Insect Hemolymph Research Articles

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcβ1–4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed β-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer—the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

Discovery of a new intravacuolar protein required for the autophagy, development and virulence of Beauveria bassiana.

High proportions of hypothetical proteins exist in genomic databases of fungi, including putative secretory proteins (PSPs) likely involved in fungal invasion and virulence. Here we characterize one of many PSPs revealed in the previous transcriptome of Beauveria bassiana (a fungal insect pathogen) infecting a global lepidopteran pest and name it vacuole-localized protein 4 (VLP4) because this small, domain-lacking protein (22.96 kDa) was specifically localized in the vacuoles of hyphal cells. Deletion of VLP4 resulted in repression of almost all genes acting in autophagy and central development pathways. Consequently, the deletion mutant formed no autophagosome in hyphal vacuoles and displayed severe defects in aerial conidiation. conidial hydrophobicity to the insect surface, and secretion of cuticle-degrading Pr1 proteases required for normal cuticle infection. Blastospore formation was inhibited in the submerged mutant culture mimic to insect haemolymph, and formation of hyphal bodies in vivo was delayed. The fungal virulence was attenuated in the absence of VLP4. These phenotypic defects were well restored by targeted gene complementation. Our findings unveil a vital role of VLP4 in B. bassiana and call attention to many more PSPs for new insights into the interactions of fungal insect pathogens with insects.

English

Trehalose-6-phosphate phosphatase (TPP) is one of the primary enzymes involved in the synthesis of trehalose, the main sugar found in insect hemolymph. In the present study, we report for the first time heterologous expression in Pichia pastoris, characterization and homology modeling of TPP from Anopheles gambiae mosquito. Purified TPP recombinant exhibited a molecular weight of approximately 36 kDa with optimum pH of 8.0, optimum temperature of 38°C, and the KM was 3.19 ± 0.10 mM. Inhibition tests revealed that CaCl2 and Ca(NO3)2 at 5 and 25 mM, respectively, were effective inhibitors of TPP activity. Homology studies and molecular modeling indicated high similarity of the TPP tridimensional structure with TPP enzymes deposited at a data bank and these homology studies confirmed that it is a trehalose phosphatase with conserved motifs of the superfamily haloacid dehydrogenase. These results may provide support for the discovering of TPP inhibitors to be used for development of insecticides to control mosquito vectors. Key words: Trehalose, trehalose-6-phosphate phosphatase, expression, enzyme characterization, homology modeling.

Fruit fly impact on an aerodynamic surface: Types of outcomes and residue components

Leading edge contamination on aircraft wings and wind turbine blades can occur through the accumulation of insect residues, which can then increase drag by causing an earlier transition of the boundary layer from laminar to turbulence. To investigate the mechanisms of residue adherence and composition, this study focuses on the impact dynamics of insects striking an airfoil with a circular leading edge placed in a wind tunnel at a wind speed of about 100 mph. To mimic in-flight conditions, live flightless fruit flies (Drosophila melanogaster) were injected into the wind tunnel sufficiently upstream such that they reached the test section air-flow speed prior to impact on the airfoil. The aerodynamic surface had a leading edge radius of 3.8 cm and was tested with coatings of various surface wettability, ranging from hydrophilic to superhydrophobic. Based on previous studies and present high-speed imaging and surface imaging, five types of insect outcomes were defined (roughly in order of increasing impact speed): 1) insect bounce without rupture and no residue, 2) insect burst with release of internal fluid with adherence of full exoskeleton, 3) insect burst with release of internal fluid with little or no exoskeleton adherence, 4) disintegration of insect yielding residue of many pieces of exoskeleton, and 5) insect burst with neither fluid nor exoskeleton residue. The fifth outcome generally occurred for a superhydrophobic surface developed in this study. When insect residue did occur on a surface, it included up to three distinct components (in order of decreasing residue heights): exoskeleton pieces, yellow hemolymph from the insect abdomen and thorax, and red fluid from the insect head. Surfaces with lower surface wettability generally yielded reduced insect hemolymph residue area per insect release. In particular, a superhydrophobic coating yielded the lowest residue area and no exoskeleton residue, while the aluminum surface resulted in the highest residue area and several exoskeletons.

