Functional expression of recombinant hybrid enzymes composed of bacterial and insect's chitinase domains in E. coli.

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect's domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one's leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other's chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi's LCL connected with BtChi's ChBD (MbLCL-BtChBD) and BtChi's LCL connected with MbChi's ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.

A Short Peptide Designed from Late Embryogenesis Abundant Protein Enhances Acid Tolerance in Escherichia coli.

Unsuitable pH is a major limiting factor for all organisms, and a low pH can lead to organism death. Late embryogenesis abundant (LEA) peptides confer tolerance to abiotic stresses including salinity, drought, high and low temperature, and ultraviolet radiation same as the LEA proteins from which they originate. In this study, LEA peptides derived from group 3 LEA proteins of Polypedilum vanderplanki were used to enhance low pH tolerance. Recombinant Escherichia coli BL21 (DE3) cells expressing the five designed LEA peptides were grown at pH 4, 3, and 2. The transformants showed higher growth capacity at low pH as compared to control cells. These results indicate that LEA peptide could prevent E. coli cell death under low pH conditions.

Recombinant yellow protein of the takeout family and albino-related takeout protein specifically bind to lutein in the desert locust.

Yellow protein of the takeout family (YPT) and albino-related takeout protein (ALTO) are involved in body-color polyphenism in Schistocerca gregaria. YPT has been proposed to bind to ß-carotene, whereas the physiological role of ALTO is unclear. Structurally, takeout proteins contain a long continuous tunnel to bind specific ligands. However, the specific ligands of YPT and ALTO have not been fully elucidated. Here, we isolated the full coding cDNAs of these proteins and successfully produced recombinant YPT and ALTO using an Escherichia coli expression system. Absorption spectral analyses of YPT with and without carotenoids revealed that this protein bound to lutein. In contrast, obvious binding of YPT to ß-carotene and astaxanthin was not detected. Similar results were obtained for ALTO. The presence of juvenile hormone only weakly affected the protein/carotenoid interactions. These results suggested that YPT and ALTO specifically bound to lutein in a juvenile hormone-independent manner.

Insights into the impact of Ivermectin on some protein aspects linked to Culex pipiens digestion and immunity.

In developing countries, low-cost control and treatment programs that offer combined approaches against diseases and their vectors are certainly needed. Ivermectin (IVM) has been well known for its role in the treatment of parasitic diseases, due to its effect on glutamate-gated chloride channels. These same channels are also present in the mosquito vector, and thus, research has focused on the insecticidal effects of this drug. Possible alternative mechanisms of IVM on the physiology of mosquitoes, however, have not been sufficiently elaborated. We assessed the protease activity, lipid peroxidation, and local expression of STAT, p53, caspase-3, and Bax markers to study the effect of this antibiotic on digestion and immunity in Culex pipiens. Sugar- and blood-feeding assays were employed to investigate the potential influence of blood feeding on the dynamics of these parameters. IVM was found to have an effect on protease activity, lipid peroxidation as well as the expression of different markers investigated in this work. The focus on the detailed effect of this drug certainly opens the gate to broadening the spectrum of IVM and expanding its health and economic benefit, especially that it is relatively more affordable than other antibiotics on the market.

Hemimetabolous insects elucidate the origin of sexual development via alternative splicing.

Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer, which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx).

Redox-Responsive Resilin-Like Hydrogels for Tissue Engineering and Drug Delivery Applications.

Resilin, a protein found in insect cuticles, is renowned for its outstanding elastomeric properties. The authors' laboratory previously developed a recombinant protein, which consisted of consensus resilin-like repeats from Anopheles gambiae, and demonstrated its potential in cartilage and vascular engineering. To broaden the versatility of the resilin-like protein, this study utilizes a cleavable crosslinker, which contains a disulfide bond, to develop smart resilin-like hydrogels that are redox-responsive. The hydrogels exhibit a porous structure and a stable storage modulus (G') of ≈3 kPa. NIH/3T3 fibroblasts cultured on hydrogels for 24 h have a high viability (>95%). In addition, the redox-responsive hydrogels show significant degradation in a reducing environment (10 mm glutathione (GSH)). The release profiles of fluorescently labeled dextrans encapsulated within the hydrogels are assessed in vitro. For dextran that is estimated to be larger than the mesh size of the gel, faster release is observed in the presence of reducing agents due to degradation of the hydrogel networks. These studies thus demonstrate the potential of using these smart hydrogels in a variety of applications ranging from scaffolds for tissue engineering to drug delivery systems that target the intracellular reductive environments of tumors.

