Export of heme by the feline leukemia virus C receptor regulates mitochondrial biogenesis and redox balance in the hematophagous insect Rhodnius prolixus.

Heme is a prosthetic group of proteins involved in vital physiological processes. It participates, for example, in redox reactions crucial for cell metabolism due to the variable oxidation state of its central iron atom. However, excessive heme can be cytotoxic due to its prooxidant properties. Therefore, the control of intracellular heme levels ensures the survival of organisms, especially those that deal with high concentrations of heme during their lives, such as hematophagous insects. The export of heme initially attributed to the feline leukemia virus C receptor (FLVCR) has recently been called into question, following the discovery of choline uptake by the same receptor in mammals. Here, we found that RpFLVCR is a heme exporter in the midgut of the hematophagous insect Rhodnius prolixus, a vector for Chagas disease. Silencing RpFLVCR decreased hemolymphatic heme levels and increased the levels of intracellular dicysteinyl-biliverdin, indicating heme retention inside midgut cells. FLVCR silencing led to increased expression of heme oxygenase (HO), ferritin, and mitoferrin mRNAs while downregulating the iron importers Malvolio 1 and 2. In contrast, HO gene silencing increased FLVCR and Malvolio expression and downregulated ferritin, revealing crosstalk between heme degradation/export and iron transport/storage pathways. Furthermore, RpFLVCR silencing strongly increased oxidant production and lipid peroxidation, reduced cytochrome c oxidase activity, and activated mitochondrial biogenesis, effects not observed in RpHO-silenced insects. These data support FLVCR function as a heme exporter, playing a pivotal role in heme/iron metabolism and maintenance of redox balance, especially in an organism adapted to face extremely high concentrations of heme.

A Novel Peptidomimetic Insecticide: Dippu-AstR-Based Rational Design and Biological Activity of Allatostatin Analogs.

Insect neuropeptides play an essential role in regulating growth, development, reproduction, nerve conduction, metabolism, and behavior in insects; therefore, G protein-coupled receptors of neuropeptides are considered important targets for designing green insecticides. Cockroach-type allatostatins (ASTs) (FGLamides allatostatins) are important insect neuropeptides in Diploptera punctata that inhibit juvenile hormone (JH) synthesis in the corpora allata and affect growth, development, and reproduction of insects. Therefore, the pursuit of novel insecticides targeting the allatostatin receptor (AstR) holds significant importance. Previously, we identified an AST analogue, H17, as a promising candidate for pest control. Herein, we first modeled the 3D structure of AstR in D. punctata (Dippu-AstR) and predicted the binding mode of H17 with Dippu-AstR to study the critical interactions and residues favorable to its bioactivity. Based on this binding mode, we designed and synthesized a series of H17 derivatives and assessed their insecticidal activity against D. punctata. Among them, compound Q6 showed higher insecticidal activity than H17 against D. punctata by inhibiting JH biosynthesis, indicating that Q6 is a potential candidate for a novel insect growth regulator (IGR)-based insecticide. Moreover, Q6 exhibited insecticidal activity against Plutella xylostella, indicating that these AST analogs may have a wider insecticidal spectrum. The underlying mechanisms and molecular conformations mediating the interactions of Q6 with Dippu-AstR were explored to understand its effects on the bioactivity. The present work clarifies how a target-based strategy facilitates the discovery of new peptide mimics with better bioactivity, enabling improved IGR-based insecticide potency in sustainable agriculture.

Verification of AKT and CDK5 Gene and RNA Interference Combined with Irradiation to Mediate Fertility Changes in Plutella xylostella (Linnaeus).

Plutella xylostella (Linnaeus) mainly damages cruciferous crops and causes huge economic losses. Presently, chemical pesticides dominate its control, but prolonged use has led to the development of high resistance. In contrast, the sterile insect technique provides a preventive and control method to avoid the development of resistance. We discovered two genes related to the reproduction of Plutella xylostella and investigated the efficacy of combining irradiation with RNA interference for pest management. The results demonstrate that after injecting PxAKT and PxCDK5, there was a significant decrease of 28.06% and 25.64% in egg production, and a decrease of 19.09% and 15.35% in the hatching rate compared to the control. The ratio of eupyrene sperm bundles to apyrene sperm bundles also decreased. PxAKT and PxCDK5 were identified as pivotal genes influencing male reproductive processes. We established a dose-response relationship for irradiation (0-200 Gy and 200-400 Gy) and derived the irradiation dose equivalent to RNA interference targeting PxAKT and PxCDK5. Combining RNA interference with low-dose irradiation achieved a sub-sterile effect on Plutella xylostella, surpassing either irradiation or RNA interference alone. This study enhances our understanding of the genes associated with the reproduction of Plutella xylostella and proposes a novel approach for pest management by combining irradiation and RNA interference.

