Expression of Phenoloxidase System in Use of Chitooligosaccharides as Immunomodulators in Insects

Effects of chitin, chitooligosaccharides, and chitin monomer N-acetyl-D-glucosamine on activities of phenoloxidases has been studied in the honeybee Apis mellifera, Colorado beetle Leptinotansa decemliniata, and housefly Musca domestica, as well as at action of bitoxybacillin. Changes of phenoloxidase activities depending on the developmental stage and age of the insects have been studied. Expression of additional molecular forms of phenoloxidases has been shown in the housefly and Colorado beetle under effects of a bacterial preparation, chitooligosaccharides, and N-acetyl-D-glucosamine.     

Relationship between Acid-catalyzed Cyclization of Citral and Deterioration of Lemon Flavor

Phenoloxidase was purified from pupae of the housefly, Musca domestica L. The purification procedures included ammonium sulfate precipitation, affinity chromatography and Sephadex G-200 gel filtration. The final preparations appear to be homogeneous based on results obtained from polyac-rylamide gel electrophoresis. The molecular weight of phenoloxidase was estimated to be 330,000, as determined by gel filtration. The kinetic properties of phenoloxidase were studied using six catecholamines as substrates. The preferred order of substrates for phenoloxidase was found to be N-β-alanyldopamine > dopamine > TV-acetyldopamine > norepinephrine > epinephrine > DOPA.     

Characterization of a new phenoloxidase inhibitor from the cuticle of Manduca sexta

Melanin, the phenolic biopolymer that serves as a skin- and hair pigment-protecting agent against harmful solar radiation and a free radical trap, is biosynthesized in animals mainly by the action of tyrosinase also known as phenoloxidase. Regulation of tyrosinase and hence melanogenesis is vital for all animals. In this report, we present the isolation and characterization of a new, heat-labile glycoprotein inhibitor of phenoloxidase from the larvae of Manduca sexta. The inhibitor was isolated from the live larval cuticle by buffer extraction and purified to homogeneity employing ammonium sulfate precipitation, dialysis, and concanavalin A-Sepharose chromatography. It migrated with a molecular weight of 380,000 on SDS-PAGE gels and inhibited the activity of insect and plant as well as fungal phenoloxidases. Inhibitor formed a tight complex with phenoloxidases, which resisted dissociation even by 1% Triton X-100 or SDS. Selective inhibition of phenoloxidase, while acting on certain but not all different substrates, was observed. The physiological importance of this newly discovered high-molecular-weight phenoloxidase inhibitor is discussed.     

Regulation of phenoloxidase activity by high- and low-molecular-weight inhibitors from the larval hemolymph of Manduca sexta

Insect phenoloxidases (POs) generate quinones and other reactive intermediates to immobilize and kill invading pathogens and parasites. Due to the presumed cytotoxicity of these compounds, PO activity and its proteolytic activation have to be regulated as a local, transient reaction against nonself in order to minimize damage to the host tissues and cells. We identified a Manduca sexta cDNA encoding a polypeptide sequence with its carboxyl-terminal 33 residues similar to the housefly phenoloxidase inhibitor (POI). The recombinant POI, secreted into the Escherichia coli periplasmic space along with its fusion partner DsbC, was released by osmotic shock and isolated by nickel affinity chromatography. Following enterokinase digestion and protein separation, the POI was purified to near homogeneity in a soluble form which inhibited M. sexta PO at a high concentration. We then produced the inhibitor using a modified baculovirus-insect cell system and isolated the glycoprotein from the conditioned medium. Deglycosylation coupled with inhibition assay revealed that O-glycosylation only moderately increased its inhibitory activity. While this led us to speculate the role of Tyr(64) hydroxylation, we were unable to modify the recombinant protein with tyrosine hydroxylase or purify M. sexta POI (Tyr(64)dopa) from the larval plasma. Instead, we isolated a low-M(r), heat-stable compound which strongly inhibited PO. The wavelength of maximum absorbance is 257 nm for the inhibitor. These data suggest that the down-regulation of PO activity in M. sexta is achieved by two mechanisms at least.     

