Hemolymph phenol oxidases in Drosophila melanogaster, Locusta migratoria, and Austropotamobius pallipes

The early enzyme-mediated reaction sequence in the biosynthesis of melanin from L-tyrosine involves an initial hydroxylation (monophenol oxidase activity, MPO) of the aromatic amino acid precursor to form L-dopa (3,4-dihydroxyphenylalanine), and the ensuing oxidation (diphenol oxidase activity, DPO) of the resultant diphenol to form dopaquinone. By means of high pressure liquid chromatography with electrochemical detection (HPLC-ED) both phenol oxidase activities were observed in the blood (hemolymph) of two species of insect, third-stage larvae of Drosophila melanogaster and adult Locusta migratoria, and in an adult fresh-water crayfish, Austropotamobius pallipes. These results establish that in each species MPO and DPO can be detected readily without the use of exogenous activators.     

Parasite-induced enhancement of hemolymph tyrosinase activity in a selected immune reactive strain of Drosophila melanogaster

Larval hemolymph tyrosinase activity in Drosophila melanogaster was detected with high performance liquid chromatography with electrochemical detection. The enzyme hydroxylated L-tyrosine, and oxidized the diphenol substrates L-dopa and dopamine. In larvae of a selected immune-reactive strain the rates of tyrosine hydroxylation, dopa oxidation, and dopamine oxidation were markedly increased during the early stages of melanotic encapsulation of the eggs of the parasitic wasp Leptopilina boulardi. Tyrosinase activity was not modified in parasitized larvae of a selected susceptible strain of D. melanogaster, in which hosts the parasitoids developed unmolested. During the same period of parasitization, the amount of free tyrosine in immune reactive larvae was approximately three times higher than in susceptible hosts. These data indicate that the tyrosinase system of the immune reactive strain is activated during parasitization, and this results in the synthesis of some precursors which ultimately produce a melanotic and sclerotic capsule around the eggs of the parasite. Based on known genetic information of the enzyme system in Drosophila, it appears that at least two genes may be involved in the activation process, one associated with the proenzyme for monophenol oxidase activity, and the second with the proenzyme for diphenol oxidase activity.     

Immulectin, an inducible C-type lectin from an insect, Manduca sexta, stimulates activation of plasma prophenol oxidase.

Immulectin, a C-type lectin from the tobacco hornworm, Manduca sexta, was cloned from a larval fat body cDNA library. The immulectin cDNA encodes a 309 residue polypeptide. Immulectin synthesis was induced by injection of killed gram-positive or gram-negative bacteria or yeast. After injection of bacteria, immulectin mRNA appeared in fat body and immulectin protein was detected in hemolymph. Immulectin contains two carbohydrate recognition domains. The carboxyl-terminal carbohydrate recognition domain is most similar (36% identity) to a lipopolysaccharide-binding protein from the American cockroach, Periplaneta americana. It also shares 26-35% identity to carbohydrate recognition domains of various mammalian C-type lectins. Two immulectin isoforms were identified in the hemolymph of bacteria-injected larvae. Recombinant immulectin agglutinated gram-positive and gram-negative bacteria and yeast. Addition of recombinant immulectin to M. sexta plasma stimulated activation of phenol oxidase. A combination of immulectin with lipopolysaccharide from E. coli activated phenol oxidase more rapidly and to a higher level than immulectin alone, whereas lipopolysaccharide by itself had little effect on phenol oxidase activation. Immulectin synthesized in response to bacterial or fungal infection may help to trigger protective responses in M. sexta in a manner similar to mannose-binding protein, a C-type lectin that functions in the mammalian innate immune system.     

Effect of ionic concentration on the higher-order structure of prophenol oxidase in Drosophila melanogaster

Phenol oxidase in Drosophila melanogaster occurs as precursors designated prophenol oxidases A₁ and A₃. Crossing experiments between isozyme variants proved that prophenol oxidase in this species is a homodimer. Prophenol oxidases were partially purified using ammonium sulfate fractionation, phenyl Sepharose, and DEAE-cellulose column chromatography. The preparations were mixed, then dialyzed against buffer containing varying salt concentrations. The resulting prophenol oxidase was analyzed by gel electrophoresis. At 20 mM KCl or NaCl, two bands of phenol oxidase were observed, corresponding to the parental ones as monomer, whereas at 200 mM KCl or NaCl, three bands appeared in the gel, one being a dimer. The monomer–dimer reversibility of the Drosophila prophenol oxidase depends on the salt concentrations. The phenol oxidase activity remained unaffected within the KCl concentrations tested. Considering the ionic concentration of Drosophila hemolymph, these results indicate that prophenol oxidase exists as a dimer in vivo, and the higher-order structure of prophenol oxidase can be altered reversibly by ionic concentrations in vitro.     