Vital role for cyclophilin B (CypB) in asexual development, dimorphic transition and virulence of Beauveria bassiana

Cyclophilin B (CypB) was previously revealed as one of many putative secretory proteins in the transcriptome of Beauveria bassiana infection to a lepidopteran pest. Here we show a main localization of CypB in hyphal cell walls and septa and its essential role in the in vitro and in vivo asexual cycles of the fungal insect pathogen. Deletion of cypB reduced colony growth by 16–42% on two rich media and 30 scant media with different carbon or nitrogen sources. The deletion mutant suffered a delayed conidiation on a standard medium and a final 47% reduction in conidial yield, accompanied with drastic transcript depression of several key genes required for conidiation and conidial maturation. The mutant conidia required 10h longer to germinate 50% at optimal 25°C than wild-type conidia. Intriguingly, cultivation of the mutant conidia in a trehalose-peptone broth mimic to insect hemolymph resulted in 83% reduction in blastospore yield but only slight decrease in biomass level, indicating severe defects in transition of hyphae to blastospores. LT50 for the deletion mutant against Galleria mellonella larvae through normal cuticle infection was prolonged to 7.4d from a wild-type estimate of 4.7d. During colony growth, additionally, the deletion mutant displayed hypersensitivity to Congo red, menadione, H2O2 and heat shock but increased tolerance to cyclosporine A and rapamycin. All of changes were restored by targeted gene complementation. Altogether, CypB takes part in sustaining normal growth, aerial conidiation, conidial germination, dimorphic transition, stress tolerance and pathogenicity in B. bassiana.

Changes in light and dark periods affect the arylalkylamine N-acetyl transferase, melatonin activities and redox status in the head and hemolymph of nocturnal insect Spodoptera litura

The present study was conducted to describe the impact of circadian rhythm on melatonin levels and redox statusunder three photoperiods (12L:12D, 0L:24D, and 24L:0D) in head and hemolymph of Spodoptera litura. Melatonin is an powerful antioxidant and controls the reproduction of organisms. In this study, melatonin levels, Arylalkylamine N-acetyltransferase(AA-NAT), and antioxidant enzyme activities were analyzed. Results showed melatonin, AA-NAT levels in hemolymph were significantly (p < 0.05) higher during the dark period than during LL regime. HPLC chromatogram of the insect head and hemolymph showed 5 peaks while hemolymph showed 6 peaks in LD, and LLregimes. The day–night changes of melatonin increased the antioxidant enzymes (GST, CAT, POX) persisted in the insect hemolymph, but were suppressed by constant light. The present study leads us to speculate that synthesis and release of melatonin in the S.litura head occur as circadian rhythm and light has an inhibitory effect on melatonin synthesis.

Adaptation to dietary conditions by trehalose metabolism in Drosophila

Trehalose is a non-reducing disaccharide that serves as the main sugar component of haemolymph in insects. Trehalose hydrolysis enzyme, called trehalase, is highly conserved from bacteria to humans. However, our understanding of the physiological role of trehalase remains incomplete. Here, we analyze the phenotypes of several Trehalase (Treh) loss-of-function alleles in a comparative manner in Drosophila. The previously reported mutant phenotype of Treh affecting neuroepithelial stem cell maintenance and differentiation in the optic lobe is caused by second-site alleles in addition to Treh. We further report that the survival rate of Treh null mutants is significantly influenced by dietary conditions. Treh mutant larvae are lethal not only on a low-sugar diet but also under low-protein diet conditions. A reduction in adaptation ability under poor food conditions in Treh mutants is mainly caused by the overaccumulation of trehalose rather than the loss of Treh, because the additional loss of Tps1 mitigates the lethal effect of Treh mutants. These results demonstrate that proper trehalose metabolism plays a critical role in adaptation under various environmental conditions.