家蝇蛹和果蝇蛹繁育的蝇蛹金小蜂对果蝇蛹的寄生比较

【目的】蝇蛹金小蜂Pachycrepoideus vindemmiae(Rondani)是杨梅园等果园果蝇类害虫蛹期常见寄生蜂种类,在对果蝇类害虫的生物防治上具有重要价值。本文旨在探讨使用家蝇蝇蛹为替代寄主繁育蝇蛹金小蜂的方法。【方法】探讨分别以家蝇蛹和果蝇蛹繁育的蝇蛹金小蜂对家蝇和果蝇蝇蛹的选择性,并比较了在两种寄主上繁育的蝇蛹金小蜂在大小、寿命、产卵期、后代产量和性比等方面的差异。【结果】结果表明与果蝇蛹相比,家蝇蛹明显较大,在家蝇蛹上发育的蝇蛹金小蜂后代个体也明显较大;家蝇蛹和果蝇蛹发育的寄生蜂雌蜂寿命为(13.4±4.11)和(3.94±2.49)d、产卵期分别为(11.4±4.11)和(3.13±2.42)d、单头雌蜂后代雌蜂数量分别为(34.31±31.83)和(7.88±3.58)头,在家蝇蛹上繁育的寄生蜂明显具有较长的寿命和产卵期、更多的雌雄蜂后代数量;在对家蝇蛹和果蝇蛹的选择上,繁育自家蝇和果蝇的蝇蛹金小蜂雌蜂选择频率的差异不大。【结论】利用家蝇蛹繁殖的蝇蛹金小蜂在寄生果蝇蛹时具有更大优势,在繁殖蝇蛹金小蜂控制杨梅园等果蝇的为害时,可以选择家蝇蛹作为替代寄主。     

A new mechanism for the control of phenoloxidase activity: inhibition and complex formation with quinone isomerase

Insect phenoloxidases participate in three physiologically important processes, viz., cuticular hardening (sclerotization), defense reactions (immune reaction), and wound healing. Arrest or even delay of any of these processes compromises the survival of insects. Since the products of phenoloxidase action, viz., quinones, are cytotoxic, uncontrolled phenoloxidase action is deleterious to the insects. Therefore, the activity of this important enzyme has to be finely controlled. A novel inhibition of insect phenoloxidases, which serves as a new regulatory mechanism for control of its activity, is described. The activity of phenoloxidases isolated from both Sarcophaga bullata and Manduca sexta is drastically inhibited by quinone isomerase (isolated from Calliphora), an enzyme that utilizes the phenoloxidase-generated 4-alkylquinones. In turn, phenoloxidase reciprocated the inhibition of isomerase. By forming a complex and controlling each other's activity, these two enzymes seem to regulate the levels of endogenously quinones. In support of this contention, an endogenous complex consisting of phenoloxidase, quinone isomerase, and quinone methide isomerase was characterized from the insect, Calliphora. This sclerotinogenic complex was isolated and purified by borate extraction of the larval cuticle, ammonium sulfate precipitation, and Sepharose 6B column chromatography. The complex exhibited a molecular mass of about 620-680 kDa, as judged by size-exclusion chromatography on Sepharose 6B and HPLC and did not even enter 3% polyacrylamide gel during electrophoresis. The phenoloxidase activity of the complex exhibited a wide substrate specificity. Incubation of the complex with N-acetyldopamine rapidly generated N-acetylnorepinephrine, dehydro-N-acetyldopamine, and its dimers. In addition, transient accumulation of N-acetyldopamine quinone was also observed. These results confirm the presence of phenoloxidase, quinone isomerase, and quinone methide isomerase in the complex. Attempts to dissociate the complex with even trace amounts of SDS ended in the total loss of quinone isomerase activity. The complex does not seems to be made up of stoichiometric amounts of individual enzymes as the ratio of phenoloxidase to quinone isomerase varied from preparation to preparation. It is proposed that the complex formation between sequential enzymes of sclerotinogenic pathway is advantageous for the organism to effectively channel various reactive intermediates during cuticular hardening.     

