Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA−box, GAPDH, AK, Actin, EF, α−tubulin, SOD, 18S rRNA, and FTZ−F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ−F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Mythimna loreyi (Lepidoptera: Noctuidae)

Histone lysine methyltransferases (HKMTs) perform vital roles in cellular life by controlling gene expression programs through the posttranslational modification of histone tails. Since many of them are intimately involved in the development of different diseases, including several cancers, understanding the molecular mechanisms that control their target recognition and activity is vital for the treatment and prevention of such conditions. RNA binding has been shown to be an important regulatory factor in the function of several HKMTs, such as the yeast Set1 and the human Ezh2. Moreover, many HKMTs are capable of RNA binding in the absence of a canonical RNA binding domain. Here, we explored the RNA binding capacity of KMT2D, one of the major H3K4 monomethyl transferases in enhancers, using RNA immunoprecipitation followed by sequencing. We identified a broad range of coding and non-coding RNAs associated with KMT2D and confirmed their binding through RNA immunoprecipitation and quantitative PCR. We also showed that a separated RNA binding region within KMT2D is capable of binding a similar RNA pool, but differences in the binding specificity indicate the existence of other regulatory elements in the sequence of KMT2D. Analysis of the bound mRNAs revealed that KMT2D preferentially binds co-transcriptionally to the mRNAs of the genes under its control, while also interacting with super enhancer- and splicing-related non-coding RNAs. These observations, together with the nuclear colocalization of KMT2D with differentially phosphorylated forms of RNA Polymerase II suggest a so far unexplored role of KMT2D in the RNA processing of the nascent transcripts. This article is protected by copyright. All rights reserved.

KMT2D preferentially binds mRNAs of the genes it regulates, suggesting a role in RNA processing.

Simple Summary Reference genes serve as a foundation for investigating the functionality of target genes in insects. Nevertheless, the selection of reference genes is highly dependent upon particular experimental circumstances. Recently, the bean bug, Riptortus pedestris (Hemiptera: Alydidae), has attracted increasing attention due to its damage on yield losses of soybean crops. However, there is a dearth of literature regarding the evaluation of reference genes for quantitative real-time PCR (qRT-PCR) in R. pedestris. The current study aimed to assess the stability of expression levels in 16 candidate reference genes in R. pedestris across six experimental conditions. This evaluation was conducted using ΔCt method, GeNorm, NormFinder, BestKeeper, and RefFinder. Our results indicate that the optimum reference genes were as follows: RPL7A and EF1 for the developmental stage, α−tubulin and EF1 for adult tissues, RPL32 and RPL7A for adult age, RPL32 and SOD for nymph age, RPS23 and RPL7A for temperature, and HSP70 and GAPDH for mating status. These findings will provide a basis for future investigations into the expression and function of target genes in bean bugs. Abstract Quantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data normalization strongly depends upon stable reference genes. The bean bug, Riptortus pedestris (Hemiptera: Alydidae), is a significant pest of leguminous crops and broadly distributed across Southeast Asia. In this study, a total of 16 candidate reference genes (RPL32, RPS23, SDHA, UBQ, UCCR, GST, TATA−box, HSP70, GAPDH, RPL7A, SOD, RPS3, Actin, α−tubulin, AK, and EF1) were carefully chosen in R. pedestris, and their expression levels were assessed across various conditions, including different developmental stages, diverse tissues, temperature treatments, adult age, molting time, and mating status. Following this, the stability of these reference genes was evaluated using four algorithms (ΔCt, GeNorm, NormFinder, and BestKeeper). Ultimately, the comprehensive rankings were determined using the online tool RefFinder. Our results demonstrate that the reference gene for qRT-PCR analysis in R. pedestris is contingent upon the specific experimental conditions. RPL7A and EF1 are optimal reference genes for developmental stages. Furthermore, α−tubulin and EF1 exhibit the most stable expression across various adult tissues. RPL32 and RPL7A exhibit the most stable expression for adult age. For nymph age, RPL32 and SOD display the most stable expression. For temperature conditions, RPS23 and RPL7A were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 (RpedOBP37) and cytochrome P450 6a2 (RpedCYP6) throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on R. pedestris, serving as a fundamental groundwork for future investigations on stable reference genes in R. pedestris as well as other organisms.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, Riptortus pedestris (Hemiptera: Alydidae)