Transcriptional Responses of the Trichoplusia ni Midgut to Oral Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus.

Baculoviruses are large double-stranded DNA viruses that are virulent pathogens of certain insect species. In a natural host, Trichoplusia ni, infection by the model baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) begins when the occluded form of the virus disassembles in the midgut and virions infect midgut epithelial cells to establish the primary phase of the infection. To better understand the primary phase of the AcMNPV infection cycle, newly molted 5th-instar T. ni larvae were orally infected with AcMNPV occlusion bodies and the transcriptional responses of the T. ni midgut were analyzed at various times from 0 to 72 h postinfection, using transcriptome sequencing analysis and a T. ni reference genome. The numbers of differentially expressed host genes increased as the infection progressed, and we identified a total of 3,372 differentially expressed T. ni transcripts in the AcMNPV-infected midgut. Genes encoding orthologs of HMG176, atlastin, and CPH43 were among the most dramatically upregulated in response to AcMNPV infection. A number of cytochrome P450 genes were downregulated in response to infection. We also identified the effects of AcMNPV infection on a large variety of genes associated with innate immunity. This analysis provides an abundance of new and detailed information on host responses to baculovirus infection during the primary phase of the infection in the midgut and will be important for understanding how baculoviruses establish productive infections in the organism.IMPORTANCE Baculoviruses are virulent pathogens of a number of important insect pest species. In the host Trichoplusia ni, infection begins in the midgut when infectious virions of the occlusion-derived virus (ODV) phenotype enter and subsequently replicate in cells of the midgut epithelium. A second virion phenotype (budded virus [BV]) is produced there, and BV mediates systemic infection of the animal. Most prior detailed studies of baculovirus infections have focused on BV infections of cultured cells. In this study, we examined the transcriptional responses of the T. ni midgut to infection by ODV of the baculovirus AcMNPV and identified a variety of host genes that respond dramatically to viral infection. Understanding the transcriptional responses of the host midgut to viral infection is critically important for understanding the biphasic infection in the animal as a whole.

Juvenile hormone promotes locust fat body cell polyploidization and vitellogenesis by activating the transcription of Cdk6 and E2f1.

Juvenile hormone (JH) is known to promote cell polyploidization for insect vitellogenesis and egg production, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that the expression of cyclin-dependent kinase 6 (Cdk6) and adenovirus E2 factor-1 (E2f1), the core mediators in cell cycle progression is regulated by JH and its receptor Methoprene-tolerant (Met). JH acts through its receptor complex comprised of Met and Taiman to directly activate the transcription of Cdk6 and E2f1. Depletion of Cdk6 or E2f1 results in significantly decreased ploidy, precocious mitotic entry and increased cell numbers in the fat body, accompanied by substantial reduction of Vitellogenin gene expression, blocked ovarian growth and arrested oocyte maturation. These findings indicate a crucial role of Cdk6 and E2f1 in JH-regulated polyploidization and vitellogenesis as well as a novel regulatory machinery for endocycling in insects.

Selection and evaluation of reference genes for expression analysis using quantitative real-time PCR in the Asian Ladybird Harmonia axyridis (Coleoptera: Coccinellidae).

Harmonia axyridis (Coleoptera: Coccinellidae) is a polyphagous insect that is an important biological agent used to control agricultural and forestry pests. The role of functional genes in H. axyridis based on quantitative real-time PCR (qRT-PCR) is increasingly well understood to investigate biology, physiology, feeding behavior and the role of important genes in physiological processes. Quantitative real-time PCR (qRT-PCR) is a powerful and reliable technique to quantify gene expression. Using qRT-PCR, expression levels of target genes are determined based on the levels of internal reference genes; therefore, reference genes need to be stably expressed under specific experimental conditions. However, there have been no studies on the stability of reference genes used in H. axyridis. In this study, we systematically investigated expression profiles of nine candidate reference genes from H. axyridis, including ß-actin (ACTIN); elongation factor 1 α (EF1A); ribosomal proteins L10, L18, L28, S13, and S15 (RPL10, RPL18, RPL28, RPS13 and RPS15); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); and superoxide dismutase (SOD). Four analytical methods (geNorm, BestKeeper, NormFinder, and the ΔCt method) were used to evaluate the suitability of these genes as internal reference genes for three biotic factors (developmental stage, tissue, and sex) and two abiotic treatments (temperature and photoperiod). RefFinder, a comprehensive evaluation platform integrating the four analytical methods, was used to rank the overall stability of these reference genes. Among the nine candidate genes, different reference genes were identified as having the most stable expression across biotic and abiotic factors. Genes encoding ribosomal proteins typically had the most stable expression, though EF1A was the most stable across developmental stages and photoperiods. To validate the suitability of these reference genes, heat shock protein 90 (HSP90) was chosen as a target. Significant up-regulation in HSP90 expression level in response to both low and high temperature was observed when using the most suitable reference genes but not when using an arbitrarily selected reference gene. The reference genes identified in this study will provide the basis for future functional genomics research in H. axyridis and will also facilitate the establishment of a standardized qRT-PCR program for other related insects.