A mutant affecting the crystal cells inDrosophila melanogaster

Summary Black cells (Bc, 2-80.6±) mutant larvae ofDrosophila melanogaster have pigmented cells in the hemolymph and lymph glands. In this report we present evidence that these melanized cells are a mutant form of the crystal cells, a type of larval hemocyte with characteristic paracrystalline inclusions.Bc larvae lack crystal cells. Furthermore, the distribution pattern of black cells inBc larvae parallels that of experimentally-blackened crystal cells in normal larvae (phenocopy).InBc/Bc zygotes black cells appear during mid embryonic development but inBc ⁺/Bc zygotes pigmented cells are not found until late in the first larval instar.Crystal cells are present in the heterozygous larvae until this time, and paracrystalline inclusions can be seen in some of the cells undergoing melanization in these larvae.The rate of phenol oxidase activity inBc ⁺/Bc larval cell-free extracts is less than half that ofBc ⁺/Bc ⁺extracts whereas enzyme activity is undetectable inBc/Bc larvae. We propose that theBc ⁺gene product is required for maintaining the integrity of the paracrystalline inclusions; inBc/Bc larvae either the product is absent or nonfunctional so an effective contact between substrate and enzyme results in melanization of the cells.Phenol oxidase itself is either destroyed or consumed in the melanization process accounting for the absence of enzyme activity inBc/Bc larvae. These studies confirm that the crystal cells store phenolic substrates and are the source of the hemolymph phenol oxidase activity in the larva ofD. melanogaster.     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Proteomics of immune-challenged Drosophila melanogaster larvae hemolymph

In the last decade, the fruit fly Drosophila melanogaster has emerged as a promising invertebrate model for the investigation of innate immunity, in part because of its well characterised genetics. The information provided by the innumerous reports on Drosophila's immune response indicates that a large number of genes, in addition to the well-known antimicrobial peptide genes, are both up- and down-regulated upon immune challenge. Nevertheless, their contribution to fighting off infection has not been seriously addressed. With the application of recent advances in proteomics, the effects of an immune challenge in the overall modification of Drosophila 2-DE protein patterns were investigated. The aim of this study was to investigate hemolymph proteins differentially expressed between control and immunised larvae sets, which could be related solely to the Drosophila immune response. The list of immune-related protein spots included heat shock proteins and other proteins with chaperone properties, serine proteases, phenol oxidase, and Drosophila antioxidant system components, which accounted for 21% of the total of 70 identified proteins, metabolic enzymes implicated in pathways such as cellular respiration, fatty-acid oxidation, protein biosynthesis, and structural proteins.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

Phenol oxidase activity: inductionin female schistosomes by in vitro incubation.

A biochemical methods has been developed for detecting phenol oxidase in female Schistosoma mansoni. Enzyme activity is observed only after incubation of the female schistosomes for an extended period of time in tissue culture media. Male S. mansoni do not contain detectable levels of phenol oxidase activity. The properties of this enzyme are similar to those identified for a phenol oxidase from Fasciola hepatica. L-DOPA, dopamine, and tyrosine were found to be good substrates for this enzyme. Vmax = 14.1, 8.1, and 6.1 mumoles O2/min/mg protein for each substrate, respectively. This enzyme appears to be associated with egg production and thus may be a useful marker for biochemical and immunological studies.     

亚热带砂岩发育森林土壤酚氧化酶活性测定方法优化

酚氧化酶在土壤有机质降解过程中起重要作用,然而,目前用于测定土壤酚氧化酶活性的方法尚未统一。本研究以亚热带地区砂岩发育的3种不同林分的森林土壤为对象,探讨底物类型、pH值、土壤储存条件、储存时间、底物浓度、水土比、培养时间和温度对土壤酚氧化酶活性的影响,以期建立统一、可比较的测定亚热带森林土壤酚氧化酶活性的方法。结果表明: 浸提液pH值显著影响土壤酚氧化酶活性,且与目前普遍使用的左旋多巴胺(L-DOPA)相比,2,2′-联氨-双(3-乙基苯并噻唑啉-6-磺酸)-二胺盐(ABTS)所测得的氧化酶活性更高、适用pH值范围更广,说明ABTS可能更适合作为测定亚热带森林酸性土壤酚氧化酶活性的底物。储存方式显著影响酚氧化酶活性,3种供试土壤样品酚氧化酶活性均随时间呈降低的趋势,降幅表现为风干> 4 ℃冷藏> -20 ℃冷冻> -80 ℃冷冻,表明在无法保证快速测定土壤酚氧化酶活性的情况下,冷冻保存方式更有利于维持土壤酚氧化酶活性。底物浓度、水土比以及培养时间和温度均影响土壤酚氧化酶活性。当土壤样品与浸提液比例为1∶100时,选择2 mmol·L^-1浓度的ABTS为底物,在25~30 ℃下培养4 h,测定酚氧化酶活性结果重复性好、灵敏度高,是测定亚热带森林酸性土壤酚氧化酶活性的最优条件。     

