The irregular developmental duration mainly caused by the broad-complex in Chilo suppressalis.

Chilo suppressalis, a critical rice stem borer pest, poses significant challenges to rice production due to its overlapping generations and irregular developmental duration. These characteristics complicate pest management strategies. According to the dynamic analysis of the overwintering adults of C. suppressalis in fields, it indicates that the phenomenon of irregular development of C. suppressalis exists widely and continuously. This study delves into the potential role of the Broad-Complex (Br-C) gene in the developmental duration of C. suppressalis. Four isoforms of Br-C, named CsBr-C Z1, CsBr-C Z2, CsBr-C Z4, and CsBr-C Z7, were identified. After CsBr-Cs RNAi, the duration of larva development spans extended obviously. And, the average developmental duration of dsCsBr-Cs feeding individuals increased obviously. Meanwhile, the average developmental duration of the dsCsBr-C Z2 feeding group was the longest among all the RNAi groups. After dsCsBr-Cs feeding continuously, individuals pupated at different instars changed obviously: the proportion of individuals pupated at the 5th instar decreased and pupated at the 7th instar or higher increased significantly. Moreover, the pupation rate of dsCsBr-Cs (except dsCsBr-C Z7) were significantly lower than that of dsGFP. The same results were obtained from the mutagenesis in CsBr-C genes mediated by CRISPR/Cas9. The average developmental duration of CsBr-Cs knockout individuals was significantly prolonged. And, the instar of pupation in knockout individuals was also delayed significantly. In conclusion, this work showed that CsBr-Cs played a crucial role in pupal commitment and affected the developmental duration of C. suppressalis significantly.

Maneuvering the Peroxidase-Like Activity of Palladium-Based Nanozymes by Alloying with Oxophilic Bismuth for Biosensing.

Engineering the catalytic performance of nanozymes is of vital importance for their broad applications in biological analysis, cancer treatment, and environmental management. Herein, a strategy to boost the peroxidase-like activity of Pd-based nanozymes with oxophilic metallic bismuth (Bi) is demonstrated, which is based on the incorporation of oxophilic Bi in the Pd-based alloy nanocrystals (NCs). To synthesize PdBi alloy NCs, a seed-mediated method is employed with the assistance of underpotential deposition (UPD) of Bi on Pd. The strong interaction of Bi atoms with Pd surfaces favors the formation of alloy structures with controllable shapes and excellent monodispersity. More importantly, the PdBi NCs show excellent peroxidase-like activities compared with pristine Pd NCs. The structure-function correlations for the PdBi nanozymes are elucidated, and an indirect colorimetric method based on cascade reactions to determine alkaline phosphatase (ALP) is established. This method has good linear range, low detection limit, excellent selectivity, and anti-interference. Collectively, this work not only provides new insights for the design of high-efficiency nanozymes, expands the colorimetric sensing platform based on enzyme cascade reactions, but also represents a new example for UPD-directed synthesis of alloy NCs.

Surface Molecular Functionalization of Unusual Phase Metal Nanomaterials for Highly Efficient Electrochemical Carbon Dioxide Reduction under Industry-Relevant Current Density.

The electrochemical carbon dioxide reduction reaction (CO2 RR) provides a sustainable strategy to relieve global warming and achieve carbon neutrality. However, the practical application of CO2 RR is still limited by the poor selectivity and low current density. Here, the surface molecular functionalization of unusual phase metal nanomaterials for high-performance CO2 RR under industry-relevant current density is reported. It is observed that 5-mercapto-1-methyltetrazole (MMT)-modified 4H/face-centered cubic (fcc) gold (Au) nanorods demonstrate greatly enhanced CO2 RR performance than original oleylamine (OAm)-capped 4H/fcc Au nanorods in both an H-type cell and flow cell. Significantly, MMT-modified 4H/fcc Au nanorods deliver an excellent carbon monoxide selectivity of 95.6% under the industry-relevant current density of 200 mA cm-2 . Density functional theory calculations reveal distinct electronic modulations by surface ligands, in which MMT improves while OAm suppresses the surface electroactivity of 4H/fcc Au nanorods. Furthermore, this method can be extended to various MMT derivatives and conventional fcc Au nanostructures in boosting CO2 RR performance.

Designer Gold-Framed Palladium Nanocubes for Plasmon-Enhanced Electrocatalytic Oxidation of Ethanol.