Endoclita signifer is a prominent wood-boring insect species in eucalyptus plantations in Guangxi, China, causing significant ecological and economic damage. A novel approach to controlling the challenging wood-boring pest involves disrupting the olfactory communication between insects and the volatile compounds emitted by plants. To identify the olfactory proteins contributing to host selection based on 11 GC-EAD-active volatiles from eucalyptus leaves and to discover the highly expressed olfactory proteins, we conducted a study on the antennal transcriptomes of adult E. signifer and screened key olfactory proteins in the antennae. We identified a total of 69 olfactory proteins. When compared to the larval transcriptomes, the antennal transcriptome of adult E. signifer revealed the presence of 17 new odorant-binding proteins (OBPs), including 2 pheromone-binding proteins (PBPs), 7 previously unreported chemosensory proteins (CSPs), 17 new odorant receptors (ORs), 4 new gustatory receptors (GRs), 11 novel ionotropic receptors (IRs), and 2 sensory neuron membrane proteins (SNMPs). Through the phylogenetic tree of OBPs and ORs, we identified EsigPBP2 and EsigPBP3 as two of the three PBPs, designated EsigOR13 as EsigOrco, and recognized EsigOR10 and EsigOR22 as the newly discovered EsigPRs in E. signifer. In the adult antennae, the expression levels of EsigGOBP14, EsigGOBP13, EsigOBP14, EsigOBP17, EsigCSP14, and EsigOR16 were notably high, indicating that these proteins could be pivotal in binding to plant volatiles.

A Study of Adult Olfactory Proteins of Primitive Ghost Moth, Endoclita signifer (Lepidoptera, Hepialidae)

Abstract Leptocybe invasa Fisher &amp; La Salle, 2004 (Hymenoptera: Eulophidae) forms galls in tips, young branches, central veins, and petioles of Eucalyptus spp. leaves. This insect reproduces primarily through thelytokous parthenogenesis, with males being rare. The parasitism of L. invasa by Quadrastichus mendeli Kim &amp; La Salle, 2008 (Hymenoptera: Eulophidae), with short life cycle and rapid dispersion, is high. The objective was to record the first occurrence of L. invasa males and its parasitoid Q. mendeli in galls of this pest in Paraguay. Branches of the grancam clone PL 113 (Eucalyptus grandis × Eucalyptus camaldulensis) with L. invasa galls were collected from February to April 2022 in the Maciel District, Caazapá Department, Paraguay. These branches were put in plastic bags, preserved in 70% ethanol and the insects emerged sent to the Laboratory of Biological Control of Forest Pests (LCBPF) at FCA/UNESP in Botucatu, São Paulo state, Brazil. Morphological characteristics confirmed the presence of L. invasa males at a ratio of 38:1 (females to males). Morphological and molecular analysis identified Q. mendeli and its samples deposited in GenBank (OR248672.1). The parasitism rate of L. invasa by Q. mendeli was 26.8%, marking the first record of this occurrence in Paraguay. 

Leptocybe invasa males and the parasitoid Quadrastichus mendeli (Hym.: Eulophidae): First report in Paraguay

Endoclita signifer Walker is the most destructive wood-boring pest of Eucalyptus in China, causing significant economic and ecological damage. As an insect of the primitive Lepidoptera family Hepialidae, E. signifer fly and mat for only 10-20 min at dusk. The courtship and mating behavior of E. signifer adults and whether male moths release sex pheromones are still unknown, especially since transitory flight survival strategies in primitive moths differ from advanced moths like noctuids. In this study, we first observed the courtship and mating behavior of E. signifer by considering the effects of space and then analyzed extracts of male hairbrushes using gas chromatography-electroantennogram detection. Our results indicated that during the courtship period, flying males form courtship fields, lekking, and chase flying females before mating with them; E. signifer were more successful in mating in larger spaces (Length × Width × Height = 9.6 × 7 × 4 m); 5 compounds in the hairbrushes of the male moths which elicited antennal responses of 2 sexes, despite at high concentrations. Combined with it, indicating that communication between male and female may rely on male sex pheromones. These findings can serve as a basis for studying the mechanisms of sex communication in E. signifer and developing sex pheromone-based trapping techniques.

Courtship and mating behavior of Endoclita signifer (Hepialidae: Lepidoptera) and the male sex pheromones in hairbrushes.