A receptor-neuron correlate for the detection of attractive plant volatiles in Helicoverpa assulta (Lepidoptera: Noctuidae).

Plant volatiles are vital cues in the location of hosts for feeding and oviposition for Lepidoptera moths. The noctuid Helicoverpa assulta is a typical polyphagous moth, regarded as a good model for studying the olfactory reception of plant volatiles. In this study, four full-length genes encoding odorant receptors HassOR24, HassOR40, HassOR41, and HassOR55 expressed in antenna in H. assulta were functionally characterized. The highly expressed HassOR40 was narrowly tuned to a few structurally-related plant volatiles: geranyl acetate, geraniol and nerolidol. By systematically analyzing responses of single neuron in both trichoid sensilla and basiconic sensilla using single sensillum recording, the specific neuron B in one type of short trichoid sensilla was found to be mainly activated by the same chemicals as HassOR40 with high sensitivity, and with no significant difference between male and female neurons. Thus, a clear "receptor-neuron" relationship in H. assulta was demonstrated here, suggesting that HassOR40/HassOrco are expressed in neuron B of short trichoid sensilla. The active tobacco volatile nerolidol, recognized by this receptor-neuron line, elicits significant behavioral attraction of both sexes in H. assulta adults. The results indicate that we identified a receptor-neuron route for the peripheral coding of a behaviorally relevant host volatile in H. assulta.

Identification of a transcription factor that functions downstream of corazonin in the control of desert locust gregarious body coloration.

Corazonin (Crz) is a neuropeptide that controls phase-dependent body color polyphenism in locusts. The Crz signaling pathway is responsible for the development of gregarious black patterns in nymphs and determination of the morphometric ratio F/C (F = hind femur length, C = maximum head width) in adults. However, little information is available regarding the mediator and effector proteins regulated by Crz. In this study, we identified a novel transcription factor, Loct, which functions downstream of Crz in Schistocerca gregaria and Locusta migratoria. In S. gregaria, we detected a variant of Loct lacking the N-terminal region. Protein-protein interaction assays showed that both the long and short Loct variants formed a complex with themselves. LOCT knockdown in gregarious nymphs reduced the intensity of their black patterning, but did not affect F/C ratios in adults. LOCT was exclusively expressed in the integument of gregarious nymphs, suggesting that Loct is involved in melanin production. In addition, we found that the melanization-associated protein Yellow (YEL) and the albino-related takeout protein (ALTO) are expressed in the integument and function downstream of Crz. However, Crz injection failed to influence LOCT, YEL, and ALTO expression. Therefore, additional factors probably cooperate with Crz to induce these genes. The gene expression profiles of YEL and ALTO in LOCT-knockdown nymphs suggest that Loct does not directly control the transcription of YEL or ALTO. In summary, we present a working model of the Crz pathway, which is active in crowded S. gregaria nymphs.

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70-80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

Ecdysis-related pleiotropic neuropeptides expression during Anopheles albimanus development / Expresión de neuropéptidos pleiotrópicos asociados con ecdisis durante el desarrollo del mosquito Anopheles albimanus

Abstract: Objective: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. Materials and methods: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. Results: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. Conclusion: The CCAP, corazonin and ETH neuropeptides are potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Resumen: Objetivo: Describir la expresión de neuropéptidos durante la ontogenia del mosquito vector de la malaria Anopheles albimanus. Material y métodos: Se midió la expresión de CCAP, corazonina, ETH, allatostatina, orcokinina, ILP2, ILP5 y bursicon en larvas de primer (2mm), segundo (4mm), tercer (5mm) y cuarto (6mm) estadio, pupas y mosquitos adultos, mediante qPCR. Resultados. A diferencia de otros insectos en donde, CCAP, corazonina y ETH se expresan principalmente en estadios pupales, en An. albimanus se expresaron mayoritariamente en larvas de cuarto estadio, CCAP tuvo 70.8% de expresión relativa, corazonina 76.5% y ETH 60.2%. ILP2 fue el neuropéptido que más se expresó en el primer, segundo y tercer estadio y orcokinina disminuyó durante el desarrollo del mosquito. Conclusión. Los péptidos estudiados se expresaron en todos los estadios de desarrollo del mosquito. Sin embargo, su expresión varió en cada uno de ellos. Los neuropéptidos CCAP, corazonina y ETH, que son esenciales para la transformación de lavas a pupas, pueden ser blancos potenciales para el diseño de estrategias de control dirigidas a interrumpir el desarrollo larvario de An. albimanus.