Contributions of β-tubulin to cellular morphology, sporulation and virulence in the insect-fungal pathogen, Metarhizium acridum

β-tubulin is an elementary subunit of microtubules that form the cytoskeleton, participating in a wide range of cellular processes. The contributions of the single β-tubulin gene in affecting cell morphology, sporulation and virulence were examined in the entomopathogenic fungus Metarhizium acridum. Targeted gene knockout of β-tubulin resulted in resistance to benomyl but impaired proper nuclear segregation, lipid droplet transport, and deposition of chitin to the cell wall. M. acridum β-tubulin mutants displayed wavy hyphal growth and densely packed, wrinkled colonies. Decreases in the rate of phialides formation and conidial yield were observed for the β-tubulin mutant, which was also impaired in virulence towards locust hosts as compared to wild type and complemented strains. Morphological analyses of infection structures revealed development of bifurcated germ tubes, with reduced appressoria formation seen in the β-tubulin mutant. M. acridum β-tubulin mutant appressoria were aberrant in morphology and displayed decreased turgor pressure. The ability of the M. acridum β-tubulin mutant to proliferate in the insect hemolymph both in vitro and in vivo was also significantly reduced. Our results indicate that in M. acridum, β-tubulin is not essential for survival but that it contributes to cellular transport of organelles and cell wall materials, impacting growth, appressorial differentiation, virulence, and sporulation.

Formation and function of intracardiac valve cells in the Drosophila heart.

Drosophila harbours a simple tubular heart that ensures haemolymph circulation within the body. The heart is built by a few different cell types, including cardiomyocytes that define the luminal heart channel and ostia cells that constitute openings in the heart wall allowing haemolymph to enter the heart chamber. Regulation of flow directionality within a tube, such as blood flow in arteries or insect haemolymph within the heart lumen, requires a dedicated gate, valve or flap-like structure that prevents backflow of fluids. In the Drosophila heart, intracardiac valves provide this directionality of haemolymph streaming, with one valve being present in larvae and three valves in the adult fly. Each valve is built by two specialised cardiomyocytes that exhibit a unique histology. We found that the capacity to open and close the heart lumen relies on a unique myofibrillar setting as well as on the presence of large membranous vesicles. These vesicles are of endocytic origin and probably represent unique organelles of valve cells. Moreover, we characterised the working mode of the cells in real time. Valve cells exhibit a highly flexible shape and, during each heartbeat, oscillating shape changes result in closing and opening of the heart channel. Finally, we identified a set of novel valve cell markers useful for future in-depth analyses of cell differentiation in wild-type and mutant animals.

Developmental changes in the levels and redox potentials of main hemolymph thiols/disulfides in the Jamaican field cricket Gryllus assimilis.

Main thiols and disulfides were determined in the hemolymph of the Jamaican field cricket Gryllus assimilis at various developmental stages. On the basis of these data, redox potentials of the glutathione, cysteine and homocysteine redox systems were calculated. The concentrations of all thiols studied decreased during development (at a stage of 6 molts) with respect to young crickets, and increased again in adult insects. Redox potentials of the glutathione and cysteine systems increased from values of -131.0±5.6 mV and -86.9±17.1 mV, respectively in young crickets to -58.0±3.6 mV and -36.1±4.2 mV, respectively, at the stage of 6 molts and decreased to values of -110.4±24.8 mV and -66.3±12.2 mV, respectively, in adult insects. Redox potentials of the glutathione and cysteine systems in the hemolymph of young and adult insects were similar to those reported for human plasma.

The process of lipid storage in insect oocytes: The involvement of β-chain of ATP synthase in lipophorin-mediated lipid transfer in the chagas’ disease vector Panstrongylus megistus (Hemiptera: Reduviidae)