Surface plasmon of coinage metal nanostructures has been employed as a powerful route in boosting the performances in heterogenous catalysis. Development of efficient plasmonic nanocatalysts with high catalytic performance and efficient light harvesting properties is of vital importance. Herein, we rationally designed and synthesized a plasmonic nanocatalyst composed of Au-framed Pd nanocubes by an Ag(I)-assisted seed-mediated growth method. In the synthesis, the incorporation of Ag(I) suppresses the reduction of Au on the {100} surface of cubic Pd seeds and leads to the formation of Au nanoframes on the Pd nanocubes. The unique Au-framed Pd nanocubes can integrate the superior electrocatalytic of Pd and the outstanding plasmonic properties of Au. Thus, these nanostructures were employed as plasmonic nanocatalysts for plasmon-enhanced electrocatalytic oxidation of ethanol with improved stability.

The impact of positive and negative information on SIR-like epidemics in delayed multiplex networks.

In order to better study the interaction between epidemic propagation and information diffusion, a new coupling model on multiplex networks with time delay is put forward in this paper. One layer represents the information diffusion about epidemics. There is not only information about the positive prevention of infectious diseases but also negative preventive information. Meanwhile, the dissemination of information at this layer will be influenced by the mass media, which can convey positive and reliable preventive measures to help the public avoid exposure to contagion. The other layer represents the transmission of infectious diseases, and the public in this layer no longer only exchange information related to infectious diseases in the virtual social network like the information layer but spread infectious diseases through contact among people. The classical SIR model is used to model for epidemic propagation. Since each infected individual needs to spend enough time to recover, the infected one at one time does not necessarily change to the recovered one at the next time, so time delay is an essential factor to be considered in the model. Based on the microscopic Markov chain approach, this paper obtains an explicit expression for epidemic threshold in the two-layered multiplex networks with time delay, which reveals some main factors affecting epidemic threshold. In particular, the time delay has a noticeable effect on the epidemic threshold to some extent. Finally, the influence of these main factors on the epidemic threshold and their interaction are proved through numerical simulations.

Time-Series Transcriptomic Analysis Reveals the Molecular Profiles of Diapause Termination Induced by Long Photoperiods and High Temperature in Chilo suppressalis.

Survival and adaptation to seasonal changes are challenging for insects. Many temperate insects such as the rice stem borer (Chilo suppressalis) overcome the adverse situation by entering diapause, wherein development changes dynamically occur and metabolic activity is suppressed. The photoperiod and temperature act as major environmental stimuli of diapause. However, the physiological and molecular mechanisms that interpret the ecologically relevant environmental cues in ontogenetic development during diapause termination are poorly understood. Here, we used genome-wide high-throughput RNA-sequencing to examine the patterns of gene expression during diapause termination in C. suppressalis. Major shifts in biological processes and pathways including metabolism, environmental information transmission, and endocrine signalling were observed across diapause termination based on over-representation analysis, short time-series expression miner, and gene set enrichment analysis. Many new pathways were identified in diapause termination including circadian rhythm, MAPK signalling, Wnt signalling, and Ras signalling, together with previously reported pathways including ecdysteroid, juvenile hormone, and insulin/insulin-like signalling. Our results show that convergent biological processes and molecular pathways of diapause termination were shared across different insect species and provided a comprehensive roadmap to better understand diapause termination in C. suppressalis.

Unveiling the Actual Catalytic Sites in Nanozyme-Catalyzed Oxidation of o-Phenylenediamine.

Nanozymes have offered remarkable advantages over natural enzymes and found widespread applications including biosensors, immunoassays, nanomedicines, and environmental remediation. Oxidation of o-phenylenediamine (OPD) by nanozymes has been listed as a standard protocol for determining nanozyme activities. Given the complexity of OPD oxidation processes, however, the mechanism of nanozyme-catalyzed oxidation of OPD remains elusive. In this report, mechanistic studies of nanozyme-catalyzed oxidation of OPD are performed and a distinguishably different mechanism from that of natural enzymes is found. A combination of Fourier transform infrared spectroscopy, nuclear magnetic resonance, electrospray ionization mass spectrometry, and electron microscopic studies provides compelling evidence that polymerization of OPD occurs on the surface of several different nanozymes. The unexpected polymerization causes a dense coating layer of poly(o-phenylenediamine) (POPD) on nanozymes renders the intrinsic properties of nanozymes. Therefore, this fundamental discovery raise serious concerns using OPD-based colorimetric method for determining nanozyme activities. Without examining the surface change of nanozymes after catalytic reactions, the use of OPD-based colorimetric method for determining nanozyme activities is strongly discouraged. Furthermore, POPD is discovered as a new oxidase mimic, and this new mechanism also provides a general and robust method to coat nanomaterials with POPD polymers of enzyme-mimicking properties.

