard ATN codons. Most of the PCGs terminated with the stop codon TAA, whereas cox2 has the incomplete stop codon T. The phylogenetic tree showed that D. yiai and other six species belong to Phycitinae, are clustered into a clade.

Insecticidal Activity of 11 Bt toxins and 3 Transgenic Maize Events Expressing Vip3Aa19 to Black Cutworm, Agrotis ipsilon (Hufnagel).

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.

Complete mitochondrial genome of Mycalesis intermedia (Lepidoptera: Nymphalidae).

The Mycalesis intermedia belongs to Nymphalidae in Lepidoptera. We described the complete mitogenome of M. intermedia, which is typical circular duplex molecules and 15,386 bp in length, containing the standard metazoan set of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and an A + T-rich region with macro-repeat sequences. All the inferred tRNA secondary structures show the common cloverleaf pattern, with the exception of trnS1(AGN) which lacks the DHU arm. The M. intermedia mitochondrial genome has the same gene order with other lepidopterans.

Wetting and adhesion behavior on apple tree leaf surface by adding different surfactants.

OBJECTIVE: This study was conducted to investigate the wetting behavior of different surfactant solutions on the leaf surfaces of apple during the fruit formation stage. METHODS: Five surfactants, including C12E5, Tween-20, Triton X-100, DTAB, and SDS were evaluated in this study. The contact angle, surface tension, adhesion tension, work of adhesion, and solid-liquid interface tension of droplets on the leaf surface were determined by the drop method. RESULTS: The results showed that the nonionic surfactants C12E5 and Triton X-100 had better wetting effects than other surfactants. Moreover, when the concentration of C12E5 and Triton X-100 was 1 × 10-3 mol/L, the leaves reached a completely wet state. Toxicity measurement showed that the incubation rate of Carposina niponensis eggs decreased gradually with increasing content of C12E5 or Triton X-100. Additionally, field efficacy analysis showed that adding C12E5 or Triton X-100 significantly improved the beta-cyfluthrin 3% water emulsion (EW) against C. niponensis. CONCLUSIONS: These results indicate that the surfactants C12E5 and Triton X-100 can significantly improve pesticide application, which will be helpful for reducing pesticide use and developing new pesticides.

Selection of Reference Genes for RT-qPCR Analysis Under Intrinsic Conditions in the Hawthorn Spider Mite, Amphitetranychus viennensis (Acarina: Tetranychidae).

Hawthorn spider mite, Amphitetranychus viennensis Zacher, is one of the most devastating pests of deciduous fruit trees. The overall goal of this research is to develop a standardized protocol for real-time quantitative reverse transcription PCR (RT-qPCR) analysis in A. viennensis following the MIQE (minimum information for publication of Quantitative real time PCR experiments) guidelines. Based on the previous knowledge, we hypothesized that internal references for RT-qPCR analysis reside in housekeeping genes (HKGs). To test this hypothesis, we examined the stability of nine HKGs from A. viennensis, including 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), Elongation factor 1-alpha (EF1A), Actin3, V-ATP vacuolar-type H+-ATPase (V-ATPase), α-tubulin (α-tubulin), Ribosomal protein L13 (RPL13), 40S ribosomal protein S9 (RPS9), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The expression profile of these candidates under intrinsic conditions was evaluated by a panel of computational programs, including geNorm, Normfinder, BestKeeper, and ΔCt method. Based on RefFinder, a comprehensive software integrating all four above-mentioned algorithms, V-ATPase, Actin3, and GAPDH are the top three reference genes, which are stably expressed across all the intrinsic conditions, including developmental stage, sex, and diapause. In addition, we compared reference genes recommended for different developmental stages among the nine cell-content feeding arthropods, including four spider mites, A. viennensis, Tetranychus urticae, Tetranychus cinnabarinus, and Panonychus citri, and five hemipterans, Myzus persicae, Aphis gossypii, Toxoptera citricida, Lipaphis erysimi, and Sogatella furcifera. Not surprisingly, rRNAs and ribosomal proteins, the most abundant RNA species, is the top choice, and follows by EF1A, Actin, GAPDH, and tubulin. Information present here lays the foundation for the genomic and functional genomic research in cell-content feeding arthropods in general and A. viennensis in particular.