Parental experiences orchestrate locust egg hatching synchrony by regulating nuclear export of precursor miRNA.

Parental experiences can affect the phenotypic plasticity of offspring. In locusts, the population density that adults experience regulates the number and hatching synchrony of their eggs, contributing to locust outbreaks. However, the pathway of signal transmission from parents to offspring remains unclear. Here, we find that transcription factor Forkhead box protein N1 (FOXN1) responds to high population density and activates the polypyrimidine tract-binding protein 1 (Ptbp1) in locusts. FOXN1-PTBP1 serves as an upstream regulator of miR-276, a miRNA to control egg-hatching synchrony. PTBP1 boosts the nucleo-cytoplasmic transport of pre-miR-276 in a "CU motif"-dependent manner, by collaborating with the primary exportin protein exportin 5 (XPO5). Enhanced nuclear export of pre-miR-276 elevates miR-276 expression in terminal oocytes, where FOXN1 activates Ptbp1 and leads to egg-hatching synchrony in response to high population density. Additionally, PTBP1-prompted nuclear export of pre-miR-276 is conserved in insects, implying a ubiquitous mechanism to mediate transgenerational effects.

Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

CRISPR/Cas9 mediated validation of spermatogenesis-related gene, tssk2 as a component of genetic pest management of fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

Invasive insect pests, currently, pose a serious economic threat to several staple crops all over the world, one such being the fall armyworm, Spodoptera frugiperda. It was first observed in Africa since 2016, outside of its natural habitat in the Americas. Subsequently, it invaded several countries in South and South East Asia and also very recently in Australia. In all the newly invaded regions, maize is the principal crop attacked causing a serious economic concern to the poor farmers, particularly in the developing countries. Owing to the innate genetic ability, it defies many of the management options that include insecticides, Bt transgenics, and so forth. This is due to its high mobility, polyphagy and ability for quick development of resistance to several classes of insecticides. At this critical juncture, CRISPR/Cas9 mediated genome editing has shown a lot of promise in developing a novel area-wide pest management strategy called precision-guided sterile insect technique (pgSIT). pgSIT was initially demonstrated in Drosophila melanogaster which holds a greater promise for the environmentally friendly management of several globally significant agricultural pests such as S. frugiperda. Therefore, before developing both sgRNA and Cas9 transgenic lines, we have validated the target gene such as tssk2 through a non-transgenic approach by microinjecting ribo nucleo protein complex (Cas9 protein and tssk2 sgRNA) into G0 eggs of S. frugiperda. In the current investigation, we have obtained five edited males with distinct mutations which were further used for crossing studies to ascertain the effect of tssk2 editing affecting egg hatchability.

Lipidation alters the phase-separation of resilin-like polypeptides.

Biology exploits biomacromolecular phase separation to form condensates, known as membraneless organelles. Despite significant advancements in deciphering sequence determinants for phase separation, modulating these features in vivo remains challenging. A promising approach inspired by biology is to use post-translational modifications (PTMs)-to modulate the amino acid physicochemistry instead of altering protein sequences-to control the formation and characteristics of condensates. However, despite the identification of more than 300 types of PTMs, the detailed understanding of how they influence the formation and material properties of protein condensates remains incomplete. In this study, we investigated how modification with myristoyl lipid alters the formation and characteristics of the resilin-like polypeptide (RLP) condensates, a prototypical disordered protein with upper critical solution temperature (UCST) phase behaviour. Using turbidimetry, dynamic light scattering, confocal and electron microscopy, we demonstrated that lipidation-in synergy with the sequence of the lipidation site-significantly influences RLPs' thermodynamic propensity for phase separation and their condensate properties. Molecular simulations suggested these effects result from an expanded hydrophobic region created by the interaction between the lipid and lipidation site rather than changes in peptide rigidity. These findings emphasize the role of "sequence context" in modifying the properties of PTMs, suggesting that variations in lipidation sequences could be strategically used to fine-tune the effect of these motifs. Our study advances understanding of lipidation's impact on UCST phase behaviour, relevant to proteins critical in biological processes and diseases, and opens avenues for designing lipidated resilins for biomedical applications like heat-mediated drug elution.