Lipophorin is the main lipoprotein in the hemolymph of insects. During vitellogenesis, lipophorin delivers its hydrophobic cargo to developing oocytes by its binding to non-endocytic receptors at the plasma membrane of the cells. In some species however, lipophorin may also be internalized to some extent, thus maximizing the storage of lipid resources in growing oocytes. The ectopic β chain of ATP synthase (β-ATPase) was recently described as a putative non-endocytic lipophorin receptor in the anterior midgut of the hematophagous insect Panstrongylus megistus. In the present work, females of this species at the vitellogenic stage of the reproductive cycle were employed to investigate the role of β-ATPase in the transfer of lipids to the ovarian tissue. Subcellular fractionation and western blot revealed the presence of β-ATPase in the microsomal membranes of the ovarian tissue, suggesting its localization in the plasma membrane. Immunofluorescence assays showed partial co-localization of β-ATPase and lipophorin in the membrane of oocytes as well as in the basal domain of the follicular epithelial cells. Ligand blotting and co-immunoprecipitation approaches confirmed the interaction between lipophorin and β-ATPase. In vivo experiments with an anti-β-ATPase antibody injected to block such an interaction demonstrated that the antibody significantly impaired the transfer of fatty acids from lipophorin to the oocyte. However, the endocytic pathway of lipophorin was not affected. On the other hand, partial inhibition of ATP synthase activity did not modify the transfer of lipids from lipophorin to oocytes. When the assays were performed at 4°C to diminish endocytosis, the results showed that the antibody interfered with lipophorin binding to the oocyte plasma membrane as well as with the transfer of fatty acids from the lipoprotein to the oocyte. The findings strongly support that β-ATPase plays a role as a docking lipophorin receptor at the ovary of P. megistus, similarly to its function in the midgut of such a vector. In addition, the role of β-ATPase as a docking receptor seems to be independent of the enzymatic ATP synthase activity.

A Mini Review of Functional Proteins in Silkworm &lt;i&gt;Bombyx mori L&lt;/i&gt; Haemolymph

This review focuses on the state of the art on haemolymph proteins in lepidopteron insect silkworm Bombyx mori L. Insects are most successful animals in the world. Their success is mostly attributed to their physiological well being and adaptability to most of the environmental conditions is an important reason to be considered evaluating their success. Proteins in insect especially the ones circulating in the blood are seen to be the major contributors for the insects successful survive, owing to their open type circulatory system the haemolymph in insects soaks all the major organs systems in the body and thus making it an important body fluid for regulation of homeostasis. This calls for an detailed study on proteomics of the haemolymph of Bombyx mori which along with being an economical insect, it is being considered as model organism for most frontier sciences like biomaterials, biomedical applications and pharmacokinetic studies, Research on lepidopteron insects has been instrumental in the discovery and the biochemical understanding of many haemolymph plasma proteins. In the present review we analyzed the haemolymph proteins based on their function and molecular characterization. We also could deduce the role of these proteins in insect physiology by comparative analysis with earlier reported proteins.

Comparative transcriptomics reveal host-specific nucleotide variation in entomophthoralean fungi.

Obligate parasites are under strong selection to increase exploitation of their host to survive while evading detection by host immune defences. This has often led to elaborate pathogen adaptations and extreme host specificity. Specialization on one host, however, often incurs a trade-off influencing the capacity to infect alternate hosts. Here, we investigate host adaptation in two morphologically indistinguishable and closely related obligate specialist insect-pathogenic fungi from the phylum Entomophthoromycota, Entomophthora muscae sensu stricto and E.muscae sensu lato, pathogens of houseflies (Musca domestica) and cabbage flies (Delia radicum), respectively. We compared single nucleotide polymorphisms within and between these two E.muscae species using 12 RNA-seq transcriptomes from five biological samples. All five isolates contained intra-isolate polymorphisms that segregate in 50:50 ratios, indicative of genetic duplication events or functional diploidy. Comparative analysis of dN/dS ratios between the multinucleate E.muscae s.str. and E.muscae s.l. revealed molecular signatures of positive selection in transcripts related to utilization of host lipids and the potential secretion of toxins that interfere with the host immune response. Phylogenetic comparison with the nonobligate generalist insect-pathogenic fungus Conidiobolus coronatus revealed a gene-family expansion of trehalase enzymes in E.muscae. The main sugar in insect haemolymph is trehalose, and efficient sugar utilization was probably important for the evolutionary transition to obligate insect pathogenicity in E.muscae. These results support the hypothesis that genetically based host specialization in specialist pathogens evolves in response to the challenge of using resources and dealing with the immune system of different hosts.

