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The Masculinizer gene, Masc, encodes a lepidopteran-specific novel CCCH-type zinc finger protein, which controls sex determination and dosage compensation in Bombyx mori. Considering the potential application of it in pest control, it is necessary to investigate the function of Masc gene in Hyphantria cunea, a globally invasive forest pest. In the present study, we identified and functionally characterized the Masc gene, HcMasc, in H. cunea. Sequence analysis revealed that HcMASC contained the conserved CCCH-type zinc finger domain, nuclear localization signal, and male determining domain, in which the last was confirmed to be required for its masculinization in BmN cell line. However, expression data showed that unlike male-biased expression in B. mori, HcMasc gene expresses in main all developmental stages or tissues in both sexes. Clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-based disruption of the common exons 1 and 3 of the HcMasc gene resulted in imbalanced sex ratio and abnormal external genitalia of both sexes. Our results suggest that the HcMasc gene is required for both male and female sexual differentiation and dosage compensation in H. cunea and provide a foundation for developing better strategies to control this pest., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

Background: The fall webworm, Hyphantria cunea, an invasive forest pest found worldwide, causes serious ecological and economic damage. Currently, the application of chemical pesticides is the most widely used strategy for H. cunea management. However, long-term pesticide use leads to pest resistance, phytotoxicity, human poisoning, and environmental deterioration. RNA interference (RNAi) technology may provide an environmentally friendly and cost-effective option for H. cunea control. However, effective RNAi targets and application methods for H. cunea are lacking., Results: We screened and obtained two highly effective RNAi targets, vATPase A (V-type proton ATPase catalytic subunit A) and Rop (Ras opposite), from 23 candidate genes, using initial and repeat screening tests with the double-stranded RNA (dsRNA) injection method. RNAi against these two genes was effective in suppressing each target messenger RNA level and interfering with larval growth, leading to significant larval mortality and pupal abnormality. For massive production of dsRNA and practical application of RNAi technology in H. cunea, transformed bacteria expressing dsRNAs of these two genes were prepared using the L4440 expression vector and HT115 strain of Escherichia coli. Oral administration of bacterially expressed dsRNA targeting vATPase A and Rop genes showed high mortality and the same malformed phenotype as the injection treatment. To further investigate the lethal effects of targeting these two genes on larval development, transcriptome sequencing (RNA-seq) was performed on RNAi samples. The results demonstrated disorders in multiple metabolic pathways, and the expression levels of most genes related to insect cuticle metabolism were significantly different, which may directly threaten insect survival. In addition, some new findings were obtained via RNA-seq analysis; for example, the progesterone-mediated oocyte maturation and oocyte meiosis processes were significantly different after silencing vATPase A, and the insect olfactory protein-related genes were significantly downregulated after dsHcRop treatment., Conclusion: vATPase A and Rop are two highly effective RNAi-mediated lethal genes in H. cunea that regulate insect growth via multiple metabolic pathways. Oral delivery of bacterially expressed dsRNA specific to vATPase A and Rop can potentially be used for RNAi-based H. cunea management. This is the first study to apply bacteria-mediated RNAi for the control of this invasive pest, which is a major step forward in the application of the RNAi technology in H. cunea. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

RNA interference (RNAi) technology is a promising approach used in pest control. The efficiency of RNAi varies considerably among different insect species, and growing evidence suggests that degradation of double-stranded RNA (dsRNA) prior to uptake is an important factor that limits RNAi efficiency in insects. Our recent work on fall webworm ( Hyphantria cunea ), an important invasive pest in China, showed a relatively low silencing efficiency of RNAi through dsRNA injection, which is considered the most feasible dsRNA delivery method for inducing RNAi, and the factors involved in the mechanism remain unknown. Herein, we first detected the dsRNA-degrading activity in the hemolymph and gut content of H. cunea in ex vivo assays and observed rapid degradation of dsRNA, especially in the hemolymph, which was complete within only 10 min. To determine whether dsRNA degradation could contribute to the low effectiveness of RNAi in H. cunea , four dsRNA nuclease ( dsRNase ) genes, HcdsRNase1, HcdsRNase2, HcdsRNase3 , and HcdsRNase4, were identified by homology searching against the H. cunea transcriptome database, and their transcript levels were subsequently investigated in different tissues, developmental stages, and after dsRNA injection. Our results show that HcdsRNases are highly expressed mainly in gut tissues and hemolymph, and the expression of HcdsRNase3 and HcdsRNase4 were significantly upregulated by dsGFP induction. RNAi-of-RNAi studies, using HcCht5 as a reporter gene, demonstrated that silencing HcdsRNase3 and HcdsRNase4 significantly increases RNAi efficacy via dsHcCht5 injection, and co-silencing these two HcdsRNase genes results in a more significant improvement in efficacy. These results confirm that the RNAi efficacy in H. cunea through dsRNA injection is certainly impaired by dsRNase activity, and that blocking HcdsRNases could potentially improve RNAi, providing a reference for related studies on insects where RNAi has low efficiency.