Genetic control of phenoloxidase in the imago of Drosophila melanogaster]

The A1 component of Drosophila melanogaster phenol oxidase is controlled by an independent structural gene which has been located by the method of recombination mapping on the second chromosome at 67.3. The Dox-A1 is the only one from the group of genes coding for phenol oxidase in Drosophila which is expressed at the imago stage. The activity of phenol oxidase depends on the level of gene Dox-A1 expression and also on the presence in haemolymph of proenzyme activators. Males have a minimum level of phenol oxidase activity, which possibly protects them from mechanical damages of cuticle and toxic effects. Females are characterized by a higher level of A1-phenol oxidase activity than males. This may be caused by their reproduction function.     

Divergence of larval resource acquisition for water conservation and starvation resistance in Drosophila melanogaster

Laboratory selection experiments have evidenced storage of energy metabolites in adult flies of desiccation and starvation resistant strains of D. melanogaster but resource acquisition during larval stages has received lesser attention. For wild populations of D. melanogaster, it is not clear whether larvae acquire similar or different energy metabolites for desiccation and starvation resistance. We tested the hypothesis whether larval acquisition of energy metabolites is consistent with divergence of desiccation and starvation resistance in darker and lighter isofemale lines of D. melanogaster. Our results are interesting in several respects. First, we found contrasting patterns of larval resource acquisition, i.e., accumulation of higher carbohydrates during 3rd instar larval stage of darker flies versus higher levels of triglycerides in 1st and 2nd larval instars of lighter flies. Second, 3rd instar larvae of darker flies showed ~40?h longer duration of development at 21°C; and greater accumulation of carbohydrates (trehalose and glycogen) in fed larvae as compared with larvae non-fed after 150?h of egg laying. Third, darker isofemale lines have shown significant increase in total water content (18%); hemolymph (86%) and dehydration tolerance (11%) as compared to lighter isofemale lines. Loss of hemolymph water under desiccation stress until death was significantly higher in darker as compared to lighter isofemale lines but tissue water loss was similar. Fourth, for larvae of darker flies, about 65% energy content is contributed by carbohydrates for conferring greater desiccation resistance while the larvae of lighter flies acquire 2/3 energy from lipids for sustaining starvation resistance; and such energy differences persist in the newly eclosed flies. Thus, larval stages of wild-caught darker and lighter flies have evolved independent physiological processes for the accumulation of energy metabolites to cope with desiccation or starvation stress.     

Endogenous protease inhibitors prevent undesired activation of prophenolase in insect hemolymph

Phenoloxidase activation in the whole hemolymph of Sarcophaga bullata and Manduca sexta larvae is shown to be achieved by proteolytic cleavage of the proenzyme. This process is inhibited by the serine protease inactivator, Diisopropyl phosphofluoridate. Endogenous protease inhibitors isolated from the larvae inhibit alpha-chymotrypsin mediated prophenoloxidase activation in the hemolymph. These observations suggest that the endogenous protease inhibitors prevent undesired activation of prophenol oxidase in the hemolymph by inhibiting the serine protease involved in the activation process.     

Effects of dietary or injected organic cations on larval Drosophila melanogaster: mortality and elimination of tetraethylammonium from the hemolymph

This study of larval Drosophila melanogaster examined the effects of injecting the prototypical organic cation tetraethylammonium (TEA) into the hemocoel or adding TEA and/or other organic cations to the diet. Mortality, hemolymph TEA levels, and Malpighian tubule TEA secretion rates were measured. The LD50 for dietary TEA was 158.4 mM and mortality increased if competitive inhibitors of organic cation transporters were also included in the diet. Mortality increased from 24% on TEA (100 mM) alone to 83 and 67% when the diet contained both TEA and quinidine (10 mM) or cimetidine (100 mM), respectively. TEA-selective microelectrode measurements indicated that hemolymph TEA concentration was approximately 3% of that in the diet for larvae maintained on TEA-enriched diet for 24 h. Malpighian tubules isolated from larvae exposed to dietary TEA excreted more TEA than did tubules from controls fed a TEA-free diet. However, the rate of decline of hemolymph TEA concentration following ingestion or injection of TEA into the hemocoel was greater than that explicable by rates of active transport by the gut and Malpighian tubules (MTs). We propose that TEA concentrations in the hemolymph are reduced not only by active transport across the MTs and gut, but also by diffusion into the gut. The latter pathway is particularly important when larvae previously maintained upon TEA-enriched diet are transferred to a TEA-free diet. The ingestion of TEA-free food not only clears the gut lumen, but also creates a TEA-free compartment into which TEA may passively diffuse from the hemolymph.     

Purification,characterization, and identification of a novel bifunctional catalase-phenol oxidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.     

Phenol oxidase is a necessary enzyme for the silkworm molting which is regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. The molting process includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis excoriation etc. Polyphenol oxidases (PPOs), dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the phenol oxidase was considered as an enzyme for epidermal layer’s tanning and melanization. This work suggested that polyphenol oxidases are one set of the key enzymes in molting, which closely related with the role of ecdysone in regulation of molting processes. The data showed that the expression peak of phenol oxidase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the phenol oxidase expression was significantly elevated compared to the control. PPO1 RNAi induced phenol oxidase expression obviously declined in the silkworm larvae, and caused the pupae incomplete pupation. Overall, the results described that the phenol oxidase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.     

Ultrastructure and Cytochemistry of the Cell-types in the Tumorous Hematopoietic Organs and the Hemolymph of the Mutant Lethal (1) Malignant Blood Neoplasm (l(1)mbn) of Drosophila Melanogaster

The fine structure and histochemistry of the neoplastic primordial blood cell-types in the larval hematopoietic organs and the mature cell-types in the hemolymph of the blood tumor mutant lethal (1) malignant blood neoplasm (l(1) mbn ) of Drosophila melanogaster were investigated. In this mutant the cell-types of the plasmatocyte-line are neoplastic while the cell-types of the crystal-cell-line are not and are much reduced in numbers (1, 2).
In contrast to the wild-type the mutant hematopoietic organs are enlarged and contain, in addition to primordial blood-cells, large numbers of mature plasmato-, podo-, and lamellocytes.
All cell-types of the plasmatocyte-line differ in their fine structure and behavior from their wild-type counterparts. The mutant blood cells show generally a numerical increase of cell organelles and acid phosphatase positive primary and secondary lysosomes. In the phenol oxidase test they showed a vigorous melanization reaction. Plasmato- and podocytes invade into the tissues of the larva and show high phagocytic activity.     

Observation of tyrosine-O-phosphate in Drosophila melanogaster larvae by 31P-NMR spectroscopy

31P-NMR spectra of intact larvae and pupae of Drosophila melanogaster have been obtained at 109.3 MHz. A major resonance in these samples has been identified as tyrosine-O-phosphate. Its chemical shift reflects the hemolymph plasma pH. Upon disruption of the organisms (necessary for chemical analyses of tyrosine-O-phosphate), phosphatases rapidly hydrolyze this phosphate ester, generating inorganic phosphate and free tyrosine.     

Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system

Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly established Bac-to-Bac/BmNPV Baculovirus expression system was used to overexpress the recombinant Mn-SOD enzyme in silkworm larvae. The hemolymph was collected from the infected larvae 96 h post-infection and subjected to a 12 % SDS-PAGE and Western blotting. A 18.0-kDa protein was visualized after rBacmid/BmNPV/SOD infection. The SOD enzyme activity was determined with a tetrazolium salt for detection of superoxide radicals generated by xanthine and xanthine oxidase and its peak appeared in 96 h post-infection with 2.7 times of the control larvae. The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for potential application in medicine.     

Genotoxicity of triasulfuron in the wing spot test of Drosophila melanogaster is modulated by winter wheat seedlings

Triasulfuron (TS) is a widely used sulfonylurea herbicide which inhibits the acetolactate synthase in broad-leaf weeds and in some wheat crop grasses (Triticum aestivum L.). Residues can be found in soil and superficial water with high toxicity to primary producers. In cereals, TS metabolism depends on cytochromes P450 (CYPs), the age of seedlings and the interaction with compounds. The genotoxicity of TS was demonstrated in the wing spot test of Drosophila melanogaster, an in vivo assay based on the loss of heterozygosity of the mwh and flr markers in the wing imaginal disk cells of larvae fed with chemical agents. Chronic treatments with analytical grade TS, commercial formulation TS (Amber) 75WG) (0.5mg/mL) and commercial formulation bentazon (Basagran) 480) (0.24mg/mL) were performed with three-day-old larvae of the standard (ST) and the high bioactivation (HB) crosses with regulated and high constitutive levels of CYPs, respectively. To demonstrate the effect of winter wheat metabolism on TS genotoxicity, T. aestivum L. seedlings were immersed for 4h in these herbicides, and aqueous extracts (AEs) of the roots were prepared to expose the larvae. TS and Amber 75WG produced similar genotoxic effects in both crosses. Wheat metabolism modulated the genotoxicity because the AEs yielded statistically significant lower spot frequencies in the HB cross than in the ST cross. Differences between the two crosses of the wing spot test in D. melanogaster must be related to CYPs levels. Basagran 480 was genotoxic only in the HB cross, and wheat metabolism did not modulate its genotoxicity.