以冷冻和新鲜家蝇蛹为寄主的蝇蛹俑小蜂实验种群生命表参数比较

【目的】为了阐明以冷冻保存家蝇Musca domestica蛹为寄主对繁殖蝇蛹俑小蜂Spalangia endius的影响。【方法】本研究分别利用新鲜和-20℃冷冻保存的家蝇蛹为寄主,记录了小蜂日存活数、日后代数量和性别等,并分别构建了实验种群生命表,分析比较了两种类型家蝇蛹对小蜂寄生率、日后代数量和性比、生命表参数等的影响。【结果】与新鲜家蝇蛹为寄主时相比,小蜂在以冷冻蛹为寄主时寄生率、后代数量和雄性百分比均较低（P<0.01）,成蜂寿命和产卵期差异不大（P>0.05）。在以新鲜和冷冻家蝇蛹为寄主时,小蜂寄生率和后代数量均随日龄的增加而显著降低（P<0.01）;以新鲜和冷冻家蝇蛹为寄主时小蜂的净生殖率（R₀）分别为34.91和20.16,种群内禀增长率（r_m）分别为0.17和0.11,均以寄生新鲜家蝇蛹时较大（P<0.01）;在以冷冻家蝇蛹为寄主时,世代时间和种群倍增时间较以新鲜家蝇蛹为寄主时有所延长（P<0.01）。【结论】蝇蛹俑小蜂可以利用冷冻家蝇蛹为寄主完成生活史;在规模化繁殖蝇蛹俑小蜂时,使用冷冻方式保存家蝇蛹的方法具有重要价值。     

Experimental climate effect on seasonal variability of polyphenol/phenoloxidase interplay along a narrow fen–bog ecological gradient in Sphagnum fallax

Extracellular phenoloxidase enzymes play an important role in the stability of soil carbon storage by contributing to the cycling of complex recalcitrant phenolic compounds. Climate warming could affect peatland functioning through an alteration of polyphenol/phenoloxidase interplay, which could lead them to becoming weaker sinks of carbon. Here, we assessed the seasonal variability of total phenolics and phenoloxidases subjected to 2–3 °C increase in air temperature using open‐top chambers. The measurements were performed along a narrow fen–bog ecological gradient over one growing season. Climate warming had a weak effect on phenoloxidases, but reduced phenolics in both fen and bog areas. Multivariate analyses revealed a split between the areas and also showed that climate warming exacerbated the seasonal variability of polyphenols, culminating in a destabilization of the carbon cycle. A negative relationship between polyphenols and phenoloxidases was recorded in controls and climate treatments suggesting an inhibitory effect of phenolics on phenoloxidases. Any significant decrease of phenolics through repeatedly elevated temperature would greatly impact the ecosystem functioning and carbon cycle through an alteration of the interaction of polyphenols with microbial communities and the production of extracellular enzymes. Our climate treatments did not have the same impact along the fen–bog gradient and suggested that not all the peatland habitats would respond similarly to climate forcing.     

Isolation of latent phenoloxidase from prepupae of the housefly,Musca domestica

Latent phenoloxidase was purified from prepupae of the housefly, Musca domestica vicina Maquart. The purification procedures included DEAE-cellulose column chromatography, sucrose density gradient centrifugation adn second sucrose density gradient centrifugation. The final preparations appear to be homogeneous based on results obtained from polyacrylamide gel electrophoresis in the presence of EDTA. Electrophoresis in the absence of EDTA caused spontaneous activation of latent phenoloxidase. While latent phenoloxidase was fairly stable over the range of temperatures between 0 and 40°C, it was quite sensitive to changes in pH, being stable only around pH 6.0. The molecular weight of latent phenoloxidase was estimated to be 178,000, as determined by gel filtration and sucrose density gradient centrifugation. Furthermore, phenoloxidase formed by the activation of latent phenoloxidase indicated a higher molecular weight (340,000) than that of latent phenoloxidase. Thus, it appears that the mechanism of the activation of latent phenoloxidase involves the association and disassociation system.     

Switch between tyrosinase and catecholoxidase activity of scorpion hemocyanin by allosteric effectors

Phenoloxidases and hemocyanins have similar type 3 copper centers although they perform different functions. Hemocyanins are oxygen carriers, while phenoloxidases (tyrosinase/catecholoxidase) catalyze the initial step in melanin synthesis. Tyrosinases catalyze two subsequent reactions, whereas catecholoxidases catalyze only the second one. Recent results indicate that hemocyanins can also function as phenoloxidases and here we show for the first time that hemocyanin can be converted to phenoloxidase. Furthermore, its substrate specificity can be switched between catecholoxidase and tyrosinase activity depending on effectors such as hydroxymethyl-aminomethan (Tris) and Mg(2+)-ions. This demonstrates that substrate specificity is not caused by a chemical modification of the active site.     