Double-Stranded RNA-Mediated Suppression of Trypsin-Like Serine Protease (t-SP) Triggers Over-Expression of Another t-SP Isoform in Helicoverpa armigera.

High diversity of digestive proteases is considered to be the key factor in the evolution of polyphagy in Helicoverpa armigera. Serine proteases (SPs) contribute ~85% of the dietary protein digestion in H. armigera. We investigated the dynamics of SP regulation in the polyphagous pest, H. armigera using RNA interference (RNAi). HaTry1, an isoform of SP, expressed irrespective of the composition of the diet, and its expression levels were directly proportional to the larval growth rate. Therefore, HaTry1 was silenced by delivering 10 and 20 µg concentrations of double-stranded RNA through semi-synthetic diet. This led to a drastic reduction in the target gene transcript levels that manifested in a significant reduction in the larval weight initially, but the larvae recovered in later stages despite continuous dsRNA treatment. This was probably due to the compensatory effect by over-expression of HaTry13 (31-folds), another isoform of SP. Phylogenetic analysis of H. armigera SPs revealed that the over-expressed isoform was closely related to the target gene as compared to the other tested isoforms. Further, silencing of both the isoforms (HaTry1 and HaTry13) caused the highest reduction in the larval weight and there was no larval growth recovery. These findings provide a new evidence of the existence of compensatory effect to overcome the effect of silencing individual gene with RNAi. Hence, the study emphasizes the need for simultaneous silencing of multiple isoforms.

Construction and characterization of mutated LEA peptides in Escherichia coli to develop an efficient protein expression system.

To develop an efficient protein expression system, we designed a late embryogenesis abundant (LEA) peptide by mutating the LEA peptide constructed in our previous study (LEA-I). The peptide is based on the repeating units of an 11mer motif characteristic of LEA proteins from Polypedilum vanderplanki larvae. In the amino acid sequence of the 13mer LEA peptide, glycine at the 6th and 12th positions was replaced with other amino acids via point mutations. Glutamic acid, lysine, leucine, and asparagine in the LEA peptide at the 6th and 12th positions increased green fluorescence protein (GFP) expression. The GFP expression of the mutated LEA peptide was 1.5 to 2.0 times higher than that without LEA peptide. In contrast, the serine-containing mutated LEA peptide has low GFP expression levels. We hypothesize that the position of amino acids and the nature of amino acid in LEA peptide are important for our coexpression system. These data suggest that the size, structure, and charge of amino acids in the LEA peptide improve the protection and expression of the target protein. The amino acid balance also plays an important role in the expression of the target protein.

Determination of the protein expression profiles of Propsilocerus akamusi (Tokunaga) Malpighian tubules response to cadmium stress by iTRAQ coupled LC-MS/MS.

Propsilocerus akamusi (Tokunaga) is an important prey species for fish and aquatic birds, which represent an important link in the aquatic food chain as other species of Chironomidae. Malpighian tubules (MT) play an important role in metabolism and detoxification of harmful compounds and metal accumulation. Our previous studies have reported that P. akamusi have an extraordinary resistance to Cd. In this paper, iTRAQ-based proteomics technology was first used to study the differential proteomics of MT of Propsilocerus akamusi under sublethal Cd stress for different time courses. 118 differential expressed proteins (DEPs) were identified through LC-MS/MS and further classified into 26 GO functional groups. Our results showed that metabolic process was the main biological functional categories. Cluster of orthologous groups of proteins (COG) analysis was also performed based on the number of unique proteins identified in each functional category, 21 metabolism-related DEPs were identified. P. akamusi adapted to Cd stress mainly by inducing the processes of lipid metabolism of MT and lipid may play a possible protection role in cadmium resistance in MT. The results provide important information for research on molecular mechanisms of Cd-stress adaption in Malpighian tubules of P. akamusi. BIOLOGICAL SIGNIFICANCE: Propsilocerus akamusi (old species name: Tokunagayusurika akamusi) is an important prey species for fish and aquatic birds, which represent an important link in the aquatic food web as other species of Chironomidae. Malpighian tubules constitute a versatile organ which is able to exert excretory of chemical compounds, acting in the insect metabolism and detoxification of harmful compounds and metal accumulation. Our previous studies have reported that P. akamusi have an extraordinary resistance to Cd. However, up to now, there has been a lack of research on the molecular mechanisms of adaption to Cd stress in the Malpighian tubules of this species. Study on the adaptive mechanisms to Cd stress in Malpighian tubules of Propsilocerus akamusi through comparative proteome analysis is one important way to solve this problem. The aim of the present study was to understand the molecular mechanisms of adaption of the Malpighian tubules of P. akamusi exposed to Cd stress and assess the high expression of proteins involved in cellular adaptation to Cd stress. This will provide information about how this organ is responding to disturbances of contaminated ecosystems.