Identification and functional study of three nAChR regulators, ubiquilin-1, PICK1, and CRELD2, in Locusta migratoria manilensis dorsal unpaired median neurons.

Insect nicotinic acetylcholine receptors (nAChRs) are not only important neurotransmitter receptors but also effective insecticide targets. The regulation of nAChRs has been mainly studied in vertebrates, especially in mammals. Here, two types of nAChRs were found present in the locust Locusta migratoria manilensis dorsal unpaired median (DUM) neurons, α-bungarotoxin (α-Bgt)-sensitive nAChRs and α-Bgt-resistant nAChRs, responding to acetylcholine (ACh) at different concentrations. The homologs to three mammalian nAChR regulators, ubiquilin-1, CRELD2 (cysteine-rich with EFG-like domain 2), and PICK1 (protein interacting with PRKCA 1), were characterized in L. migratoria, and their functions on regulating native nAChRs were investigated via RNAi followed by membrane potential measurement with DiBAC4 (3) and agonist-evoked macroscopic current recording in cultured L. migratoria DUM neurons. Ubiquilin-1 and PICK1 negatively regulated nAChRs because silencing of ubiquilin-1 and PICK1 both resulted in increased membrane potential and increased inward currents in DUM neurons, while CRELD2 positively regulated nAChRs as decreased membrane potential and inward currents were observed in DUM neurons. In addition, ubiquilin-1 regulated both α-Bgt-sensitive and α-Bgt-resistant types of nAChRs whereas PICK1 and CRELD2 regulated only the α-Bgt-resistant nAChRs. The present study broadened our understanding on the regulation of insect nAChRs and will benefit pest management given the important role of nAChRs in insect neurons and insecticide science. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Two distinctive ß subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensis.

Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one ß subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third ß subunits (Locß3) was identified in this study, which reveals at least three ß subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with Kd values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locß1, Locß2 and Locß3 respectively. Specific immunodepletion of Locß1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locß3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locß2 had no significant effect on the specific [3H]imidacloprid binding. Taken together, these data indicated that Locß1 and Locß3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlß1 was in two binding sites for imidacloprid. The involvement of two ß subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in ß subunits in L. migratoria.

Heterologous formation of neonicotinoid-sensitive nAChRs containing UNC-38 and UNC-29 subunits from Bursaphelenchus xylophilus.

Nicotinic acetylcholine receptor (nAChR) subunits are encoded by a large multigene family and generate a large number of pentameric receptors with various properties. At present, nematode species, such as Caenorhabditis elegans, have the largest number of nAChR subunits. In this study, two nAChR subunits (Bxy-Unc-38 and Bxy-Unc-29) were cloned from Bursaphelenchus xylophilus, a fatal nematode pest on pine trees causing pine wilt disease. When Bxy-Unc-38 and Bxy-Unc-29 were co-expressed in Xenopus oocytes, constructed functional nAChRs showed agonist responses to acetylcholine and imidacloprid, a neonicotinoid insecticide. When complementary RNAs (cRNAs) of Bxy-Unc-38 and Bxy-Unc-29 were injected at different ratios, the assembled nAChRs showed different pharmacological subtypes, especially in terms of the sensitivity to imidacloprid and another two neonicotinoids. At cRNA ratios 1:1 and 1:5 (Bxy-Unc-38: Bxy-Unc-29), nAChRs showed low sensitivity to test neonicotinoids, which were partial agonists on the receptors. In contrast, at cRNA ratio 5:1, the three test neonicotinoids were full agonists and showed much higher potency compared to that on the receptors with cRNA ratio 1:1 and 1:5. For example, EC50 values of the three neonicotinoids on the receptors with cRNA ratio 1:5 were 170-222 times of those of receptors with cRNA ratio 5:1. The results showed that the subunit stoichiometry of Bxy-Unc-38/Bxy-Unc-29 receptor dramatically affected the agonist potency of neonicotinoids, and even altered the action property. Due to the high sensitivity of the constructed nAChRs at cRNA ratio 5:1, the construct would serve as an important model to study the interaction between invertebrate nAChRs and neonicotinoids.