Background: Micromelalopha siversi (Staudinger) (Lepidoptera: Notodontidae) is a defoliator of poplar trees, Populus spp. (Salicaceae). In our previous study, 13,15-octadecadienal has been conformed as a female-produced candidate sex pheromone component of M. siversi, but the Z/E stereochemistry of the 1,3-diene system has not been identified so far., Results: Four unsaturated aliphatic aldehydes, Z13,E15-18:Ald, Z13,Z15-18:Ald, E13,E15-18:Ald, and E13,Z15-18:Ald, were synthesized from the commercially available 12-bromo-1-decanol mainly by alkylation of lithium alkyne, normal Wittig or Wittig-Schlosser olefination, and hydroboration-protonolysis. According to gas chromatography (GC) analysis of pheromone gland extracts, Z13,E15-18:Ald was the main component, and a small amount of Z13,Z15-18:Ald was also detected, with a ratio of approximately 7:3. However, the results of GC-electroantennographic detection (GC-EAD) showed that Z13,E15-18:Ald was the only compound with electrophysiological activity, whereas Z13,Z15-18:Ald elicited no activity. In the field, traps baited with only Z13,E15-18:Ald resulted in much superior results to those with Z13,Z15-18:Ald as well as the Z13,E15-18:Ald and Z13,Z15-18:Ald binary mixture., Conclusions: Based on geometrically selective synthesis and bioactivity tests, the active sex pheromone component of M. siversi has been identified as Z13,E15-18:Ald, the pheromone component that has not been identified in Lepidoptera before. The synthetic component was attractive to male moths in preliminary field traps, which provides novel technologies to monitor and control this pest., (© 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Dendrolimus punctatus Walker (Lepidoptera: Lasiocampidae) is a pine caterpillar moth distributed in most areas of southern China and is an economically important pest of pine, due to its defoliation activity. Understanding fundamental sex pheromone perception mechanisms in D. punctatus may provide effective and sustainable options for novel control strategies. However, the identification and function of pheromone receptors, key genes that receipt the pheromone of this pest, are both unclear now. Previous researches suggested several candidate pheromone receptors whose expression levels were male antennae bias in D. punctatus. In this study, we cloned six candidate pheromone receptors (DpunOR 20/45/46/51/54/58) and Orco from D. punctatus. Phylogenetic tree analysis showed that lepidopteran PRs tend to be conserved and clustered together; however, D. punctatus candidate PRs were located in a distinct clade. Motif analysis of PRs showed clear sequences differences between Dendrolimus spp. and other tested moth species. To illustrate the ligand response properties of the candidate PRs of D. punctatus, each of the six genes was expressed with an Orco gene in Xenopus oocytes and using two-electrode voltage-clamp recordings. Finally, we successfully identified two sex pheromone receptors (PR45 and PR46). Our study, which identified a novel lineage of PRs tuned to Type I pheromones in Lepidoptera, provides evidence for the new evolution origin of sex pheromone communication in moths, and lays a foundation for the development of novel control strategies of D. punctatus., Competing Interests: Declaration of competing interest The authors declare they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dendrolimus spp. are important destructive pests of conifer forests, and Dendrolimus punctatus Walker (Lepidoptera; Lasiocampidae) is the most widely distributed Dendrolimus species. During periodic outbreaks, this species is said to make "fire without smoke" because large areas of pine forest can be quickly and heavily damaged. Yet, little is known about the molecular mechanisms that underlie the unique ecological characteristics of this forest insect. Here, we combined Pacific Biosciences (PacBio) RSII single-molecule long reads and high-throughput chromosome conformation capture (Hi-C) genomics-linked reads to produce a high-quality, chromosome-level reference genome for D. punctatus. The final assembly was 614 Mb with contig and scaffold N50 values of 1.39 and 22.15 Mb, respectively, and 96.96% of the contigs anchored onto 30 chromosomes. Based on the prediction, this genome contained 17,593 protein-coding genes and 56.16% repetitive sequences. Phylogenetic analyses indicated that D. punctatus diverged from the common ancestor of Hyphantria cunea, Spodoptera litura and Thaumetopoea pityocampa ~ 108.91 million years ago. Many gene families that were expanded in the D. punctatus genome were significantly enriched for the xenobiotic biodegradation system, especially the cytochrome P450 gene family. This high-quality, chromosome-level reference genome will be a valuable resource for understanding mechanisms of D. punctatus outbreak and host resistance adaption. Because this is the first Lasiocampidae insect genome to be sequenced, it also will serve as a reference for further comparative genomics., (© 2020 John Wiley & Sons Ltd.)

Erannis ankeraria Staudinger, 1861 (Lepidoptera: Geometridae) is one of the major pests causing serious damages on Larix spp., Quercus L., and Picea Mill. To investigate the conceivable functions of antennal sensilla associated with pheromone detection, we observed the ultrastructure of antennae in female and male of E. ankeraria moths by scanning electron microscopy. Six types (including two subtypes and a new type) of sensilla were recorded and characterized, including Sensilla trichodea (ST I and ST II), Böhm bristles, Sensilla squamiformia, Sensilla chaetica, Sensilla auricillic (SAU) and newly observed serrate-like sensilla. ST I and SAU were abundant on male antennae, displaying a sexual dismorphism. Serrate-like sensilla were also peculiar to male antennae. In summary of reported functions of corresponding sensilla, we presumed putative functions of the recorded sensilla in E. ankeraria, providing the morphological basis for sensory mechanisms related to pest management., (© 2019 Wiley Periodicals, Inc.)

Syntheses of (11 Z ,13 Z )-hexadecadienal ( 1 ), (11 Z ,13 Z )-hexadecadienol ( 2 ), (11 Z ,13 Z )-hexadecadien-1-yl acetate ( 3 ), and (Z)-13-hexadecen-11-ynal ( 4 ) from commercially available starting material 10-bromo-1-decanol are reported. These ( Z , Z )-dienes and conjugated en-yne moieties are common in sex pheromone and attractant components for many Notodontide insect pests. The synthetic scheme, using the C10 + C3 + C3 strategy, was mainly based on three key steps: alkylation of lithium alkyne under a low temperature, cis -Wittig olefination of the aldehyde with propylidentriphenylphosphorane, and hydroboration-protonolysis of alkyne. This synthetic route provided (11 Z ,13 Z )-hexadecadienal ( 1 ) in a 23.0% total yield via an eight-step sequence, alcohol ( 2 ) in a 21.9% total yield, acetate ( 3 ) in a 21.4% total yield, and (Z)-13-hexadecen-11-ynal ( 4 ) in a 34.7% total yield. This simple strategy provides a new way to achieve syntheses of the key sex pheromones of Notodontide insect pests.

Invasive species cause considerable ecological and economic damage. Despite decades of broad impacts of invasives on diversity and agriculture, the genetic adaptations and near-term evolution of invading populations are poorly understood. The fall webworm, Hyphantria cunea, a highly successful invasive species that originated in North America, spread throughout the Northern Hemisphere during the past 80 years. Here, we use whole-genome sequencing of invasive populations and transcriptome profiling to probe the underlying genetic bases for the rapid adaptation of this species to new environments and host plants. We find substantial reductions in genomic diversity consistent with founder effects. Genes and pathways associated with carbohydrate metabolism and gustatory receptors are substantially expanded in the webworm genome and show strong signatures of functional polymorphisms in the invasive population. We also find that silk-yielding-associated genes maintained a relatively low level of functional diversity, and identify candidate genes that may regulate the development of silk glands in fall webworms. These data suggest that the fall webworm's ability to colonize novel hosts, mediated by plasticity in their gustatory capabilities along with an increased ability to utilize novel nutrition sources and substrates, has facilitated the rapid and successful adaptation of the species throughout its range.

Pheromone-binding proteins (PBPs) play an important role in the recognition of pheromones by insects. However, the abilities of these PBPs to discriminate pheromone components and recognize the isomers are unclear. Dendrolimus houi and Dendrolimus kikuchii are two sympatric coniferous pests whose pheromones have cis-trans isomers. We used these insect species to detect the precise recognition abilities of PBPs. The four PBPs examined showed male-biased antenna-intensive expression patterns, whereas PBP1 showed higher expression than PBP2 in the antenna. DhouPBP1 only bound to a minor interspecific pheromone component, whereas DhouPBP2 bound to all three intraspecific components and another minor interspecific component. DkikPBP1 and DkikPBP2 could recognize all three intraspecific components with affinities negatively correlated with their ratios, and they bound to interspecific pheromones with affinity that was positively correlated with the ratios. The four PBPs have different cis-trans isomer discrimination abilities, i.e., DhouPBP1 and DkikPBP1 could not discriminate the two cis-trans isomer pairs of pheromones from the two species, whereas DhouPBP2 could discriminate between both pairs, and DkikPBP2 could only discriminate one pair. Overall, PBPs from D. houi and D. kikuchii use different strategies to help the moths to discriminate the intra- and interspecific pheromone components. Our work will contribute to better understanding of the sex pheromone recognition mechanism in these two sister species of moths and provide insights into more effective management practices of these pest species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Yunnan pine and Simao pine caterpillar moths, Dendrolimus houi Lajonquière and Dendrolimus kikuchii Matsumura (Lepidoptera: Lasiocampidae), are two closely related and sympatric pests of coniferous forests in southwestern China, and olfactory communication systems of these two insects have received considerable attention because of their economic importance. However, there is little information on the molecular aspect of odor detection about these insects. Furthermore, although lepidopteran species have been widely used in studies of insect olfaction, few work made comparison between sister moths on the olfactory recognition mechanisms. In this study, next-generation sequencing of the antennal transcriptome of these two moths were performed to identify the major olfactory genes. After comparing the antennal transcriptome of these two moths, we found that they exhibit highly similar transcripts-associated GO terms. Chemosensory gene families were further analyzed in both species. We identified 23 putative odorant binding proteins (OBP), 17 chemosensory proteins (CSP), two sensory neuron membrane proteins (SNMP), 33 odorant receptors (OR), and 10 ionotropic receptors (IR) in D. houi; and 27 putative OBPs, 17 CSPs, two SNMPs, 33 ORs, and nine IRs in D. kikuchii. All these transcripts were full-length or almost full-length. The predicted protein sequences were compared with orthologs in other species of Lepidoptera and model insects, including Bombyx mori, Manduca sexta, Heliothis virescens, Danaus plexippus, Sesamia inferens, Cydia pomonella, and Drosophila melanogaster. The sequence homologies of the orthologous genes in D. houi and D. kikuchii are very high. Furthermore, the olfactory genes were classed according to their expression level, and the highly expressed genes are our target for further function investigation. Interestingly, many highly expressed genes are ortholog gene of D. houi and D. kikuchii. We also found that the Classic OBPs were further separated into three groups according to their motifs, which will help future functional researches. Surprisingly, no pheromone receptor was identified in the two Dendrolimus species, which may indicate a special pheromone identification mechanism in Dendrolimus. Our work allows for further functional studies of pheromones and host volatile recognition genes, and give novel candidate targets for pest management., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Odorant binding proteins (OBPs) play important roles in insect olfactory processes. The Chinese pine caterpillar moth, Dendrolimus tabulaeformis (Lepidoptera, Lasiocampidae) is a serious economic pest in China, and the pheromones of this species have been identified to monitor their presence. However, the molecular mechanisms by which D. tabulaeformis perceive pheromones and host volatiles remain unknown. In this study, we identified and characterized three new OBPs, including one pheromone binding protein (PBP1) and two general odor binding proteins (GOBPs), from antennal cDNA of D. tabulaeformis. The deduced amino acid sequences of DtabPBP1, DtabGOBP1, and DtabGOBP2 revealed mature proteins of 140, 147, and 140 amino acids, respectively. Each has six cysteine residues in conserved positions relative to other known OBPs. Amino-acid alignments indicated that the two GOBPs are more conserved (DtabGOBP1 is 52.9-67.4 % identical to orthologs from other Lepidoptera, and DtabGOBP2 is 55.2-81.8 % identical) than the PBP (32.5-46.0 %). Real-time PCR indicated tissue- and sex-specific expression patterns of the three genes. DtabPBP1 was mainly expressed in the antennae of males, whereas female antennae had only 1.09 % the expression in male antennae. Both DtabGOBP1 and DtabGOBP2 were more highly expressed in antennae than in other tissues, while DtabGOBP1 was more abundant in male antennae and DtabGOBP2 in female antennae. In addition, the binding specificities of the three proteins were investigated, and all three OBPs exhibited high binding affinities for the pheromone component (5Z,7E)-5,7-dodecadien-1-yl propionate (Z5,E7-12:OPr). This suggests a role in binding pheromone for GOBPs, as well as PBP1, in D. tabulaeformis.