Self-Powered Environmental Monitoring Using Insect-Mountable Biofuel Cell

This paper reports the first demonstration of a self-powered environmental monitoring robot which backpacked a biofuel cell (BFC) and a micro wireless sensor module on an insect. Electric power was generated from blood sugar in its hemolymph by the BFC and temperature and humidity was monitored by the wireless sensor module. Insect cyborgs, which are micro robots controlled by electric stimulation for their brain and muscles, are desired to be utilized as a search robot and an environmental monitoring robot. However, coin batteries which are currently used for power sources of insect cyborgs have issues for practical use of insect cyborgs. They not only interfere with insect’s locomotion because of their size and weight but also they have to be recharged or replaced to drive an integrated circuit for a long time. Our group has already reported a BFC using trehalose found in insect hemolymph and succeeded in driving some IC devices. In this paper, we proposed an insect-mountable BFC (imBFC) and developed the self-powered and autonomous distribute sensor robot by mounting the BFC and the wireless sensor module onto insect. The imBFC consisted of two chambers (top: length, 18 mm; width, 9mm; depth, 0.5 mm, bottom: length, 18 mm; width, 9mm; depth, 1 mm) separated by dialysis membrane (MWCO: 500-1000) and enzymatic electrodes (surface area, 118 mm2). The chambers were fabricated with a 3D printer (RVS-S1, Real Vision Systems Inc.) and a 500 nm thick parylene layer was deposited on the surface of the chambers. Trehalose molecules found in insect hemolymph are curried to the chamber connected to insects by diffusion and the trehalose molecules are decomposed to glucose molecules by trehalase. Then, the glucose molecules are oxidized by glucose dehydrogenase (GDH) immobilized on an anode and oxygen is reduced by bilirubin oxidase (BOD) immobilized on a cathode. As a result of these processes, electricity is generated. A maximum power output of 333 µW (285 µW/cm2) was obtained at 0.5 V in the imBFC. Because electrodes were not implanted in the insect but mounted on the insect, larger electrodes could be used in the imBFC and the maximum power output reached 333 µW. Then, we developed the micro wireless sensor module and demonstrated to monitor environmental conditions by mounting the sensor and the BFC on the insect. The wireless sensor module consisted of a power circuit (ADP5090, Analog Devices Inc.) which boosted the voltage from around 400 mV to 2.5 V, a wireless transmitter (carrier frequency, 315 MHz), a temperature and humidity sensor (SHT20, Sensirion AG) and a microcomputer (PIC16LF1824, Microchip Technology Inc.). The voltage of the imBFC was boosted to 2.5 V by the power circuit and charged to tantalum capacitors (total capacity, 2200 µF). First, the capacitor was charged until the start-up voltage of the wireless sensor module and the microcomputer. Then, the microcomputer and the sensor were driven and temperature and humidity were measured. These processes were repeated until the power output of the imBFC was reduced bellow the energy consumption of the wireless sensor device. The size and weight of the wireless sensor are 10 mm × 10 mm × 8 mm and 1.3 g, respectively. The insect, which backpacked the BFC and the wireless sensor module, walked autonomously and first temperature and humidity data were transmitted about 26 min after the imBFC was connected to the wireless sensor module. Then, the temperature and humidity data were transmitted 3 times for 8 min. We succeeded in the first demonstration of the self-powered environmental monitoring robot which generates electric power from its body fluid and activates the wireless sensor by the electric power. This study investigated the self-powered bio-hybrid robots which generate electric power from their body fluid and monitor the environmental conditions by the wireless sensor backpacked insect. The maximum power output of the imBFC reached 333 µW at 0.5 V. To the best of our knowledge, our imBFC has the highest performance of any living battery. Furthermore, we succeeded in monitoring the environmental conditions by using the insect which was mounted the BFC and the micro wireless sensor module. These results indicate that self-powered sensor robots have potential to be applied an autonomous distributed sensor network for environmental monitoring. Furthermore, the power generation mechanism of the imBFC can be applied to all living organisms because all living organisms utilize sugar as energy for their activity. In future work, the BFC might be utilized as a battery of biomedical devices, sensors and robots implanted into human beings. Figure 1

Method development for the determination of biogenic amines in insect hemolymph by HPLC with electrochemical detection.