Phenoloxidase Activity and Fruiting Body Formation in Schizophyllum commune

Studies of induced haploid fruiting in the Basidiomycete Schizophyllum commune suggested that polyphenoloxidase enzymes may participate in the formation of fruiting bodies. Reported inhibitors of phenoloxidases were found to affect both the activity of these enzymes and the fruiting response. Fertile mycelia were found to give a positive Bavendamm reaction in situ, and cell-free extracts from fertile mycelia exhibited polyphenoloxidase activity when studied spectrophotometrically. Sterile mycelia, on the other hand, were devoid of these activities. Various kinds of mutant homo- and dikaryotic mycelia were studied, and the results suggested a correlation between phenoloxidase activity and fruiting body formation in S. commune.     

Functional and phylogenetic analyses of phenoloxidases from brachyuran (Cancer magister) and branchiopod (Artemia franciscana, Triops longicaudatus) crustaceans

Arthropod phenoloxidases catalyze the melanization and sclerotization of the new postmolt exoskeleton, and they function in the immune response. Hemocyanin, phylogenetically related to phenoloxidase, can function as a phenoloxidase under certain conditions. We investigated the relative contributions of hemocyte phenoloxidase and hemocyanin in the brachyuran crab Cancer magister, using the physiological ratio at which they occur in the hemolymph, and found that hemocyte phenoloxidase has higher activity. They both convert diphenols to o-quinones, but only the hemocyte phenoloxidase is able to catalyze the conversion of monophenols to diphenols. The quaternary structure of hemocyanin affects its reactivity as phenoloxidase. We suggest that prophenoloxidase is released from hemocytes and moves across epidermis into new exoskeleton during premolt and is activated in early postmolt. In addition to functional studies, we have determined the complete cDNA sequence of C. magister hemocyte prophenoloxidase and partial sequences from the branchiopods Artemia franciscana and Triops longicaudatus. We also sequenced C. magister cryptocyanin 2 and a hemocyanin from the amphipod Cyamus scammoni and used these and other members of the arthropod hemocyanin superfamily for phylogenetic analyses. The phylogenies presented here are consistent with the possibility that a common ancestral molecule had both phenoloxidase and reversible oxygen-binding capabilities.     

Laccase and tyrosinase activities in lichens

Phenoloxidase activity was found in lichenized ascomycetes belonging to different taxonomic groups. Most of the epigeic and epilithic lichens of the order Peltigerales were found to possess both laccase and tyrosinase activities; the lichens of the order Lecanorales possessed only laccase activity, which was an order of magnitude lower than that of Peltigerales. Water-soluble phenoloxidases were present only in peltigerous lichens: activity that could be washed out from intact thalli comprised 10% of that released from disrupted thalli. The activity of the peltigerous lichens and the release of soluble phenoloxidases into the medium increased when the thalli were rehydrated quickly. In some of the lichens tested, the phenoloxidase activity was stimulated by desiccation-rehydration cycles. The oxidases discovered may play an important role in the phenolic metabolism of lichens and be involved in the biochemical reaction of humus synthesis during primary soil formation, which may be a previously unknown geochemical function of these symbiotic microorganisms.     

A novel endogenous inhibitor of phenoloxidase from Musca domestica has a cystine motif commonly found in snail and spider toxins