Characterization of the Fifth Putative Acetylcholinesterase in the Wolf Spider, Pardosa pseudoannulata.

Background: Acetylcholinesterase (AChE) is an important neurotransmitter hydrolase in invertebrate and vertebrate nervous systems. The number of AChEs is various among invertebrate species, with different functions including the 'classical' role in terminating synaptic transmission and other 'non-classical' roles. Methods: Using rapid amplification of cDNA ends (RACE) technology, a new putative AChE-encoding gene was cloned from Pardosa pseudoannulata, an important predatory natural enemy. Sequence analysis and in vitro expression were employed to determine the structural features and biochemical properties of this putative AChE. Results: The cloned AChE contained the most conserved motifs of AChEs family and was clearly clustered with Arachnida AChEs. Determination of biochemical properties revealed that the recombinant enzyme had the obvious preference for the substrate ATC (acetylthiocholine iodide) versus BTC (butyrylthiocholine iodide). The AChE was highly sensitive to AChE-specific inhibitor BW284C51, but not butyrylcholinesterase-specific inhibitor tetraisopropyl pyrophosphoramide (ISO-OMPA). Based on these results, we concluded that a new AChE was identified from P. pseudoannulata and denoted as PpAChE5. Conclusion: Here we report the identification of a new AChE from P. pseudoannulata and increased the AChE number to five in this species. Although PpAChE5 had the biggest Vmax value among five identified AChEs, it showed relatively low affinity with ATC. Similar sensitivity to test insecticides indicated that this AChE might serve as the target for both organophosphorus and carbamate insecticides.

The roles of CYP6AY1 and CYP6ER1 in imidacloprid resistance in the brown planthopper: Expression levels and detoxification efficiency.

Two P450 monooxygenase genes, CYP6AY1 and CYP6ER1, were reported to contribute importantly to imidacloprid resistance in the brown planthopper, Nilaparvata lugens. Although recombinant CYP6AY1 could metabolize imidacloprid efficiently, the expression levels of CYP6ER1 gene were higher in most resistant populations. In the present study, three field populations were collected from different countries, and the bioassay, RNAi and imidacloprid metabolism were performed to evaluate the importance of two P450s in imidacloprid resistance. All three populations, DOT (Dongtai) from China, CNA (Chainat) from Thailand and HCM (Ho Chi Minh) from Vietnam, showed high resistance to imidacloprid (57.0-, 102.9- and 89.0-fold). CYP6AY1 and CYP6ER1 were both over expressed in three populations, with highest ratio of 13.2-fold for CYP6ER1 in HCM population. Synergism test and RNAi analysis confirmed the roles of both P450 genes in imidacloprid resistance. However, CYP6AY1 was indicated more important in CNA population, and CYP6AY1 and CYP6ER1 were equal in HCM population, although the expression level of CYP6ER1 (13.2-fold) was much higher than that of CYP6AY1 (4.11-fold) in HCM population. Although the recombinant proteins of both P450 genes could metabolize imidacloprid efficiently, the catalytic activity of CYP6AY1 (Kcat=3.627 pmol/min/pmol P450) was significantly higher than that of CYP6ER1 (Kcat=2.785 pmol/min/pmol P450). It was supposed that both P450 proteins were important for imidacloprid resistance, in which CYP6AY1 metabolized imidacloprid more efficiently and CYP6ER1 gene could be regulated by imidacloprid to a higher level.

Selective actions of Lynx proteins on different nicotinic acetylcholine receptors in the locust, Locusta migratoria manilensis.

Nicotinic acetylcholine receptors (nAChRs) are major neurotransmitter receptors and targets of neonicotinoid insecticides in the insect nervous system. The full function of nAChRs is often dependent on associated proteins, such as chaperones, regulators and modulators. Here, three Lynx (Ly-6/neurotoxin) proteins, Loc-lynx1, Loc-lynx2 and Loc-lynx3, were identified in the locust, Locusta migratoria manilensis. Co-expression with Lynx resulted in a dramatic increase in agonist-evoked macroscopic currents on nAChRs Locα1/ß2 and Locα2/ß2 in Xenopus oocytes, but no changes in agonist sensitivity. Loc-lynx1 and Loc-lynx3 only modulated nAChRs Locα1/ß2 while Loc-lynx2 modulated Locα2/ß2 specifically. Meanwhile, Loc-lynx1 induced a more significant increase in currents evoked by imidacloprid and epibatidine than Loc-lynx3, and the effects of Loc-lynx1 on imidacloprid and epibatidine were significantly higher than those on acetylcholine. Among three lynx proteins, only Loc-lynx1 significantly increased [(3) H]epibatidine binding on Locα1/ß2. The results indicated that Loc-lynx1 had different modulation patterns in nAChRs compared to Loc-lynx2 and Loc-lynx3. Taken together, these findings indicated that three Lynx proteins were nAChR modulators and had selective activities in different nAChRs. Lynx proteins might display their selectivities from three aspects: nAChR subtypes, various agonists and different modulation patterns. Insect Lynx (Ly-6/neurotoxin) proteins act as the allosteric modulators on insect nicotinic acetylcholine receptors (nAChRs), the important targets of insecticides. We found that insect lynx proteins showed their selectivities from at least three aspects: nAChR subtypes, various agonists and different modulation patterns.

Validating the importance of two acetylcholinesterases in insecticide sensitivities by RNAi in Pardosa pseudoannulata, an important predatory enemy against several insect pests.

The pond wolf spider (Pardosa pseudoannulata) is an important predatory enemy against several insect pests and showed relative different sensitivities to organophosphate and carbamate insecticides compared to insect pests. In our previous studies, two acetylcholinesterases were identified in P. pseudoannulata and played important roles in insecticide sensitivities. In order to understand the contributions of the two acetylcholinesterases to insecticide sensitivities, we firstly employed the RNAi technology in the spider. For a suitable microinjection RNAi method, the injection site, injection volume and interference time were optimized, which then demonstrated that the injection RNAi method was applicable in this spider. With the new RNAi method, it was revealed that both Pp-AChE1 and Pp-AChE2, encoded by genes Ppace1 and Ppace2, were the targets of organophosphate insecticides, but Pp-AChE1 would be more important. In contrast, the carbamate acted selectively on Pp-AChE1. The results showed that Pp-AChE1 was the major catalytic enzyme in P. pseudoannulata and the major target of organophosphate and carbamate insecticides. In a word, an RNAi method was established in the pond wolf spider, which further validated the importance of two acetylcholinesterases in insecticide sensitivities in this spider.

Identification of polymorphisms in Cyrtorhinus lividipennis RDL subunit contributing to fipronil sensitivity.

As one of the most important predatory enemies, the miridbug, Cyrtorhinus lividipennis, plays an important role in rice planthoppers control, such as Nilaparvata lugens (brown planthopper). In order to compare insecticide selectivity between C. lividipennis and N. lugens, the contact acute toxicities of six insecticides (diazoxon, paraoxon, carbaryl, fenobucarb, fipronil and ethofenprox) were monitored. The results showed that all tested insecticides were more toxic to C. lividipennis than to N. lugens and fipronil had the biggest difference. The RDL subunit (Cl-RDL) was cloned from C. lividipennis and a RDL isoform (Cl-RDL-In) was also found with 31 amino acids insertion in RDL intracellular region. In order to understand the role of the insertion on insecticide sensitivities, three subunits (Nl-RDL, Cl-RDL and Cl-RDL-In) were constructed to obtain the functional receptors in Xenopus oocytes and the fipronil sensitivities were detected by the voltage-clamp technique. Nl-RDL (IC50=32.36 ± 4.07 µM) was more insensitive to fipronil than Cl-RDL (IC50=6.47 ± 1.12 µM). The insertion in Cl-RDL significantly reduced fipronil sensitivity with IC50 value in Cl-RDL-In of 16.83 ± 2.30 µM. Interestingly, after the elution of fipronil, the current response of Cl-RDL-In appeared obvious recovery, which were not observed in Cl-RDL and Nl-RDL. It might imply that the insertion played a special role in fipronil sensitivity.

Chromosome-level genome of spider Pardosa pseudoannulata and cuticle protein genes in environmental stresses.

Spiders are representative arthropods of adaptive radiation. The high-quality genomes have only been reported in several web weaver spider species, leaving the wandering spiders' genomic information scarce. The pond wolf spider, Pardosa pseudoannulata, is a representative species in the retrolateral titial apophysis (RTA) clade. We present a chromosome-level P. pseusoannulata genome assembly of 2.42 Gb in size with a scaffold N50 of 169.99 Mb. Hi-C scaffolding assigns 94.83% of the bases to 15 pseudo-chromosomes. The repeats account for 52.79% of the assembly. The assembly includes 96.2% of the complete arthropod universal single-copy orthologs. Gene annotation predicted 24,530 protein-coding genes with a BUSCO score of 95.8% complete. We identified duplicate clusters of Hox genes and an expanded cuticle protein gene family with 243 genes. The expression patterns of CPR genes change in response to environmental stresses such as coldness and insecticide exposure. The high-quality P. pseudoannulata genome provides valuable information for functional and comparative studies in spiders.

Event-Triggered Quantized Quasisynchronization of Uncertain Quaternion-Valued Chaotic Neural Networks With Time-Varying Delay for Image Encryption.

In this article, a class of quaternion-valued master-slave neural networks (NNs) with time-varying delay and parameter uncertainties was first established by conducting the extension from real-valued chaotic NNs to the quaternion field. Then, based on logarithmic quantized output feedback, the quasisynchronization issue of the NNs was investigated via devising a neoteric dynamic event-triggered controller. In virtue of the classical Lyapunov method and a generalized Halanay inequality, not only corresponding synchronization criteria were obtained to realize the quasisynchronization of master-slave NNs but also a precise upper bound was provided. Moreover, Zeno behavior can be eliminated under the presented scheme in this article. The accuracy of the theoretical outcomes was demonstrated by means of Chua's circuit. Ultimately, some experimental results of pragmatic application in image encryption/decryption were exposed to substantiate the feasibility and efficacy of the current algorithm for the proposed quaternion-valued NNs.

Uniform Stability of Complex-Valued Neural Networks of Fractional Order With Linear Impulses and Fixed Time Delays.

As a generation of the real-valued neural network (RVNN), complex-valued neural network (CVNN) is based on the complex-valued (CV) parameters and variables. The fractional-order (FO) CVNN with linear impulses and fixed time delays is discussed. By using the sign function, the Banach fixed point theorem, and two classes of activation functions, some criteria of uniform stability for the solution and existence and uniqueness for equilibrium solution are derived. Finally, three experimental simulations are presented to illustrate the correctness and effectiveness of the obtained results.

Global Mittag-Leffler Stability of the Delayed Fractional-Coupled Reaction-Diffusion System on Networks Without Strong Connectedness.

In this article, we mainly consider the existence of solutions and global Mittag-Leffler stability of delayed fractional-order coupled reaction-diffusion neural networks without strong connectedness. Using the Leary-Schauder's fixed point theorem and the Lyapunov method, some criteria for the existence of solutions and global Mittag-Leffler stability are given. Finally, the correctness of the theory is verified by a numerical example.

Preferential binding of DAP-PGs by major peptidoglycan recognition proteins found in cell-free hemolymph of Manduca sexta.

Peptidoglycan recognition proteins (PGRPs) detect invading bacteria to trigger or modulate immune responses in insects. While these roles are established in Drosophila, functional studies are not yet achieved at the PGRP family level in other insects. To attain this goal, we selected Manduca sexta PGRP12 and five of the nine secreted PGRPs for recombinant expression and biochemical characterization. We cloned PGRP2-5, 12 and 13 cDNAs, produced the proteins in full (PGRP2-5, 13) or in part (PGRP3s, 12e, 13N, 13C) in Sf9 cells, and tested their bindings of two muramyl pentapeptides by surface plasmon resonance, two soluble peptidoglycans by competitive ELISA, and four insoluble peptidoglycans and eight whole bacteria by a pull-down assay. Preferential binding of meso-diaminopimelic acid-peptidoglycans (DAP-PGs) was observed in all the proteins containing a peptidoglycan binding domain and, since PGRP6, 7 and 9 proteins were hardly detected in cell-free hemolymph, the reportoire of PGRPs (including PGRP1 published previously) in M. sexta hemolymph is likely adapted to mainly detect Gram-negative bacteria and certain Gram-positive bacteria with DAP-PGs located on their surface. After incubation with plasma from naïve larvae, PGRP2, 3f, 4, 5, 13f and 13N considerably stimulated prophenoloxidase activation in the absence of a bacterial elicitor. PGRP3s and 12e had much smaller effects. Inclusion of the full-length PGRPs and their regions in the plasma also led to proHP8 activation, supporting their connections to the Toll pathway, since HP8 is a Spӓtzle-1 processing enzyme in M. sexta. Together, these findings raised concerns on the common belief that the Toll-pathway is specific for Gram-positive bacteria in insects.