An HPLC method was developed for the quantification of biogenic amines in insect hemolymph. The major challenge in the analysis is that a typical sample only contains a few hundred pictograms of the amine. A C18 column and electrochemical detection was used. The eluent was an aqueous 0.06 M phosphate buffer (pH- 6.7) mixed with 10-15% methanol and an ion-pairing reagent. With this method, the amines can be detected at picogram levels. However, there are hundreds of compounds in a typical biological fluid and, in some cases, another electro-active species can co-elute with the compounds of interest. To address this problem we have developed methods to selectively remove some potentially interfering compounds by pretreating the sample by solid phase extraction (SPE). Previously, we had used weak cation SPE columns to simplify the mixture but found that the method was too time consuming. We have now developed a method that uses weak anion SPE columns. The samples were collected in a syringe preloaded with an anticoagulant, either trichloroacetic acid or formic acid. The proteins were removed by centrifuging with a 10K Da filter. The solid phase extraction followed. This pretreatment was found to be effective for selective removal many interfering peaks. DHBA (3,4-Dihydroxybenzylamine hydrobromide) was used as an internal standard.

Regulation of antimicrobial peptide synthesis in larvae of Calliphora vicina (Diptera, Calliphoridae): a dose-dependent effect of ecdysteroids

Ecdysteroids are multifunctional hormones regulating virtually all morphogenetic processes in insects. Their role in stress and immune response regulation is less known. Here we studied 20-hydroxyecdysone effect on synthesis of the antimicrobial peptides in larvae of Calliphora vicina. An inverse correlation was found between 20-hydroxyecdysone titer and the concentration of antimicrobial peptides in the hemolymph of unaffected and bacteria-immunized insects. High and low doses of 20-hydroxyecdysone, injected simultaneously with bacterial cells, had an opposite effect on antimicrobial peptide synthesis in the diapausing larvae. Morphogenetically effective doses of 20-hydroxyecdysone demonstrated immuno-suppressive activity. Low doses of 20-hydroxyecdysone, on the contrary, moderately stimulated synthesis of the antimicrobial peptides. These data suggest that ecdysteroids are directly involved in regulation of the immune system activity and the final effect is dose-dependent.

The Na+ /H+ antiporter Nhx1 controls vacuolar fusion indispensible for life cycles in vitro and in vivo in a fungal insect pathogen.

The sole Na+ /H+ antiporter Nhx1 has been generally unexplored in filamentous fungi. We characterized Nhx1 in the entomopathogenic fungus Beauveria bassiana. An eGFP-tagged Nhx1 fusion accumulated in small punctuate structures, presumably endosomal and trans-Golgi network compartments, between septum and tubular vacuole of each wild-type cell stained with a vacuole-specific dye. Deletion of nhx1 resulted in significant acidification and severe fusion defect in vacuoles, which were fragmented and distinct from large or tubular wild-type vacuoles. The deletion also caused a drastic reduction in aerial conidiation or submerged blastospore production and more severe defect in vegetative growth than in conidial germination. The Δnhx1 mutant became more sensitive to high osmolarity, heat shock and several metal ions during growth but its conidia showed increased UV-B tolerance. Intriguingly, Δnhx1 was unable to infect a model insect through cuticle penetration or intrahaemocoel injection because it produced much less biomass and cuticle-degrading enzymes in a minimal broth and failed to form blastospores in the insect haemolymph. All changes were completely or largely restored by targeted nhx1 complementation. Our results provide novel insight into an indispensability of Nhx1 for not only vacuolar fusion but also life cycles in vitro and in vivo in B. bassiana.

Binding of lysine residues of apolipophorin III to phosphatidylglycerol membranes