Phenoloxidase inhibitor (POI), found in the hemolymph of housefly pupae, is a novel dopa-containing and cystine-rich peptide that competitively inhibits phenoloxidase with a Ki in the nanomolar range. [Tyr32]POI is a potential precursor molecule also found in the hemolymph that may be posttranslationally oxidized to the dopa-containing peptide after creation of a rigid structure. By employing both a solid-phase peptide synthesis system based on a 9-fluorenylmethoxycarbonyl strategy and a specific air oxidation technique to ensure correct folding, we have been able to synthesize [Tyr32]POI. The synthetic [Tyr32]POI was confirmed to be identical to the native [Tyr32]POI by coelution high-performance liquid chromatography analysis and by enzymatic analysis using the phenoloxidase inhibition assay. To determine the disulfide pairings within the peptides, a series of enzyme hydrolyses and partial reduction/alkylation steps were performed. Three cystine pairs (Cys11-Cys25, Cys18-Cys29, and Cys24-Cys36) were determined by identification of the resulting peptides. The disulfide pairings of the two adjacent Cys residues (Cys11-Cys25 and Cys24-Cys36) were unambiguously assigned by comparing the derived fragments with the two possible isomers synthesized through a novel disulfide-linking technique. The arrangement of the disulfide bridges in POI was found to be topologically identical to those found for several peptides within the inhibitor cystine knot structural family. Although these peptides share a low primary sequence homology and display a diversity of biological functions, they nonetheless share similarities in their cystine motifs and tertiary structure. The tertiary structure model of POI, which was derived through molecular dynamics and energy minimization studies using restraints with determined disulfide connectivities, suggests that POI is a new class member of the inhibitor cystine-knot structural family.     

Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica)

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR?DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.     

Early season dispersal of Muscidifurax zaraptor (Hymenoptera: Pteromalidae) utilizing freeze-killed housefly pupae as hosts

The pteromalid wasp, Muscidifurax zaraptor Kogan and Legner, was released at three locations at a dairy in May before housefly and stable fly breeding had begun. Freeze-killed housefly pupae were placed adjacent to the emerging parasites at biweekly intervals for a 6-week period. Hosts placed out weeks 0 and 2 were heavily parasitized. Decreased parasitism in hosts placed out at week 4 suggested that many of the M. zaraptor had dispersed or died. High parasitism of hosts placed in the field at week 6 was the result of second generation parasites emerging from pupae placed out at week 0. Parasitism of freeze-killed housefly pupae placed 6 m and in the four cardinal directions from the release points was similar but lower than for hosts placed adjacent to the emerging parasites. The study demonstrated that emerging M. zaraptor readily utilized nearby freeze-killed housefly pupae but the availability of these hosts did not deter the parasites from searching for additional hosts.     

蝇蛹金小蜂对家蝇蛹的寄生策略

蝇蛹金小蜂Pachycrepoideus vindemmiae Rondani是家蝇Musca domestica蛹期常见寄生蜂种类。本文探讨蝇蛹金小蜂对寄主日龄的选择策略以及该寄生蜂的寿命、产卵历期和后代数量等规律。结果表明寄生蜂可利用各日龄的蝇蛹,寄生高龄期蝇蛹时,寄生蜂后代产量显著降低,既未出蜂也未出蝇的死亡蝇蛹比例显著增加;寄生蜂寿命为(11.89±6.99)d,产卵历期为(9.58±6.67)d,单个雌蜂后代产量为(33.74±18.08)头,雄性后代的发育历期显著短于雌性后代,随着寄生蜂产卵历期的延长,寄生蜂后代产量下降,雄性后代比例增加。     

Phenoloxidase of peach (Prunus persica) endocarp: Its relationship with peroxidases and lignification

A preliminary characterization of a phenoloxidase from extracts of soluble and ionically-bound cell wall proteins of peach ( Prunus persica L. Batsch, cv. Redhaven) endocarp is described in the present study to establish differences with peroxidases from the same plant tissue. The phenoloxidase activity was detected mainly in the first stage of peach fruit growth, while peroxidase activity and lignin content increased along the second stage of growth. There were clear differences between the two enzymes. The phenoloxidase had a pI value of 5.6, different from those of peroxidases isoenzymes with various pIs ranging from 3.6 to 9.6. The oxidase molecular mass was 112 kDa, similar to other phenoloxidases described in the literature, while all peroxidase isoenzymes showed a molecular mass of around 40 kDa. The specific activities of phenoloxidase against different substrates and its inhibition by various effectors suggest that the endocarp oxidase described here is probably a metal-dependent polyphenol oxidase, displaying attributes of both catechol oxidase (EC 1.10.3.1) and laccase (EC 1.10.3.2).