Micellar Self-Assembly of Recombinant Resilin-/Elastin-Like Block Copolypeptides.

Reported here is the synthesis of perfectly sequence defined, monodisperse diblock copolypeptides of hydrophilic elastin-like and hydrophobic resilin-like polypeptide blocks and characterization of their self-assembly as a function of structural parameters by light scattering, cryo-TEM, and small-angle neutron scattering. A subset of these diblock copolypeptides exhibit lower critical solution temperature and upper critical solution temperature phase behavior and self-assemble into spherical or cylindrical micelles. Their morphologies are dictated by their chain length, degree of hydrophilicity, and hydrophilic weight fraction of the ELP block. We find that (1) independent of the length of the corona-forming ELP block there is a minimum threshold in the length of the RLP block below which self-assembly does not occur, but that once that threshold is crossed, (2) the RLP block length is a unique molecular parameter to independently tune self-assembly and (3) increasing the hydrophobicity of the corona-forming ELP drives a transition from spherical to cylindrical morphology. Unlike the self-assembly of purely ELP-based block copolymers, the self-assembly of RLP-ELPs can be understood by simple principles of polymer physics relating hydrophilic weight fraction and polymer-polymer and polymer-solvent interactions to micellar morphology, which is important as it provides a route for the de novo design of desired nanoscale morphologies from first principles.

Elastic and pH-Responsive Hybrid Interfaces Created with Engineered Resilin and Nanocellulose.

We investigated how a genetically engineered resilin fusion protein modifies cellulose surfaces. We characterized the pH-responsive behavior of a resilin-like polypeptide (RLP) having terminal cellulose binding modules (CBM) and showed its binding to cellulose nanofibrils (CNF). Characterization of the resilin fusion protein at different pHs revealed substantial conformational changes of the protein, which were observed as swelling and contraction of the protein layer bound to the nanocellulose surface. In addition, we showed that employment of the modified resilin in cellulose hydrogel and nanopaper increased their modulus of stiffness through a cross-linking effect.

Expression of the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana.

The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between the insect hosts and their natural pathogens. In this report, we analyze the expression of a gene encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We have used a natural infection, i.e. covering larval integument with fungal aerospores, as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, whose expression reflects the stimulation of Galleria mellonella defense mechanisms. Also, gene expression is analyzed in the light of survival of animals after spore injection.

Synthesis and biological activity of FGLamide allatostatin analogs with Phe(3) residue modifications.

A FGLamide allatostatin neuropeptide mimic (H17) is a potential insect growth regulator which inhibits the production of juvenile hormone by the corpora allata. To find more evidence to reveal the structure-activity relationships of the Phe(3) residue in the C-terminal conserved pentapeptide and search for novel analogs with high activity, a series of Phe(3) residue-modified analogs were designed and synthesized using H17 as the lead compound. Bioassay using juvenile hormone (JH) production by corpora allata of the cockroach Diploptera punctata indicated that analogs 4, 11, and 13 showed strong ability to inhibit JH production in vitro, with IC50 of 38.5, 22.5, and 26 nM, respectively. As well, the activity of analog 2 (IC50 : 89.5 nM) proved roughly equivalent to that of H17. Based on the primary structure-activity relationships of Phe(3) residue, we suggest that for analogs containing six-membered aromatic rings, removing the methylene group of Phe(3) or an o-halogen or p-halogen-substituted benzene ring could increase the ability to inhibit biosynthesis of JH. This study will be useful for the design of new allatostatin analogs for insect management. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.