Gustatory Receptor 206 Participates in the Foraging Behavior of Larvae of Polyphagous Pest Spodoptera litura.

Insect gustatory receptors (GRs) aid in the precise identification of deterrent or stimulant compounds associated with food, mating, and egg-laying. Thus, they are promising targets for developing efficient insecticides. Here, 61 GRs in the chemosensory organs of Spodoptera litura larvae and adults were identified. Among them, SlitGR206 exhibited larval labium (LL)-specific expression characteristics. To explore the role of SlitGR206, a bacterial expression system was established to produce high-quality double-stranded RNA (dsRNA) and suppress SlitGR206 expression in LL. Subsequent behavioral assessments revealed that SlitGR206 silencing influenced larval feeding preferences and absorption. Moreover, it was found to reduce the ability of larvae to forage the five crucial host odorants. These findings demonstrate that SlitGR206 likely plays an indirect regulatory role in host recognition, consequently affecting foraging behavior. This provides a crucial foundation for the analysis of functional diversity among insect GRs and the precise development of nucleic acid pesticides in the future.

Exploring the binding affinity and characteristics of DcitOBP9 in citrus psyllids.

Odorant-binding proteins (OBPs) are crucial in insect olfaction. The most abundant expressed OBP of citrus psyllids, DcitOBP9 encodes 148 amino acids. DcitOBP9 lacks a transmembrane structure and possesses a 17-amino acid signal peptide at the N-terminus. Characterized by the six conserved cysteine sites, DcitOBP9 is classified as the Classical-OBP family. RT-qPCR experiments revealed ubiquitous expression of DcitOBP9 across all developmental stages of the citrus psyllid, with predominant expression in adults antennae. Fluorescence competitive binding assays demonstrated DcitOBP9's strong affinity for ocimene, linalool, dodecanoic acid, and citral, and moderate affinity for dimethyl trisulfide. Additionally, it binds to myrcia, (-)-trans-caryophyllene, (±)-Citronellal, nonanal, and (+)-α-pinene. Among them, ocimene, linalool, and dodecanoic acid were dynamically bound to DcitOBP9, while citral was statically bound to DcitOBP9. Molecular docking simulations with the top five ligands indicated that amino acid residues V92, S72, P128, L91, L75, and A76 are pivotal in the interaction between DcitOBP9 and these odorants. These findings suggest DcitOBP9's involvement in the citrus psyllid's host plant recognition and selection behaviors, thereby laying a foundation for elucidating the potential physiological and biological functions of DcitOBP9 and developing attractants.

Salivary Protein Cyclin-Dependent Kinase-like from Grain Aphid Sitobion avenae Suppresses Wheat Defense Response and Enhances Aphid Adaptation.

Aphids are insect pests that suck phloem sap and introduce salivary proteins into plant tissues through saliva secretion. The effector of salivary proteins plays a key role in the modulation of host plant defense responses and enhancing aphid host adaptation. Based on previous transcriptome sequencing results, a candidate effector cyclin-dependent kinase-like (CDK) was identified from the grain aphid Sitobion avenae. In this study, the function of SaCDK in wheat defense response and the adaptation of S. avenae was investigated. Our results showed that the transient overexpression of SaCDK in tobacco Nicotiana benthamiana suppressed cell death triggered by mouse pro-apoptotic protein-BAX or Phytophthora infestans PAMP-INF1. SaCDK, delivered into wheat cells through a Pseudomonas fluorescens-mediated bacterial type III secretion system, suppressed callose deposition in wheat seedlings, and the overexpression of SaCDK in wheat significantly decreased the expression levels of salicylic acid and jasmonic acid signaling pathway-related genes phenylalanine ammonia lyase (PAL), pathogenesis-related 1 protein (PR1), lipoxygenase (LOX) and Ω-3 fatty acid desaturase (FAD). In addition, aphid bioassay results showed that the survival and fecundity of S. avenae were significantly increased while feeding on the wheat plants carrying SaCDK. Taken together, our findings demonstrate that the salivary protein SaCDK is involved in inhibiting host defense response and improving its host adaptation, which lays the foundation to uncover the mechanism of the interaction of cereal aphids and host plants.

Functional analysis of nuclear receptor genes in molting and metamorphosis of the cigarette beetle, Lasioderma serricorne.