Abstract Background Endoclita signifer causes severe damage to eucalyptus plantations, and the larvae transfer to and damage eucalyptus accurately in mixed forests, suggesting that the larval olfactory system contributes to host selection. The olfactory proteins in the head and tegument of E. signifer larvae were previously identified. To identify the relationship between olfactory protein expression in the larval head the larvae head and the developmental expression dynamics, and its functions in further recognition of plant volatiles, the head transcriptomes of two instar larvae and the expression profiles of olfactory proteins in the instars after exposure to volatiles were studied. Results Eight odorant-binding proteins, six chemosensory proteins, three odorant receptors, three gustatory receptors, and 18 ionotropic receptors were identified. Half of the olfactory proteins were the most highly expressed in the young (5th) larval head, and EsigGOBP2, EsigGOBP4, EsigGOBP5, EsigCSP1, EsigCSP3, EsigGR1 and EsigGR3 were highly expressed and showed a specific expression pattern. In addition, after exposure to o-cymene, α-phellandrene, n-butyl ether, and 4-ethylacetophenone, EsigGR3 was downregulated significantly, and exposure to n-butyl ether caused EsigGR1 to be downregulated significantly. Conclusions Seven specific olfactory proteins may be important genes in larval olfactory recognition. Furthermore, based on the receptors that were downregulated after exposure to volatiles and the previous electrophysiological activity in the third larvae, we speculated that the ligand of EsigGR1 was n-butyl ether, and the ligands of the newly identified EsigGR3 are all electrophysiologically active compounds, which demonstrated host recognition in the third larvae of E . signifer . These results provide a way to find key plant volatiles recognized by the key olfactory proteins as new targets for pest control. Graphical Abstract 

/pdf/olfactory-proteins-of-endoclita-signifer-larvae-and-their-1ypju2ij.pdf

Olfactory proteins of Endoclita signifer larvae and their roles in host recognition

/pdf/olfactory-proteins-of-endoclita-signifer-larvae-and-their-172oe65z.pdf

Simple Summary In gene expression investigations, the first crucial step is choosing appropriate housekeeping genes. However, the choice of reference genes is not absolute but relative and varies with different experimental conditions. It is vital to note that using unvalidated or unscreened internal reference genes can lead to erroneous inferences. This study was conducted on Leptocybe invasa to calculate the stability of eight housekeeping genes across various test conditions, such as sexes, somites, temperatures, diets, and pesticides. The relative expression of HSP90 at different temperature settings was evaluated to validate the results. This study aims to assist future gene expression research on this invasive species and lay the groundwork for further investigations into the gene function of this pest. Abstract Leptocybe invasa (Hymenoptera: Eulophidae) is a globally intrusive pest. Despite extensive research into the physiological responses of this pest, our understanding of the molecular mechanisms still needs to be improved. We want to accurately investigate the expression of L. invasa’s target genes, so it is imperative to select fitting reference genes. In this study, eight housekeeping genes’ stability (RPS30, ACTR, 18S rRNA, ACT, RPL18, GAPDH, 28S rRNA, and TUB) was tested under five different experimental conditions, including male or female adults, somites (head, thorax, and abdomen), temperatures (0 °C, 25 °C, and 40 °C), diets (starvation, clear water, 10% honey water, Eucalyptus sap), and pesticides (acetone was used as a control, imidacloprid, monosultap). Gene stability was calculated using RefFinder, which integrates four algorithms (the ∆Ct method, geNorm, NormFinder, and BestKeeper). The findings implied that ACT and ACTR were the most accurate when comparing sexes. For analyzing different somites, 28S rRNA and RPL18 were ideal; the 28S rRNA and RRS30 were perfect for analyzing at different temperatures. The combination of ACT and GAPDH helped to analyze gene expression in different diets, and GAPDH and 28S rRNA were suitable for various pesticide conditions. Overall, this research offers a complete list of reference genes from L. invasa for precise analysis of target gene expression, which can improve the trustworthiness of RT-qPCR and lay the foundation for further investigations into the gene function of this pest.

/pdf/identification-and-validation-of-reference-genes-for-sp3wyzgk.pdf

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae)

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Courtship and mating behavior of Endoclita signifer (Hepialidae: Lepidoptera) and the male sex pheromones in hairbrushes.

Olfactory proteins of Endoclita signifer larvae and their roles in host recognition

Olfactory proteins of Endoclita signifer larvae and their roles in host recognition

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae)