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Benzyl alcohol (E1519) is an aromatic alcohol used in the pharmaceutical and food industry. It is used to protect food products against microorganisms during storage, as a flavoring in the production of chocolate and confectionery products, as an important ingredient in fragrance, and as a preservative in medical products. However, little is known of its effect on insects. The main aim of this study was to determine the influence of benzyl alcohol on the defense systems of the wax moth Galleria mellonella , i.e., its cuticular lipid composition and critical elements of its immune system. A gas chromatography/mass spectrometry (GC/MS) analysis found benzyl alcohol treatment to elicit significant quantitative and qualitative differences in cuticular free fatty acid (FFA) profiles. Our findings indicate that benzyl alcohol treatment increased the levels of HSP70 and HSP90 and decreased those of HSF1, histamine, and cysteinyl leukotriene. Benzyl alcohol application also increased dismutase level in the hemolymph and lowered those of catalase and 8-OHdG. The treatment also had negative effects on G. mellonella hemocytes and a Sf9 cell line in vitro: 48-h treatment resulted in morphological changes, with the remaining cells being clearly spindle-shaped with numerous granules. The high insecticidal activity of compound and its lack of toxicity towards vertebrates suggest it could be an effective insecticide.

Vitellogenin (Vg), a yolk protein precursor, plays an important role in the oocyte development of insects and is an important target of genetic pest management. Vg is synthesized in the fat body, transported through haemolymph and accumulates in developing oocytes. In this regard, the eggplant shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major pest in South and South East Asia and a serious concern for farmers. Therefore, in the present study, we have cloned and characterized Vg from L. orbonalis (LoVg) for further applications. The cloned Vg consisted of 5,370 base pairs encoding 1,790 amino acid residues long protein. Further, sequence alignment revealed that LoVg has three conserved domains: a Vitellogenin N domain (LPD-N), a domain of unknown function protein families (DUF1943), and a von Willebrand factor type D domain (VWD). Using phylogenetic analysis, it was found that LoVg evolved alongside homologous proteins from different insects. The real-time expression levels of LoVg were significantly greater in female adults followed by the pupal stage. This suggests that Vg production and absorption in L. orbonalis occurs in the later pupal stage. Our studies showed that editing LoVg using CRISPR/Cas9 did not affect the total number of eggs laid but affected egg hatchability. These studies help us to design newer approaches in insect pest management through genetic suppression for sustainable pest management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Histone acetylation is an important epigenetic mechanism that has been shown to play a role in diapause regulation. To explore the physiological and molecular mechanisms of histone deacetylase in the diapause process, LC-MS/MS analysis was used to perform TMT proteomic and metabolomic analysis on non-diapause (ND), pre-diapause (PreD), diapause (D), cold treatment (CT), and post-diapause (RD) stages of the meadow moth. A total of 5367 proteins were identified by proteomics, including 1179 differentially expressed proteins. We found 975 (602 up-regulated and 373 down-regulated), 997 (608 up-regulated and 389 down-regulated), 1119 (726 up-regulated and 393 down-regulated), 1179 (630 up-regulated and 549 down-regulated), 94 (51 up-regulated and 43 down-regulated), 111 (63 up-regulated and 48 down-regulated), 533 (243 up-regulated and 290 down-regulated), 58 (31 up-regulated and 27 down-regulated), and 516 (228 up-regulated and 288 down-regulated) proteins in ND and PreD, ND and D, ND and CT, ND and RD, PreD and D, PreD and CT, PreD and RD, D and CT, D and RD, and CT and RD stages, respectively. A total of 1255 differentially expressed metabolites were annotated by metabolomics. Through KEGG analysis and time series analysis of differentially expressed metabolites, we found that phospholipids were annotated in significantly different modules, demonstrating their important role in the diapause process of the meadow moth. Using phospholipids as an indicator for weighted gene co-expression network analysis, we analyzed the most relevant differentially expressed proteins in the module and found that ribosomal 40s and 60s subunits were the most relevant proteins for diapause. Because there have been studies that have shown that histone deacetylase is associated with the diapause of meadow moths, we believe that histone deacetylase regulates the 40s and 60s subunits of ribosomes, which in turn affects the diapause of meadow moths. This finding expands our understanding of the regulation of meadow moth diapause and provides new insights into its control mechanism.

Paralipsa gularis (Zeller) is a storage pest; however, in recent years it has evolved into a considerable maize pest during the late growth stage in the border region between China and other Southeast Asian countries. Bt transgenic insect-resistant maize is an effective measure in controlling a wide range of lepidopteran pests, but there is a lack of research on the toxic effects of storage pests. We tested the toxicity of Bt-Cry1Ab, Vip3Aa, and their complex proteins against P. gularis via bioassay and investigated the efficiency of Bt-(Cry1Ab+Vip3Aa) maize in controlling P. gularis during the late growth stage of maize in the period 2022-2023. The bioassay results show that the susceptibilities of P. gularis to the two Bt proteins and their complex proteins were significantly different. The LC 50 values of DBNCry1Ab ("DBN9936" event), DBNVip3Aa ("DBN9501" event), DBN Cry1Ab+Vip3Aa ("DBN3601T" event), and Syngenta Cry1Ab+Vip3Aa ("Bt11" event × "MIR162" event) were 0.038 μg/g, 0.114 μg/g, 0.110 μg/g, and 0.147 μg/g, and the GIC 50 values were 0.014 μg/g, 0.073 μg/g, 0.027 μg/g, and 0.026 μg/g, respectively. Determination of the expression content of the insecticidal protein in different tissues of Bt-(Cry1Ab+Vip3Aa) maize shows that the total Bt protein content in different tissues was in the following order: stalk > bract > cob > kernel. However, the bioassay results show that the mortalities of P. gularis feeding on Bt-(Cry1Ab+Vip3Aa) maize in different tissues at different growth stages were all above 93.00%. The field trial indicates that the occurrence density of larvae and plant damage rate for conventional maize were 422.10 individuals/100 plants and 94.40%, respectively, whereas no larvae were found on Bt-(Cry1Ab+Vip3Aa) maize. In summary, this study implies that Bt-(Cry1Ab+Vip3Aa) maize has a high potential for control of P. gularis , providing a new technical measure for the management of the pest.

Trehalose is synthesized in insects through the trehalose 6-phosphate synthase and phosphatase (TPS/TPP) pathway. TPP dephosphorylates trehalose 6-phosphate to release trehalose. Trehalose is involved in metamorphosis, but its relation with body weight, size, and developmental timing is unexplored. The expression and activity of TPS/TPP fluctuate depending on trehalose demand. Thus, TPS/TPP inhibition can highlight the significance of trehalose in insect physiology. TPS/TPP transcript levels are elevated in the pre-pupal and pupal stages in Helicoverpa armigera. The inhibition of recombinantly expressed TPP by N-(phenylthio)phthalimide (NPP), is validated by in vitro assays. In vivo inhibition of trehalose synthesis reduces larval weight and size, hampers metamorphosis, and reduces its overall fitness. Insufficient trehalose leads to a shift in glucose flux, reduced energy, and dysregulated fatty acid oxidation. Metabolomics reaffirms the depletion of trehalose, glucose, glucose 6-phosphate, and suppressed tricarboxylic acid cycle. Reduced trehalose hampers the energy level affecting larval vitality. Through trehalose synthesis inhibition, the importance of trehalose in insect physiology and development is investigated. Also, in two other lepidopterans, TPP inhibition impedes physiology and survival. NPP is also found to be effective as an insecticidal formulation. Overall, trehalose levels affect the larval size, weight, and metabolic homeostasis for larval-pupal transition in lepidoptera., (© 2023 Wiley-VCH GmbH.)

The sophisticated olfactory system of insects is plays a critical role in detecting chemical signals and guiding insect behaviors, such as selecting mates, finding hosts, evading predators, and discovering oviposition sites. Therefore, exploring and clarifying the molecular processes of this system is crucial for developing new insecticides or efficient pest control methods. Plodia interpunctella (Hübner) is a disruptive insect pest damaging the stored grains over the world. However, the olfactory processes of P. interpunctella remain unclear. Herein, we employed a transcriptome analysis to identify olfactory and differentially expressed genes to characterize their expression patterns in different developmental stages and antennal tissue. Subsequently, a total of 172 potential olfactory-related genes included 42 odorant-binding proteins, 12 chemosensory proteins, 51 odorant receptors, 13 gustatory receptors, three sensory neuron membrane proteins, and 51 ionotropic receptors. Furthermore, phylogenetic analysis and BLASTx best-hit analyses showed that these olfactory genes were closely linked with those identified in other lepidopterans. Transcriptome analysis revealed 49 differentially expressed olfactory-related genes, and a semiquantitative reverse transcription polymerase chain reaction showed that 11 olfactory genes were particularly expressed in the legs and wings of female P. interpunctella. Meanwhile, PintOBP29 was notably expressed in female antennae and legs. Genes with high expression levels in the abdomen showed high expression in the legs, but low expression in the antennae. Our findings provide the candidate genetic factors for analysis of the olfactory processes in P. interpunctella., (© 2023 Wiley Periodicals LLC.)

Insect growth regulators (IGRs) are important insecticides that reduce the harm caused by insects to crops by controlling pest population growth. Chitinases are closely associated with insect growth and are among the most important glycoside hydrolases. Thus, Chitinase is an attractive target for the development of novel insecticides. In this study, we designed and synthesized a series of novel and highly potent insecticides targeting Of ChtI and Of Chi-h in insects. Enzymatic activity tests showed that most compounds exhibited a potent inhibitory activity against Of Ch-h. Binding mode analysis revealed that the target compounds bound to the -1 active subsite of Chitinase through the key pharmacophore N -methylcarbamoylguanidino. Compounds 6e , 6g , 6j , and 6o significantly affected the growth and development of Plutella xylostella at 200 mg/L. Our study provides novel insights for the development of potent insecticide-targeted Chitinase combinations based on receptors and ligands.

Herbivorous insects are well known for detoxifying a broad range of the defense compounds produced by the plants that they feed on, but knowledge of the mechanisms of detoxification is still very limited. Here, we describe a system in which two species of lepidopteran caterpillars metabolize an abietane diterpene from the plants of Nepeta stewartiana Diels to an oxygenated derivative that is less active biologically. We found that this transformation could be catalyzed by a cytochrome P450 enzyme in caterpillars, which are associated with molting. Most interestingly, abietane diterpene targets the molting-associated gene CYP306A1 to alter the content of molting hormones in the insect at specific developmental stages and competitively inhibit molting hormone metabolism. These findings identify the mechanism by which caterpillars are able to detoxify abietane diterpenoid through hydroxylation at the C-19 position, which may be opening up exciting research questions into the mechanisms of interaction between plants and insects.

The tachinid fly, Exorista sorbillans, is a notorious ovolarviparous endoparasitoid of the silkworm, Bombyx mori, causing severe damage to silkworm cocoon industry. Silkworm larvae show typically precocious wandering behavior after being parasitized by E. sorbillans; however, the underlying molecular mechanism remains unexplored. Herein, we investigated the changes in the levels of 20-hydroxyecdysone (20E) and juvenile hormone (JH) titer, and they both increased in the hemolymph of parasitized silkworms. Furthermore, we verified the expression patterns of related genes, which showed an upregulation of 20E signaling and biosynthesis genes but a significant downregulation of ecdysone oxidase (EO), a 20E inactivation enzyme, in parasitized silkworms. In addition, related genes of the JH signaling were activated in parasitized silkworms, while related genes of the JH degradation pathway were suppressed, resulting in an increase in JH titer. Notably, the precocious wandering behavior of parasitized silkworms was partly recoverable by silencing the transcriptions of BmCYP302A1 or BmCYP307A1 genes. Our findings suggest that the developmental duration of silkworm post parasitism could be shortened by regulation of 20E and JH titers, which may help silkworm to resist the E. sorbillans infestation. These findings provide a basis for deeper insight into the interplay between silkworms and E. sorbillans and may serve as a reference for the development of a novel approach to control silkworm myiasis., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America.)

The Asian corn borer Ostrinia furnacalis (Lepidoptera: Crambidae) is a serious corn pest with widespread distribution in East Asia. Its olfactory mechanism is a focus of scientific study, aiming to find good ways to control this pest. Molecular events are considered to be important in olfactory mechanism. Current understandings of olfactory molecular events in O. furnacalis, mainly involving sex pheromones and olfactory proteins, were summarized to provide a reference for further studies. O. furnacalis sex pheromone contains two components E-12-tetradecenyl acetate and Z-12-tetradecenyl acetate, which may be recognized and bound by the pheromone binding proteins OfurPBP3 and OfurPBP2, and then transported to the odorant receptors (ORs) OfurOR4 and OfurOR6 to activate them. The ORs OfurOR8, OfurOR7 and OfurOR5b mainly respond to the sex pheromone components of other Ostrinia species, E-11-tetradecenyl acetate, Z-11-tetradecenyl acetate and Z-9-tetradecenyl acetate. The OR OfurOR27 responds strongly to plant odorants nonanal, octanal and 1-octanol. Much work remains to be done to fully understand odorants with olfactory activity to O. furnacalis and the functions of its olfactory proteins. These studies will help to reveal olfactory mechanism in O. furnacalis, with the aim of regulating its behaviors to control this pest., (© 2022 Wiley Periodicals LLC.)

Chitin degradation is a vital process for the growth of insects. Chitin hydrolase Of ChtI and β- N -acetylhexosaminidase Of Hex1 are two key enzymes involved in hydrolyzing the chitin of insects' cuticles. Thus, they are considered promising targets for preventing and controlling agricultural pests. In this study, we designed and synthesized a series of compounds bearing N -methylcarbamoylguanidinyl and N -methoxycarbonylguanidinyl as dual-target inhibitors of Of ChtI and Of Hex1. The most potent dual-target inhibitor, compound 10d , exhibited half-maximal inhibitory concentration (IC 50 ) values of 27.1 and 249.1 nM against Of ChtI and Of Hex1, respectively. Furthermore, the insecticidal activity studies showed that compounds 10a - c , 10k , and 10l bear significant effects on the growth and development of Plutella xylostella . This work provides a promising method for the development of novel chitin hydrolase inhibitors as potential pest control and management agents.

MicroRNAs (miRNAs) are small noncoding RNAs (sRNAs) that regulate gene expression by inhibiting translation or degrading mRNA. Although the functions of miRNAs in many biological processes have been reported, there is currently no research on the possible roles of miRNAs in Micromelalopha troglodyta (Graeser) involved in the response of plant allelochemicals. In this article, six sRNA libraries (three treated with tanic acid and three control) from M. troglodyta were constructed using Illumina sequencing. From the results, 312 known and 43 novel miRNAs were differentially expressed. Notably, some of the most abundant miRNAs, such as miR-432, miR-541-3p, and miR-4448, involved in important physiological processes were also identified. To better understand the function of the targeted genes, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The results indicated that differentially expressed miRNA targets were involved in metabolism, development, hormone biosynthesis, and immunity. Finally, we visualized a miRNA-mRNA regulatory module that supports the role of miRNAs in host-allelochemical interactions. To our knowledge, this is the first report on miRNAs responding to tannic acid in M. troglodyta . This study provides indispensable information for understanding the potential roles of miRNAs in M. troglodyta and the applications of these miRNAs in M. troglodyta management.

Ostrinia furnacalis (Guenée) is a major insect pest in maize production that is highly adaptable to the environment. Small heat shock proteins (sHsps) are a class of chaperone proteins that play an important role in insect responses to various environmental stresses. The present study aimed to clarify the responses of six O. furnacalis sHsps to environmental stressors. In particular, we cloned six sHsp genes, namely, OfHsp24.2, OfHsp21.3, OfHsp20.7, OfHsp21.8, OfHsp29.7, and OfHsp19.9, from O. furnacalis. The putative proteins encoded by these genes contained a typical α-crystallin domain. Real-time quantitative polymerase chain reaction was used to analyze the differences in the expression of these genes at different developmental stages, in different tissues of male and female adults, and in O. furnacalis under UV-A and extreme temperature stresses. The six OfsHsp genes were expressed at significantly different levels based on the developmental stage and tissue type in male and female adults. Furthermore, all OfsHsp genes were significantly upregulated in both male and female adults under extreme temperature and UV-A stresses. Thus, O. furnacalis OfsHsp genes play important and unique regulatory roles in the developmental stages of the insect and in response to various environmental stressors., (© Crown copyright 2022.)

The divergence of sister orders Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) from a silk-spinning ancestor occurred around 290 million years ago. Trichoptera larvae are mainly aquatic, and Lepidoptera larvae are almost entirely terrestrial-distinct habitats that required molecular adaptation of their silk for deployment in water and air, respectively. The major protein components of their silks are heavy chain and light chain fibroins. In an effort to identify molecular changes in L-fibroins that may have contributed to the divergent use of silk in water and air, we used the ColabFold implementation of AlphaFold2 to predict three-dimensional structures of L-fibroins from both orders. A comparison of the structures revealed that despite the ancient divergence, profoundly different habitats, and low sequence conservation, a novel 10-helix core structure was strongly conserved in L-fibroins from both orders. Previously known intra- and intermolecular disulfide linkages were accurately predicted. Structural variations outside of the core may represent molecular changes that contributed to the evolution of insect silks adapted to water or air. The distributions of electrostatic potential, for example, were not conserved and present distinct order-specific surfaces for potential interactions with or modulation by external factors. Additionally, the interactions of L-fibroins with the H-fibroin C-termini are different for these orders; lepidopteran L-fibroins have N-terminal insertions that are not present in trichopteran L-fibroins, which form an unstructured ribbon in isolation but become part of an intermolecular β-sheet when folded with their corresponding H-fibroin C-termini. The results are an example of protein structure prediction from deep sequence data of understudied proteins made possible by AlphaFold2.

Micromelalopha troglodyta (Graeser) has been one of the most serious pests on poplars in China. We used Illumina HiSeq 2000 sequencing to construct an antennal transcriptome and identify olfactory-related genes. In total, 142 transcripts were identified, including 74 odorant receptors (ORs), 32 odorant-binding proteins (OBPs), 13 chemosensory proteins (CSPs), 20 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs). The genetic relationships were obtained by the phylogenetic tree, and the tissue-specific expression of important olfactory-related genes was determined by quantitative real-time PCR (qRT-PCR). The results showed that most of these genes are abundantly expressed in the antennae and head. In most insects, olfaction plays a key role in foraging, host localization, and searching for mates. Our research lays the foundation for future research on the molecular mechanism of the olfactory system in M. troglodyta. In addition, this study provides a theoretical basis for exploring the relationship between M. troglodyta and their host plants, and for the biological control of M. troglodyta using olfactory receptor as targets., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.)

Endoclita signifer larvae show olfactory recognition towards volatiles of eucalyptus trunks and humus soils. Further, EsigGOBP1 was identified through larval head transcriptome and speculated as the main odorant-binding proteins in E. signifer larvae. In this study, the highest expression of EsigGOBP1 was only expressed in the heads of 3rd instar larvae of E. signifer , compared with the thorax and abdomen; this was consistent with the phenomenon of habitat transfer of 3rd instar larvae, indicating that EsigGOBP1 was a key OBP gene in E. signifer larvae. Results of fluorescence competition binding assays (FCBA) showed that EsigGOBP1 had high binding affinities to eight GC-EAD active ligands. Furthermore, screening of key active odorants for EsigGOBP1 and molecular docking analysis, indicated that EsigGOBP1 showed high binding activity to alpha-phellandrene in 3rd instar larvae of E. signifer . Conformational analysis of the EsigGOBP1 -alpha-phellandrene complex, showed that MET49 and GLU38 were the key sites involved in binding. These results demonstrated that EsigGOBP1 is a key odorant-binding protein in E. signifer larvae, which recognizes and transports eight key volatiles from eucalyptus trunk, especially the main eucalyptus trunks volatile, alpha-phellandrene. Taken together, our results showed that EsigGOBP1 is involved in host selection of E. signifer larvae, which would aid in developing EsigGOBP1 as molecular targets for controlling pests at the larval stage.

Neuropeptides and neuropeptide receptors are crucial regulators to insect physiological processes. The 21.0 Gb bases were obtained from Illumina sequencing of two libraries representing the female and male heads of Phauda flammans (Walker) (Lepidoptera: Phaudidae), which is a diurnal defoliator of ficus plants and usually outbreaks in the south and south-east Asia, to identify differentially expressed genes, neuropeptides and neuropeptide receptor whose tissue expressions were also evaluated. In total, 99,386 unigenes were obtained, in which 156 up-regulated and 61 down-regulated genes were detected. Fifteen neuropeptides (i.e., F1b, Ast, NP1, IMF, Y, BbA1, CAP2b, NPLP1, SIF, CCH2, NP28, NP3, PDP3, ARF2 and SNPF) and 66 neuropeptide receptor genes (e.g., A2-1, FRL2, A32-1, A32-2, FRL3, etc.) were identified and well-clustered with other lepidopteron. This is the first sequencing, identification neuropeptides and neuropeptide receptor genes from P. flammans which provides valuable information regarding the molecular basis of P. flammans., (© 2022. The Author(s).)

Magnetic resonance imaging (MRI) is the key whole-body imaging technology for observing processes within a living object providing excellent resolution and contrast between soft tissues. In the present work, we exploited the non-destructive properties of MRI to track longitudinally the dynamic changes that take place in developing pupae of the Emperor Moth (Saturnia pavonia) during the last days before eclosion. While in diapause pupae, body fluid was almost homogeneously distributed over the internal compartments, as soon as wings, legs, flight muscles and the head region were fully developed, a significant redistribution of water levels occurred between thoracic and abdominal regions. During the last two days before eclosion, the developing moths transferred substantial amounts of liquid into the gut and the labial gland, and in case of females, into developing eggs. Concomitantly, the volume of the air sacs increased drastically and their expansion/compression became clearly visible in time-resolved MR images. Furthermore, besides ventilation of the tracheal system, air sacs are likely to serve as volume reservoir for liquid transfer during development of the moths inside their pupal case. In parallel, we were able to monitor noninvasively lipid consumption, cardiac activity and haemolymph circulation during final metamorphosis., (© 2022. The Author(s).)

<b>Background and Objective:</b> The tomato leaf miner, <i>Tuta absoluta</i> (Meyrick) is being a serious pest to tomato cultivations in Egypt since 2009. The present study was carried out to calculate the developmental parameters of insects based on temperature degree. <b>Materials and Methods:</b> The influence of 3 tested temperatures (20, 24, 28°C) were examined to evaluate its effect on the developmental stages of <i>T. absoluta</i>. Developmental thresholds and needed heat units for insect stages were mathematically calculated according to developmental rates. <b>Results:</b> Developmental threshold for egg stage and mean thermal units were calculated to be 7°C and 86.2 DD's. The developmental threshold for the larval stage was 10°C, while mean thermal units were calculated to be 310.8 DD's. Percentages mortality of larval stage were 52, 74, 74 and 100% at 20, 24, 28 and 32°C, respectively. For the pupal stage developmental threshold and mean thermal units required for completing the pupal stage was 11.2°C and 132.2 DD's. For an adult, zero of the developmental threshold female and of male were 11.2 and 9.8°C, respectively. The mean required heat units for female and male was 142.3 and 136.7 DD's Life table parameters such as net Reproduction Rate (R<sub>◦</sub>), Mean Generation Time (Gt), Intrinsic Rate of Increase (r<sub>m</sub>), Finite Rate of Increase (λ) and Population Double Time (Dt) were calculated at three tested temperatures. <b>Conclusion:</b> Estimating thermal heat units of <i>T. absoluta</i> help in predicting the field generations of the insect and improve planning the integrated pest management.

During metamorphosis, the wings of a butterfly sprout hundreds of thousands of scales with intricate microstructures and nano-structures that determine the wings' optical appearance, wetting characteristics, thermodynamic properties, and aerodynamic behavior. Although the functional characteristics of scales are well known and prove desirable in various applications, the dynamic processes and temporal coordination required to sculpt the scales' many structural features remain poorly understood. Current knowledge of scale growth is primarily gained from ex vivo studies of fixed scale cells at discrete time points; to fully understand scale formation, it is critical to characterize the time-dependent morphological changes throughout their development. Here, we report the continuous, in vivo, label-free imaging of growing scale cells of Vanessa cardui using speckle-correlation reflection phase microscopy. By capturing time-resolved volumetric tissue data together with nanoscale surface height information, we establish a morphological timeline of wing scale formation and gain quantitative insights into the underlying processes involved in scale cell patterning and growth. We identify early differences in the patterning of cover and ground scales on the young wing and quantify geometrical parameters of growing scale features, which suggest that surface growth is critical to structure formation. Our quantitative, time-resolved in vivo imaging of butterfly scale development provides the foundation for decoding the processes and biomechanical principles involved in the formation of functional structures in biological materials., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

During the past decade, antennal transcriptome sequencing has been applied to at least 50 species from 16 families of the Lepidoptera order of insects, emphasizing the identification and characterization of chemosensory-related genes. However, little is known about the chemosensory genes in the Zygaenidae family of Lepidoptera. Herein, we report the transmembrane protein gene repertoires involved in chemoreception from Achelura yunnanensis (Lepidoptera: Zygaenidae) through transcriptome sequencing, bioinformatics, phylogenetics and polymerase chain reaction (PCR) approaches. Transcriptome analysis led to the generation of 555.47 million clean reads and accumulation of 83.30 gigabases of data. From this transcriptome, 132 transcripts encoding 69 odorant receptors (ORs), 33 gustatory receptors (GRs), 26 ionotropic receptors (IRs), and four sensory neuron membrane proteins (SNMPs) were identified, 69 of which were full-length sequences. Notably, the number of SNMPs in A. yunnanensis was the largest set in Lepidoptera to date. Phylogenetic analysis combined with sequence homology highlighted several conserved groups of chemoreceptors, including pheromone receptors (a so-called pheromone receptor (PR) clade: AyunOR50 and novel PR members: AyunOR39 and OR40), a phenylacetaldehyde-sensing OR (AyunOR28), carbon dioxide receptors (AyunGR1-3), and antennal IRs (13 A-IRs). In addition, a Zygaenidae-specific OR expansion was observed, including 15 A. yunnanensis members. Expression profiles revealed 99 detectable chemosensory genes in the antennae and 20 in the reproductive tissues, some of which displayed a sex-biased expression. This study identifies potential olfactory molecular candidates for sensing sex pheromones, phenylacetaldehyde or other odorants, and provides preliminary evidence for the putative reproductive function of chemosensory membrane protein genes in A. yunnanensis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Genome-wide analysis of cytochrome P450 monooxygenase (CYP) genes from the advanced genome project of the Leucinodes orbonalis and the expression analysis provided significant information about the metabolism-mediated insecticide resistance. A total of 72 putative CYP genes were identified from the genome and transcriptome of L. orbonalis. The genes were classified under 30 families and 46 subfamilies based on the standard nomenclature. In the present study, a novel CYP gene, CYP324F1, was identified and it has not been reported from any other living system so far. Biochemical assays showed enhanced titers (5.81-18.5-fold) of O-demethylase of CYP in five field-collected populations. We selected 34 homologous CYP gene sequences, seemed to be involved in insecticide resistance for primer design and quantitative real-time PCR studies. Among the many overexpressed genes (>10 fold), the expression levels of CYP324F1 and CYP306A1 were prominent across all the field populations as compared with the susceptible iso-female line. Oral delivery of ds-CYP324F1 and ds-CYP306A1 directed against CYP324F1 and CYP306A1 to the larvae of one of the insecticide resistance populations caused reduced expression of these two transcripts in a dose-dependent manner (53.4%-85.0%). It appears that the increased titer of O-demethylase is the result of increased transcription level of CYP genes in resistant populations. The data provide insight for identifying the novel resistance management strategies against L. orbonalis., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Members of the insect ATP binding cassette transporter subfamily C2 (ABCC2) in several moth species are known as receptors for the Cry1Ac insecticidal protein from Bacillus thuringiensis (Bt). Mutations that abolish the functional domains of ABCC2 are known to cause resistance to Cry1Ac, although the reported levels of resistance vary widely depending on insect species. In this study, the function of the ABCC2 gene as a putative Cry1Ac receptor in Helicoverpa zea , a major pest of over 300 crops, was evaluated using CRISPR/Cas9 to progressively eliminate different functional ABCC2 domains. Results from bioassays with edited insect lines support that mutations in ABCC2 were associated with Cry1Ac resistance ratios (RR) ranging from 7.3- to 39.8-fold. No significant differences in susceptibility to Cry1Ac were detected between H. zea with partial or complete ABCC2 knockout, although the highest levels of tolerance were observed when knocking out half of ABCC2. Based on >500-1000-fold RRs reported in similar studies for closely related moth species, the low RRs observed in H. zea knockouts support that ABCC2 is not a major Cry1Ac receptor in this insect.

Aquaporin (AQP) transport solutes across cell membranes in both unicellular and multicellular organisms. In this study, the aquaporin CsPrip was identified in Chilo suppressalis, an important pest of rice. CsPrip was comprised of two variants, CsPrip&#95;v1 and CsPrip&#95;v2; the former variant was <103 bp was shorter than the latter, although both exhibited the same open reading frame (ORF). Transmembrane topology and protein structure analyses showed that CsPrip retained the conserved features of water-selective insect AQPs, including six transmembrane domains, two conserved hydrophobic asparagine-proline-alanine motifs and the aromatic/arginine constriction region. Expression in Xenopus oocytes revealed that CsPrip preferentially transported water and urea instead of trehalose and glycerol. The CsPrip transcript was expressed in multiple organs and tissues of C. suppressalis larvae and was most abundant in the hindgut and Malpighian tubules. CsPrip transcription was highest in male adults and was relatively stable throughout development. CsPrip expression in larvae was significantly altered by thermal stress, and relative humidity levels impacted CsPrip transcription in 3rd and 5th instar larvae. This study confirms that the aquaporin CsPrip performs multiple critical functions in maintaining water equilibrium in C. suppressalis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor ( pkf ), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Maize with enhanced β-carotene production was engineered to counteract pervasive vitamin A deficiency in developing countries. Second-generation biofortified crops are being developed with additional traits that confer pest resistance. These include crops that can produce Bacillus thuringiensis Berliner (Bt) insecticidal proteins. Currently, it is unknown whether β-carotene can confer fitness benefits through to insect pests, specifically through altering Ostrinia nubilalis foraging behaviour or development in the presence of Bt insecticidal toxin. Therefore the effects of dietary β-carotene plus Bt insecticidal protein on feeding behaviour, mortality, and physiology in early and late instars of O. nubilalis larvae were investigated. The results of two-choice experiments showed that irrespective of β-carotene presence, at day five 68%-90% of neonates and 69%-77% of fifth-instar larvae avoided diets with Cry1A protein. Over 65% of neonate larvae preferred to feed on diets with β-carotene alone compared to 39% of fifth-instar larvae. Higher mortality (65%-97%) in neonates fed diets supplemented with β-carotene alone and in combination with Bt protein was found, whereas <36% mortality was observed when fed diets without supplemented β-carotene or Bt protein. Diets with both β-carotene and Bt protein extended 25 days the larval developmental duration from neonate to fifth instar (compared to Bt diets) but did not impair larval or pupal weight. Juvenile hormone and 20-hydroxyecdysone regulate insect development and their levels were at least 3-fold higher in larvae fed diets with β-carotene for 3 days. Overall, these results suggest that the effects of β-carotene and Bt protein on O. nubilalis is dependent on larval developmental stage. This study is one of the first that provides insight on how the interaction of novel traits may modulate crop susceptibility to insect pests. This understanding will in turn inform the development of crop protection strategies with greater efficacy., Competing Interests: The authors have declared that no competing interests exist.

Insect pheromone binding proteins (PBPs) are believed to have a high degree of pheromone selectivity, acting as the first filter to discriminate specific pheromones from other volatile compounds. Herein, we provide evidence using homology-based model for the pheromone discrimination of Plutella xylostella pheromone binding protein 3 (PxPBP3). Combining molecular dynamics simulations and in vitro binding assays, two dominant sites are determined to be essential for the PxPBP3 to discriminate (Z)-11-hexadecenyl acetate (Hexadecenyl) from (Z)-11-hexadecenal (Hexadecenal). As the first key site for pheromone discrimination, Arg111 is indispensable to the PxPBP3-Hexadecenyl interaction. However, its importance in the binding of Hexadecenal to PxPBP3 is greatly reduced. A second site where pheromone discrimination occurs is a small loop (residues 34-38) in PxPBP3. It is shown that the hydrophobic strength provided by three hydrophobic residues (Phe34, Tyr37, and Trp38) in the small loop is significantly biased in the two complexes formed by PxPBP3 and the two pheromones. The discrimination capacity of PxPBP3 indicates that the P. xylostella pheromones may not share the same peri-receptor pathway, although they both show high affinity to PxPBP3., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

&lt;b&gt;Background and Objective:&lt;/b&gt; One of the Nymphalidae butterfly species found in West Sumatra in &lt;i&gt;Hypolimnas bolina&lt;/i&gt;. Currently, research on the artificial diet for the Nymphalidae butterfly is relatively rare in Padang, West Sumatra. The objectives of this study were to analyze the preferences of &lt;i&gt;H. bolina&lt;/i&gt; larvae, duration of the immature stage and mortality of &lt;i&gt;H. bolina&lt;/i&gt; in artificial diet treatment. &lt;b&gt;Materials and Methods:&lt;/b&gt; Some biological aspects of &lt;i&gt;H. bolina&lt;/i&gt; in corresponding to artificial diet and its effect were investigated in the laboratory. &lt;b&gt;Results:&lt;/b&gt; The result showed that there was no significant difference in the frequency of visits of the larvae in the two diet treatments namely natural (&lt;i&gt;Laportea interrupta&lt;/i&gt; leaves) and artificial diets (Sig = 0.289, p>0.05) but the duration of the visit of &lt;i&gt;H. bolina&lt;/i&gt; larvae was significantly different (Sig = 0.000, p<0.05). The visit duration of the immature stage of &lt;i&gt;H. bolina &lt;/i&gt;was significantly different, except the prepupa and pupal stage. There was no mortality of instar larvae and prepupa stage observed in both of the two-diet treatments. However, the mortality of pupae in an artificial diet was 4%. Of the total of 24 individual larvae fed with artificial diet, all of them successfully emerged, consisted of 12 males and 12 females but there was one male with abnormal wings. The average living period in the artificial diet of imago was 14.82 days for males and 16.77 days for a female. The average larval weight was no significant difference (Sig = 0.981, p>0.05) but the average pupal weight of the natural diet was slightly higher than the artificial diet. &lt;b&gt;Conclusion:&lt;/b&gt; The formulation of an artificial diet is suitable for &lt;i&gt;H. bolina&lt;/i&gt; larvae based on the results of immature mortality and adult emergences. Therefore, the formulation of an artificial diet is suitable for &lt;i&gt;H. bolina&lt;/i&gt; with its composition almost similar to &lt;i&gt;L. interrupta&lt;/i&gt; leaves (natural diet).

The diverse colors and patterns found in Lepidoptera are important for success of these species. Similar to the wings of adult butterflies, lepidopteran larvae exhibit diverse color variations to adapt to their habitats. Compared with butterfly wings, however, less attention has been paid to larval body colorations and patterns. In the present study, we focus on the yellow-y gene, which participates in the melanin synthesis pathway. We conducted CRISPR/Cas9-mediated targeted mutagenesis of yellow-y in the tobacco cutworm Spodoptera litura. We analyzed the role of S. litura yellow-y in pigmentation by morphological observation and discovered that yellow-y is necessary for normal black pigmentation in S. litura. We also showed species- and tissue-specific requirements of yellow-y in pigmentation in comparison with those of Bombyx mori yellow-y mutants. Furthermore, we found that almost none of the yellow-y mutant embryos hatched unaided. We provide evidence that S. litura yellow-y has a novel important function in egg hatching, in addition to pigmentation. The present study will enable a greater understanding of the functions and diversification of the yellow-y gene in insects., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Molting is an essential biological process occurring characteristic times throughout the life cycle of holometabolous insects. However, it is not clear how insects determine the direction of molting to remain status quo or to initiate metamorphosis. To explore the functional factors that determine the direction of molts, liquid chromatography-mass spectrometry was used to identify the molecules involved in larval and metamorphic molting, and the differentially expressed proteins (DEPs) were compared in the two processes., Results: There were 321 and 1140 DEPs identified in larval and metamorphic molting process, respectively. Bioinformatics analyses show that the amino sugar pathway was up-regulated in both processes. The up-regulated protease contributed to the metamorphosis. In addition, several proteins with different expression patterns in larval-larval and larval-pupal transitions, including Endochitinase, GRIM-19 (Genes associated with retinoid-IFN-induced mortality-19), IDE (Insulin-degrading enzyme), Sorcin (Soluble resistance related calcium binding protein), OBP (Odorant-binding protein-2 precursor), TRAP1(Tumor necrosis factor receptor associated protein-1), etc., were further identified by parallel reaction monitoring, which may play diverse functions in larval-larval and larval-pupal transitions., Conclusions: These results provide a proteomic insight into molecules involved in larval and metamorphic molts, and will likely improve the current understanding of determination of direction of molts.

Diatraea saccharalis constitutes a threat to the sugarcane productivity, and obtaining borer tolerant cultivars is an alternative method of control. Although there are studies about the relationship between the interaction of D. saccharalis with sugarcane, little is known about the molecular and genomic basis of defense mechanisms that confer tolerance to sugarcane cultivars. Here, we analyzed the transcriptional profile of two sugarcane cultivars in response to borer attack, RB867515 and SP80-3280, which are considered tolerant and sensitive to the borer attack, respectively. A sugarcane genome and transcriptome were used for read mapping. Differentially expressed transcripts and genes were identified and termed to as DETs and DEGs, according to the sugarcane database adopted. A total of 745 DETs and 416 DEGs were identified (log 2 |ratio| > 0.81; FDR corrected P value ≤ 0.01) after borer infestation. Following annotation of up- and down-regulated DETs and DEGs by similarity searches, the sugarcane cultivars demonstrated an up-regulation of jasmonic acid (JA), ethylene (ET), and defense protein genes, as well as a down-regulation of pathways involved in photosynthesis and energy metabolism. The expression analysis also highlighted that RB867515 cultivar is possibly more transcriptionally activated after 12 h from infestation than SP80-3280, which could imply in quicker responses by probably triggering more defense-related genes and mediating metabolic pathways to cope with borer attack.

Cell lines derived from Trichoplusia ni (Tn) are widely used as hosts in the baculovirus-insect cell system (BICS). One advantage of Tn cell lines is they can produce recombinant proteins at higher levels than cell lines derived from other insects. However, Tn cell lines are persistently infected with an alphanodavirus, Tn5 cell-line virus (TnCLV), which reduces their utility as a host for the BICS. Several groups have isolated TnCLV-negative Tn cell lines, but none were thoroughly characterized and shown to be free of other adventitious viruses. Thus, we isolated and extensively characterized a new TnCLV-negative line, Tn-nodavirus-negative (Tn-NVN). Tn-NVN cells have no detectable TnCLV, no other previously identified viral contaminants of lepidopteran insect cell lines, and no sequences associated with any replicating virus or other viral adventitious agents. Tn-NVN cells tested negative for >60 species of Mycoplasma, Acholeplasma, Spiroplasma, and Ureaplasma. Finally, Tn-NVN cells grow well as a single-cell suspension culture in serum-free medium, produce recombinant proteins at levels similar to High Five™ cells, and do not produce recombinant glycoproteins with immunogenic core α1,3-fucosylation. Thus, Tn-NVN is a new, well-characterized TnCLV-negative cell line with several other features enhancing its utility as a host for the BICS., (© 2020 Wiley Periodicals LLC.)

The sugarcane borer (Diatraea saccharalis, Fabricius, 1794) is a devastating pest that causes millions of dollars of losses each year to sugarcane producers by reducing sugar and ethanol yields. The control of this pest is difficult due to its endophytic behavior and rapid development. Pest management through biotechnological approaches has emerged in recent years as an alternative to currently applied methods. Genetic information about the target pests is often required to perform biotechnology-based management. The genomic and transcriptomic data for D. saccharalis are very limited. Herein, we report a tissue-specific transcriptome of D. saccharalis larvae and a differential expression analysis highlighting the physiological characteristics of this pest in response to two different diets: sugarcane and an artificial diet. Sequencing was performed on the Illumina HiSeq 2000 platform, and a de novo assembly was generated. A total of 27,626 protein-coding unigenes were identified, among which 1,934 sequences were differentially expressed between treatments. Processes such as defence, digestion, detoxification, signaling, and transport were highly represented among the differentially expressed genes (DEGs). Furthermore, seven aminopeptidase genes were identified as candidates to encode receptors of Cry proteins, which are toxins of Bacillus thuringiensis used to control lepidopteran pests. Since plant-insect interactions have produced a considerable number of adaptive responses in hosts and herbivorous insects, the success of phytophagous insects relies on their ability to overcome challenges such as the response to plant defences and the intake of nutrients. In this study, we identified metabolic pathways and specific genes involved in these processes. Thus, our data strongly contribute to the knowledge advancement of insect transcripts, which can be a source of target genes for pest management., Competing Interests: The authors have declared that no competing interests exist.

Evolution of insect resistance to Bt toxins challenges the use of Cry toxins to control agricultural pests. In lepidopterans, Cry toxin affinity towards multiple midgut epithelial receptors has become a matter of dispute. Cry1Ah toxin-binding proteins were identified in the larval midgut of susceptible (ACB-BtS) and resistant (ACB-AhR) strains of the Asian corn borer (ACB). A pull-down assay was performed using biotinylated Cry1Ah toxin, and the binding proteins were identified by employing liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study aimed to find the binding consistency of the midgut epithelial protein to the Cry1Ah toxin. The binding proteins from different fractions of SDS-PAGE showed a different pattern. We observed an isoform of prophenoloxidase PPO1b (UniProt Acc No. A0A1Q1MKI0), which was found only in the ACB-AhR fractions. Prophenoloxidase (proPO) is an extraordinary defense molecule activated in insect species during pathogen invasion and the wound healing process. Importantly, this prophenoloxidase might have direct/indirect interaction with the Cry1Ah toxin. Our data also suggest that factors like techniques, enrichment of binding proteins in the sample and the reversible and irreversible nature of the brush border membrane vesicles (BBMVs) to Cry toxins could cause the inconsistency in the protein-protein interactions. Moreover, inside the larva midgut, the influence of the Cry toxins under physiological conditions might be different from the laboratory procedures.

To understand the effects of micronutrients have particular biological functions that are involved mainly in the antioxidant system, which has essential implications for the development of diseases, this study investigated how vitamin E, selenium, and their combination affect lipid, protein, carbohydrate, and malondialdehyde (MDA) content; antioxidant enzyme (catalase [CAT], superoxide dismutase [SOD], glutathione-S-transferase [GST]) activity; and the total hemocyte count (THC) in larvae of Galleria mellonella L. fed different diets. Diet 1 (100 µg of selenium) significantly decreased carbohydrate and lipid content. Diets 2 (100 µg of vitamin E), 3 (100 µg of selenium and vitamin E each), and 5 (Tween 80) did not significantly affect protein and carbohydrate content. Diet 2 significantly increased the lipid content compared to diet 4 (control). Diet 1 increased CAT, SOD, and GST activity and MDA content (highest at 27.64 nmol/mg protein). Diet 2 significantly decreased SOD activity and MDA content compared to other diets. Diet 1 significantly decreased the THC compared to other diets. These results suggested that selenium changes oxidative stress parameters, energy reserves, and THC in G. mellonella. These changes could be a physiological adaptation against selenium-induced oxidative stress. Vitamin E could play a protective role in selenium toxicity.

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125 I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 ( HvmALP1 ) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1 .

Cellular immunity is evolutionarily conserved in invertebrates and vertebrates. In insects, cellular immune response is provided by the hemocytes, and its molecular mechanisms are currently not fully understood. Here, we identified a CD109 antigen-like gene ( HaCD109 ) from Helicoverpa armigera which is highly expressed in the hemocytes of larvae. Stimulation by Escherichia coli and chromatography beads significantly upregulated HaCD109 expression. In vivo HaCD109 silencing significantly increased bacterial load in larval hemolymphs and reduced the hemocyte spread. 20-Hydroxyecdysone (20E) can induce HaCD109 expression through its receptors, EcR and USP. In vivo HaCD109 silencing nearly abolished 20E-induced bacterial clearance and hemocyte spread. These results suggested that HaCD109 plays an important role in cellular immunity, and the 20E-induced cellular immune response in H. armigera requires HaCD109 involvement. Our study contributes to the understanding of regulatory mechanisms for innate immune response and provides new insights into the interaction between innate immunity and steroid hormone signaling.

Herbivory insects can discriminate the quality of a host plant for food or oviposition, by detecting the volatile organic compounds (VOC's) released by the plant, however, damaged plants may release a different VOC's profile modifying the insects' response. We tested if the VOC's profile from damaged plants affected the response of Copitarsia decolora as these moths oviposit preferably around undamaged host plants. We assessed the response in wind tunnel conditions of C. decolora mated females to volatiles collected by dynamic headspace from 30-40 d old cabbage undamaged plants and mechanical and larval damaged plants. Headspace volatile compounds from undamaged cabbage plants were more attractive to mated females than those from larval and mechanical damaged cabbage plants. Moths stimulated with headspace volatiles from undamaged plants performed more complete flight and ovipositor displays than those moths stimulated with headspace volatiles from damaged cabbage plants. A mixture of synthetic compounds identified from undamaged cabbages elicited similar antennal and wind tunnel responses in mated females as headspace volatiles from undamaged cabbage plants. C. decolora females may discriminate between damaged and undamaged host plants by detecting their VOC's profiles as a strategy to avoid unsuitable plants for their offspring increasing their fitness.

Anticarsia gemmatalis (Hübner, 1818) and Chrysodeixis includens (Walker, 1858) are species of Lepidoptera that cause great damages in the soybean plantations of Brazil. Despite the importance they have in this regard, there are no studies on the chromosomal organization of these species and recently, A. gemmatalis, which belonged to the Noctuidae family, was allocated to the Erebidae family. Therefore, the objective of this paper was to analyze, through conventional and molecular cytogenetic markers, both species of Lepidoptera. A 2n = 62 was observed, with ZZ/ZW sex chromosome system and holokinetic chromosomes for both species. There was homogeneity in the number of 18S rDNA sites for both species. However, variations in heterochromatin distribution were observed between both species. The cytogenetic analyses enabled separation of the species, corroborating the transference of A. gemmatalis, from the family Noctuidae to the family Erebidae, suggesting new cytotaxonomic characteristics., Competing Interests: The authors have declared that no competing interests exist.

Conogethes pinicolalis (Lepidoptera: Crambidae), a major pine pest, possesses a sensitive olfactory system to locate its host. Pheromone binding proteins (PBPs) and general odorant binding proteins (GOBPs) are two types of proteins involved in the process. In this work, we used phylogenetic analysis, gene expression, fluorescence competitive binding assay, and molecular docking to characterize PBPs and GOBPs in C. pinicolalis. The phylogenetic unrooted tree revealed the C. pinicolalis GOBPs and PBPs amino acid sequences showed very close relation with Conogethes punctiferalis (yellow peach moth). Meanwhile, both the PBPs and GOBPs were specifically expressed in the antennae. Binding affinities of PBPs and GOBPs to 19 volatile compounds were tested. PBP2 shows the strongest binding to E10-16: Ald with a K i value of 0.28/1.66 μM; GOBP1 to Z10-16:Ald with a K i value of 3.11/4.24 μM. Furthermore, molecular docking reveals potential active sites in PBP2 and GOBP1 to interact with most of the tested volatiles. Finally, we demonstrate that PBP2 and GOBP1 are the dominant genes in their respective families using several different assays. In conclusion, PBP2 and GOBP1 genes may play similar roles in detecting and transporting sex pheromones and host plant volatiles in C. pinicolalis., (Copyright © 2019. Published by Elsevier B.V.)

Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3 (also called CENP-A), which physically interacts with components of the inner kinetochore constitutive centromere-associated network (CCAN). Unexpectedly, regarding its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3 while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components, including a divergent CENP-T homolog, that are required for accurate mitotic progression. Our study focuses on CENP-T, which we found to be sufficient to recruit the Mis12 and Ndc80 outer kinetochore complexes. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T and additional CCAN homologs in other independently derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, contributing to the emerging picture of an unexpected plasticity to build a kinetochore., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.

Sex pheromone receptors (PRs) are key players in chemical communication between mating partners in insects. In the highly diversified insect order Lepidoptera, male PRs tuned to female-emitted type I pheromones (which make up the vast majority of pheromones identified) form a dedicated subfamily of odorant receptors (ORs). Here, using a combination of heterologous expression and in vivo genome editing methods, we bring functional evidence that at least one moth PR does not belong to this subfamily but to a distantly related OR lineage. This PR, identified in the cotton leafworm Spodoptera littoralis , is highly expressed in male antennae and is specifically tuned to the major sex pheromone component emitted by females. Together with a comprehensive phylogenetic analysis of moth ORs, our functional data suggest two independent apparitions of PRs tuned to type I pheromones in Lepidoptera, opening up a new path for studying the evolution of moth pheromone communication., Competing Interests: LB, SC, FK, GC, SR, CM, MF, TR, AM, TC, Ad, WW, GW No competing interests declared, Ad, EJ, NM The intellectual property rights of SlitOR5 have been licensed by Inra, Sorbonne Université and CNRS for the purpose of developing novel insect control agents., (© 2019, Bastin-Héline et al.)

In insects, male and female pheromone signals are detected by olfactory sensory neurons (OSNs) expressing the "sensory neuron membrane protein type 1". SNMP1 is supposed to function as a co-receptor involved in the transfer of pheromones to adjacent pheromone receptors. In the moth Heliothis virescens, we previously found OSNs that project their dendrites into pheromone-responsive trichoid sensilla and are associated with cells containing transcripts for the HvirSNMP1-related protein HvirSNMP2. Like HvirSNMP1, HvirSNMP2 belongs to the CD36-family of two-transmembrane domain receptors and transporters for lipophilic compounds, but its role in the olfactory system is unknown. Here, we generated polyclonal anti-peptide antibodies against HvirSNMP2 as well as HvirSNMP1 and conducted an in-depth immunohistochemical analysis of their subcellular localization in the antenna of both sexes. In line with a function in pheromone detection, HvirSNMP1 was immunodetected in the somata and the dendrites of distinct OSNs in subsets of trichoid sensilla. These trichoid sensilla contained only one α-SNMP1-positive OSN in males and clusters of 2-3 labeled cells in females. In contrast, experiments with α-SNMP2-antibodies revealed a broad labeling of non-neuronal support cells (SCs) that are associated with OSNs in likely all trichoid and basiconic sensilla of the antenna with no differences between sexes. Detailed confocal microscope examinations of olfactory sensilla revealed SNMP2-like immunoreactivity close to the apical membrane of SCs and interestingly inside the sensillum. Together, these findings indicate a potential function of SNMP2 in pheromone- as well as general odorant-responsive sensilla and a role fundamentally different from SNMP1.

Lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into 16, 17, and 18 carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing 16 carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13 C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel 16 carbon terpenes by GC-QTOF. On the basis of the available farnesyl pyrophosphate analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.

Oligomerization of stromal interacting molecule 1 (STIM1) promotes store-operated calcium entry (SOCE); however, the mechanism of STIM1 aggregation is unclear. Here, using the lepidopteran insect and agricultural pest cotton bollworm ( Helicoverpa armigera ) as a model and immunoblotting, RT-qPCR, RNA interference (RNAi), and ChIP assays, we found that the steroid hormone 20-hydroxyecdysone (20E) up-regulates STIM1 expression via G protein-coupled receptors (GPCRs) and the 20E nuclear receptor (EcRB1). We also identified an ecdysone-response element (EcRE) in the 5'-upstream region of the STIM1 gene and also noted that STIM1 is located in the larval midgut during metamorphosis. STIM1 knockdown in larvae delayed pupation time, prevented midgut remodeling, and decreased 20E-induced gene transcription. STIM1 knockdown in a H. armigera epidermal cell line, HaEpi, repressed 20E-induced calcium ion influx and apoptosis. Moreover, 20E-induced STIM1 clustering to puncta and translocation toward the cell membrane. Inhibitors of GPCRs, phospholipase C (PLC), and inositol trisphosphate receptor (IP3R) repressed 20E-induced STIM1 phosphorylation, and we found that two GPCRs are involved in 20E-induced STIM1 phosphorylation. 20E-induced STIM1 phosphorylation on Ser-485 through protein kinase C (PKC), and we observed that Ser-485 phosphorylation is critical for STIM1 clustering, interaction with calcium release-activated calcium channel modulator 1 (Orai1), calcium ion influx, and 20E-induced apoptosis. These results suggest that 20E up-regulates STIM1 phosphorylation for aggregation via GPCRs, followed by interaction with Orai1 to induce SOCE, thereby promoting apoptosis in the midgut during insect metamorphosis., (© 2019 Chen et al.)

Background: Ryanodine receptor (RyR), a calcium-release channel located in the sarcoplasmic reticulum membrane of muscles, is the target of insecticides used against a wide range of agricultural pests. Mammalian RyRs have been shown to be under the regulatory control of several kinases and phosphatases, but little is known about the regulation of insect RyRs by phosphorylation., Results: Here we present the crystal structures of wild-type and phospho-mimetic RyR Repeat34 domain containing PKA phosphorylation sites from diamondback moth (DBM), a major lepidopteran pest of cruciferous vegetables. The structure has unique features, not seen in mammalian RyRs, including an additional α-helix near the phosphorylation loop. Using tandem mass spectrometry, we identify several PKA sites clustering in the phosphorylation loop and the newly identified α-helix. Bioinformatics analysis shows that this α-helix is only present in Lepidoptera, suggesting an insect-specific regulation. Interestingly, the specific phosphorylation pattern is temperature-dependent. The thermal stability of the DBM Repeat34 domain is significantly lower than that of the analogous domain in the three mammalian RyR isoforms, indicating a more dynamic domain structure that can be partially unfolded to facilitate the temperature-dependent phosphorylation. Docking the structure into the cryo-electron microscopy model of full-length RyR reveals that the interface between the Repeat34 and neighboring HD1 domain is more conserved than that of the phosphorylation loop region that might be involved in the interaction with SPRY3 domain. We also identify an insect-specific glycerol-binding pocket that could be potentially targeted by novel insecticides to fight the current resistance crisis., Conclusions: The crystal structures of the DBM Repeat34 domain reveals insect-specific temperature-dependent phosphorylation sites that may regulate insect ryanodine receptor function. It also reveals insect-specific structural features and a potential ligand-binding site that could be targeted in an effort to develop green pesticides with high species-specificity.

Most protein molecules do not adsorb onto ice, one of the exceptions being so-called antifreeze proteins. In this paper, we describe that there is a force pushing an antifreeze protein molecule away from the ice surface when it is not oriented with its ice-binding plane toward the ice and that this pushing force may be also present even when the protein is oriented with its ice-binding plane toward the ice. This force is absent only when certain specific distance criteria are met, regarding the surface of ice and the protein. It acts at early stages of adsorption, prior to the solidification of water between the ice and the protein molecule nearby. We propose the water-originating mechanism of the generation of this force and also the mechanism of remote attachment of an antifreeze molecule to the ice surface. In liquid water, there exist locally favored structures, ordered and of high specific volume. The presence of a protein molecule usually shifts the equilibrium that exists in liquid water toward increasing the number of high-density, disordered structures and diminishing the number of low-density structures. Creation of the locally favored structures may be hampered not only near the non-ice-binding surfaces but also between the ice surface and the protein surface, if the distance between these surfaces does not allow these structures to develop because the available space is not sufficient for their proper formation. This conclusion is supported by the analysis of the mean geometry of a single hydrogen bond, as well as of the hydrogen bond network in the solvation layer and a structural order parameter that characterizes the separation between the first and second solvation shells of a water molecule.

Sex pheromones are crucial for communication between females and males in moths, and pheromone receptors (PRs) play a key role in peripheral coding of sex pheromones. During the last decade, many PR candidates have been identified based on transcriptome sequencing and bioinformatic analysis, but their detailed functions remain mostly unknown. Here, focusing on four PR candidates of Athetis dissimilis (AdisOR1, AdisOR6, AdisOR11 and AdisOR14) identified in a previous study, we first cloned the full-length cDNAs and determined the tissue expression profiles by quantitative real-time PCR (qPCR). The results revealed that expression of three of these genes were male antennae-specific, while AdisOR11 was similar in expression between male and female antennae. Furthermore, the expression level of AdisOR1 was much higher than those of the other three genes. Then, functional analysis was conducted using Xenopus oocyte system. AdisOR1 responded strongly to the sex pheromone component Z9-14:OH and the potential pheromone component Z9,E12-14:OH, suggesting its important role in the sex pheromone perception; AdisOR14 showed specificity for Z9,E12-14:OH; while AdisOR6 and AdisOR11 did not respond to any of the pheromone components and analogs tested. Taken together, this study contributes to elucidate the molecular mechanism of sex pheromone reception and provides potential targets for development of OR based pest control techniques in A. dissimilis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Excretion in insects is accomplished by the combined actions of the Malpighian tubules (MTs) and hindgut, which together form the functional kidney. MTs of many insect groups consist of principal cells (PC) and secondary cells (SC). In most insect groups SCs are reported to secrete ions from haemolymph into the tubule lumen. Paradoxically, SCs in the MTs of the lepidopteran cabbage looper T. ni are used to reabsorb Na + and K + back into haemolymph. The current study was designed to investigate the effects and mode of action of the lepidopteran kinin, Helicokinin (HK), on ion transport in the SC-containing region of MT of T. ni. We identified a HK receptor (HK-R) homologue in T. ni and detected its expression in the SC-containing region of the MTs. The mRNA abundance of hk-r altered in response to changes in dietary K + and Na + content. HK-R immunolocalized to both PCs and SCs. Ramsay assays of preparations of the isolated distal ileac plexus (DIP) indicated that [HK] = 10 -8 M: (i) decreased fluid secretion rate in unstimulated and serotonin-stimulated preparations, and (ii) increased [Na + ]/[K + ] ratio in the secreted fluid. Scanning ion-selective electrode technique measurements revealed that HK reduced: (i) K + secretion by the PCs, and (ii) Na + reabsorption by the SCs in intact tubules. In vitro incubation of the DIP with HK resulted in reduced mRNA abundance of hk-r as well as Na + /K + -ATPase subunit α (NKAα), Na + /K + /Cl - co-transporter (nkcc), Na + /H + exchangers (nhe) 7 and 8, and aquaporin (aqp) 1. Taken together, results of the current study suggest that HK is capable of altering fluid secretion rate and [Na + ]/[K + ] ratio of the fluid, and that HK targets both PCs and SCs in the DIP of T. ni., (Copyright © 2018 Elsevier Inc. All rights reserved.)