Nuclear receptors (NRs) are ligand-regulated transcription factors that are important for the normal growth and development of insects. However, systematic function analysis of NRs in the molting process of Lasioderma serricorne has not been reported. In this study, we identified and characterized 16 NR genes from L. serricorne. Spatiotemporal expression analysis revealed that six NRs were mainly expressed in 3-d-old 4th-instar larvae; five NRs were primarily expressed in 5-d-old adults and four NRs were predominately expressed in prepupae. All the NRs were highly expressed in epidermis, fat body and foregut. RNA interference (RNAi) experiments revealed that knockdown of 15 NRs disrupted the larva-pupa-adult transitions and caused 64.44-100 % mortality. Hematoxylin-eosin staining showed that depletion of 12 NRs prevented the formation of new cuticle and disrupted apolysis of old cuticle. Silencing of LsHR96, LsSVP and LsE78 led to newly formed cuticle that was thinner than the controls. The 20E titer and chitin content significantly decreased by 17.67-95.12 % after 15 NR dsRNA injection and the gene expression levels of 20E synthesis genes and chitin metabolism genes were significantly reduced. These results demonstrated that 15 NR genes are essential for normal molting and metamorphosis of L. serricorne by regulating 20E synthesis and chitin metabolism.

New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

Functional characterization of Helicoverpa assulta CYP6B6 in insecticide metabolism.

The oriental tobacco budworm Helicoverpa assulta (Lepidoptera: Noctuidae) is a specialist pest that may cause serious damages to important crops such as chili pepper and tobacco. Various man-made insecticides have been applied to control the infestation of this pest. To understand how this pest copes with insecticides, it is required to identify key players involved in insecticide transformation. In this study, a P450 gene of CYP6B subfamily was identified in the oriental tobacco budworm, and its expression pattern was revealed. Moreover, the activities of HassCYP6B6 against 12 insecticides were explored using recombinant enzymes produced in the facile Escherichia coli. Data from metabolic experiments showed that HassCYP6B6 was able to metabolize conventional insecticides including organophosporates (diazinon, malathion, phoxim), carbamate propoxur, and pyrethroid esfenvalerate, while no significant metabolism was observed towards new-type pesticides such as neonicotinoids (acetamiprid, imidacloprid), diamides (chlorantraniliprole, cyantraniliprole), macrocyclic lactone (emamectin benzoate, ivermectin), and metaflumizone. Structures of metabolites were proposed based on mass spectrometry analyses. The results demonstrate that HassCYP6B6 plays important roles in the transformation of multiple insecticides via substrate-dependent catalytic mechanisms including dehydrogenation, hydroxylation and oxidative desulfurization. The findings have important applied implications for the usage of insecticides.

Supreme glutathione-dependent ketosteroid isomerase in the yellow-fever transmitting mosquito Aedes aegypti.

The steroid hormone ecdysone is essential for the reproduction and survival of insects. The hormone is synthesized from dietary sterols such as cholesterol, yielding ecdysone in a series of consecutive enzymatic reactions. In the insect orders Lepidoptera and Diptera a glutathione transferase called Noppera-bo (Nobo) plays an essential, but biochemically uncharacterized, role in ecdysteroid biosynthesis. The Nobo enzyme is consequently a possible target in harmful dipterans, such as disease-carrying mosquitoes. Flavonoid compounds inhibit Nobo and have larvicidal effects in the yellow-fever transmitting mosquito Aedes aegypti, but the enzyme is functionally incompletely characterized. We here report that within a set of glutathione transferase substrates the double-bond isomerase activity with 5-androsten-3,17-dione stands out with an extraordinary specific activity of 4000 µmol min-1 mg-1. We suggest that the authentic function of Nobo is catalysis of a chemically analogous ketosteroid isomerization in ecdysone biosynthesis.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

The dual role of Spn-E in supporting heterotypic ping-pong piRNA amplification in silkworms.

The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping-pong cycle, is mediated by the two PIWI proteins, Siwi and BmAgo3, in silkworms. However, the detailed molecular mechanism of the ping-pong cycle remains largely unclear. Here, we show that Spindle-E (Spn-E), a putative ATP-dependent RNA helicase, is essential for BmAgo3-dependent production of Siwi-bound piRNAs in the ping-pong cycle and that this function of Spn-E requires its ATPase activity. Moreover, Spn-E acts to suppress homotypic Siwi-Siwi ping-pong, but this function of Spn-E is independent of its ATPase activity. These results highlight the dual role of Spn-E in facilitating proper heterotypic ping-pong in silkworms.

The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 µm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.

Improved reference quality genome sequence of the plastic-degrading greater wax moth, Galleria mellonella.

Galleria mellonella is a pest of honeybees in many countries because its larvae feed on beeswax. However, G. mellonella larvae can also eat various plastics, including polyethylene, polystyrene, and polypropylene, and therefore, the species is garnering increasing interest as a tool for plastic biodegradation research. This paper presents an improved genome (99.3% completed lepidoptera_odb10 BUSCO; genome mode) for G. mellonella. This 472 Mb genome is in 221 contigs with an N50 of 6.4 Mb and contains 13,604 protein-coding genes. Genes that code for known and putative polyethylene-degrading enzymes and their similarity to proteins found in other Lepidoptera are highlighted. An analysis of secretory proteins more likely to be involved in the plastic catabolic process has also been carried out.

Ecdysone-controlled nuclear receptor ERR regulates metabolic homeostasis in the disease vector mosquito Aedes aegypti.

Hematophagous mosquitoes require vertebrate blood for their reproductive cycles, making them effective vectors for transmitting dangerous human diseases. Thus, high-intensity metabolism is needed to support reproductive events of female mosquitoes. However, the regulatory mechanism linking metabolism and reproduction in mosquitoes remains largely unclear. In this study, we found that the expression of estrogen-related receptor (ERR), a nuclear receptor, is activated by the direct binding of 20-hydroxyecdysone (20E) and ecdysone receptor (EcR) to the ecdysone response element (EcRE) in the ERR promoter region during the gonadotropic cycle of Aedes aegypti (named AaERR). RNA interference (RNAi) of AaERR in female mosquitoes led to delayed development of ovaries. mRNA abundance of genes encoding key enzymes involved in carbohydrate metabolism (CM)-glucose-6-phosphate isomerase (GPI) and pyruvate kinase (PYK)-was significantly decreased in AaERR knockdown mosquitoes, while the levels of metabolites, such as glycogen, glucose, and trehalose, were elevated. The expression of fatty acid synthase (FAS) was notably downregulated, and lipid accumulation was reduced in response to AaERR depletion. Dual luciferase reporter assays and electrophoretic mobility shift assays (EMSA) determined that AaERR directly activated the expression of metabolic genes, such as GPI, PYK, and FAS, by binding to the corresponding AaERR-responsive motif in the promoter region of these genes. Our results have revealed an important role of AaERR in the regulation of metabolism during mosquito reproduction and offer a novel target for mosquito control.

Identification of immunity-related genes distinctly regulated by Manduca sexta Spӓtzle-1/2 and Escherichia coli peptidoglycan.

The immune system of Manduca sexta has been well studied to understand molecular mechanisms of insect antimicrobial responses. While evidence supports the existence of major immune signaling pathways in this species, it is unclear how induced production of defense proteins is specifically regulated by the Toll and Imd pathways. Our previous studies suggested that diaminopimelic acid-type peptidoglycans (DAP-PG) from Gram-negative and some Gram-positive bacteria, more than Lys-type peptidoglycans (Lys-PG) from other Gram-positive bacteria, triggers both pathways through membrane-bound receptors orthologous to Drosophila Toll and PGRP-LC. In this study, we produced M. sexta proSpätzle-1 and proSpätzle-2 in Sf9 cells, identified their processing enzymes, and used prophenoloxidase activating protease-3 to activate the cytokine precursors. After Spätzle-1 and -2 were isolated from the reaction mixtures, we separately injected the purified cytokines into larval hemocoel to induce gene transcription in fat body through the Toll pathway solely. On the other hand, we treated a M. sexta cell line with E. coli DAP-PG to only induce the Imd pathway and target gene expression. RNA-Seq analysis of the fat body and cultured cells collected at 0, 6, and 24 h after treatment indicated that expression of diapausin-4, -10, -12, -13, cecropin-2, -4, -5, attacin-5, -11, and lebocin D is up-regulated predominantly via Toll signaling, whereas transcription of cecropin-6, gloverin, lysozyme-1, and gallerimycin-2 is mostly induced by DAP-PG via Imd signaling. Other antimicrobial peptides are expressed in response to both pathways. Transcripts of most Toll-specific genes (e.g., lebocin D) peaked at 6 h, contrasting the gradual increase and plateauing of drosomycin mRNA level at 24-48 h in Drosophila. We also used T (oll)-I (md) ratios to estimate relative contributions of the two pathways to transcriptional regulation of other components of the immune system. The differences in pathway specificity and time course of transcriptional regulation call for further investigations in M. sexta and other insects.