Apolipoproteins are the protein components of lipoproteins, which are responsible for transport of lipids to target tissues through the aqueous environment of the body. Apolipophorin III (apoLp‐III) is an abundant apolipoprotein of the hemolymph of insects. The 18 kDa protein is made of a bundle of five amphipathic α‐helices, which are the structural elements responsible for lipid binding. ApoLp‐III facilitates the lipoprotein mediated transport of diacylglycerols, which are released from fat body tissue for transport to the flight muscles during flight. Another novel function performed by apoLp‐III is modulating the cellular and humoral immune response. The protein up‐regulates expression of antimicrobial proteins, neutralizes lipopolysaccharides, and binds and deforms bacterial membranes. Lipopolysaccharides and phosphatidylglycerol (PG) are abundant components of the bacterial membrane, and our studies aim to better understand the antimicrobial properties of apoLp‐III of Locusta migratoria. This eventually may lead to improved strategies to treat bacterial infection and sepsis. ApoLp‐III contains 8 lysine residues, and the objective of this study was to characterize the ionic interactions between lysine residues of apoLp‐III and the negatively charged membrane components of gram‐negative bacteria, lipopolysaccharides and PG. To mimic bacterial membranes, PG vesicles with encapsulated calcein were prepared by extrusion. Protein‐induced membrane perturbation causes release of calcein which fluoresces intensively. When lysine side‐chains were neutralized by acetylation, binding to PG membranes was greatly reduced as indicated by the large reduction in calcein release from 80 ± 3.3% (unmodified apoLp‐III) to 21 ± 0.2% (acetylated apoLp‐III). In contrast, no effect on lipopolysaccharide binding was observed. To identify the lysine residues responsible for PG binding, site‐directed mutagenesis was used to change lysine into glutamine at position 52, 54, 63, 68 and 121. The proteins were expressed in bacteria and purified to homogeneity. The lysine variants were all able to release calcein, with values for most mutants around 70 to 80%. Since this is similar to the wild‐type protein, this indicates that these residues are not critical for PG binding. The remaining lysine residues at positions 143,145 and 161 were also changed into glutamine; the mutant proteins are currently expressed and purified. In addition, studies are underway to specifically acetylate the N‐terminal amino group by lowering the pH since the pKa for α amino groups is lower than the lysine ɛ‐amino group and hence making it a better nucleophile.

Peptidoglycan recognition protein-S5 functions as a negative regulator of the antimicrobial peptide pathway in the silkworm, Bombyx mori

Prophenoloxidase (proPO), immune deficiency (IMD), and Toll are the major signaling pathways leading to melanization and antimicrobial peptide production in insect hemolymph. Peptidoglycan recognition proteins (PGRPs) act as receptors and negative regulators in these pathways, and some PGRPs exhibit antimicrobial activity. Previously, we demonstrated that silkworm PGRP-S5 recognizes peptidoglycans (PGs) and triggers activation of the proPO pathway. It also acts as a bactericide, via its amidase activity (Chen et al., 2014). Here, we generated a C177S site-mutated silkworm PGRP-S5 protein that lacked amidase activity but retained its PG-binding capacity. Functional studies showed that the mutation caused loss of its receptor function for activation of the proPO pathway, suggesting that processing of PG by PGRP-S5 is necessary for formation of the pathway initiation complex. Further, we found that PGRP-S5 negatively regulates antimicrobial peptides generation in an amidase-dependent manner, likely through the IMD pathway. Thus, silkworm PGRP-S5 acts as a sensor, a modulator, and an effector in the silkworm humoral immune system.

Differential Susceptibility to High Temperature and Variation of Seasonal Occurrence between Spodoptera exigua and Plutella xylostella

Climate change has been regarded as one of main factors to change Korean insect pest fauna. Especially, a global warming model predicts to expand habitat for insect pests originated from tropical or subtropical regions. Two insect pests, the beet armyworm (Spodoptera exigua) and the diamondback moth (Plutella xylostella), are known to overwinter in some greenhouse conditions without diapause induction in Korea. There was a clear difference between these two insects in seasonal occurrence. P. xylostella occurred only at early spring and fall seasons, but did not occur during summer. In contrast, S. exigua maintained their occurrence from late spring to fall seasons. This study set up a hypothesis that the difference in the seasonal occurrence may be resulted from variation in susceptibility to high temperature. To test the hypothesis, heat tolerance was compared between these two insects. Exposure to for 40 min killed 100% individuals of P. xylostella larvae. However, most larvae of S. exigua survived in response to even for 80 min. Heat tolerance varied among developmental stages in both insects. Highest tolerant stages were instar larvae and adults for P. xylostella, but instar larvae for S. exigua. Pre-exposure to for 30 min significantly increased heat tolerance in both insects. Induction of heat tolerance accompanied with significant increase of glycerol contents in the hemolymph in both insects and up-regulation of three heat shock protein expressions in S. exigua. These results suggest that the differential susceptibility to high temperature explains the disappearance of P. xylostella during summer, at which S. exigua maintains its occurrence.